Supercritical carbon dioxide extraction of astaxanthin from Corynebacterium glutamicum

Supercritical carbon dioxide extraction of astaxanthin from Corynebacterium glutamicum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Supercritical carbon dioxide extraction of astaxanthin from Corynebacterium glutamicum Jan Seeger, Maximilian Zäh, Volker F. Wendisch, Christoph Brandenbusch, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5798823/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 May, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted 5 You are reading this latest preprint version Abstract Astaxanthin, a red carotenoid with potent antioxidant properties, holds significant value in the feed, cosmetics, and nutraceutical industries. While traditionally sourced from microalgae, Corynebacterium glutamicum , a well-established industrial microorganism, has been engineered to serve as an efficient host for astaxanthin production. As astaxanthin integrates into the cellular membrane, effective extraction methods are essential to access this valuable compound. In this study, a sustainable batch extraction process using supercritical carbon dioxide (scCO₂) as a green solvent was developed. The effects of cosolvent concentration (0–9% ( w / w )), temperature (50–75°C), and pressure (450–650 bar) were investigated with regard to the extraction yield. An optimized extraction was achieved with 9% ( w / w ) ethanol as a cosolvent, at 68°C and 550 bar, allowing the extraction of 67.5 ± 3.7% of the cellular astaxanthin within 0.5 hours. Prolonging the extraction time further increased the recovery to 93.3%, which is comparable to processes that have been established for the extraction of astaxanthin from microalgae and yeast. This approach provides a scalable and environmentally friendly solution for industrial astaxanthin recovery. Astaxanthin supercritical carbon dioxide extraction Corynebacterium glutamicum Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Although more than 1,100 carotenoids occur in nature (Yabuzaki 2017), only a few are industrially relevant (Sathasivam and Ki 2018). Among them is the red-colored marine carotenoid astaxanthin with a current market size of USD 2.83 bn and an expected compound annual growth rate of 17.1% until 2030 (Astaxanthin Market 2024). Besides its application as an animal feed additive (Lim et al. 2018), astaxanthin is being more and more used in the nutraceutical and cosmetics industries due to its various health promoting effects (Galasso et al. 2017; Barreiro and Barredo 2018; Stachowiak and Szulc 2021). However, only natural astaxanthin can be used for the latter, as the usage of synthetic astaxanthin is not considered for human consumption (Li et al. 2011). The most common microbial production systems for natural astaxanthin are the microalgae Haematococcus pluvialis (Rodríguez-Sifuentes et al. 2020; An et al. 2024), the red yeast Xanthophyllomyces dendrorhous (Zhuang and Zhu 2021), and the Gram-negative bacterium Paracoccus carotinifaciens (Hayashi et al. 2021). Based on its lipophilic nature, astaxanthin (and its esterified derivatives) are either incorporated into the cellular membrane (Kishimoto et al. 2016) or stored in intracellular lipid droplets (Ota et al. 2018). Due to the relatively rigid cell envelope of microalgae and yeasts, it is generally necessary to permeabilize or disrupt the cells before extraction (Rodríguez-Sifuentes et al. 2020). This can be achieved using physical methods like bead milling (Molino et al. 2018b; Irshad et al. 2019) or high-pressure homogenization (Praveenkumar et al. 2020), chemical methods such as acid treatment (Sarada et al. 2006; Wu et al. 2011), or biological methods like enzymatic lysis (Machado et al. 2016; Harith et al. 2020). Different extraction processes have been developed, comprising (pressurized) organic solvents, e.g., acetone, ethanol, and ethyl acetate (Molino et al. 2018b; Irshad et al. 2019; Praveenkumar et al. 2020), vegetable oils (Kang and Sim 2008), ionic liquids (Desai et al. 2016), and eutectic solvents (Pitacco et al. 2022). Another solvent-based method for the extraction of astaxanthin is the supercritical fluid extraction (SFE). By reaching the supercritical point, the properties of the gas and the liquid converge, leading to a state characterized by (low) gas-like surface tension, diffusivity and viscosity, and liquid-like density (Knez et al. 2014). Being non-toxic, non-flammable, chemically stable as well as readily affordable, carbon dioxide (CO 2 ) is considered as a green solvent (Wu and Han 2019) making it the most common used solvent for SFE in food processing (Picot-Allain et al. 2021). The rather low supercritical point ( T C = 31.1°C, p C = 73.8 bar) of CO 2 (Wu and Han 2019) enables an extraction under mild conditions, thus avoiding thermal or chemical degradation of the extract (Picot-Allain et al. 2021). The main factor affecting the solvent power of supercritical CO 2 (scCO 2 ) is its density, which can be adjusted by pressure and temperature (Knez and Lütge 2023). Increasing the pressure increases the density, favoring the solubility of the solute. Conversely, higher temperatures elevate the solute's vapor pressure while reducing the solvent's density. Therefore, optimization of the process conditions is required for an efficient extraction process (Knez et al. 2014; Wang et al. 2021). The polarity of the scCO 2 can be modified by cosolvents (polar modifier) like polar organic solvents or plant oils (Krichnavaruk et al. 2008; Wang et al. 2021). After the extraction, simple pressure reduction enables the residue-free removal of the CO 2 from the extract (Kang et al. 2024). By employing multiple separators and gradually reducing pressure (and thus solvent power of scCO 2 ), different fractions of the extract can be collected (Knez and Lütge 2023). Recovery of the gas after expansion allows its recycling without the need of solvent purification (Knez et al. 2014; Kang et al. 2024). Apart from being used for the extraction of essential oils, phenolic compounds, and alkaloids from natural sources (Wang et al. 2021), scCO 2 extraction has been applied and optimized for the extraction of astaxanthin from H. pluvialis in numerous studies (Valderrama et al. 2003; Machmudah et al. 2006; Nobre et al. 2006; Krichnavaruk et al. 2008; Pan et al. 2012; Reyes et al. 2014; Di Sanzo et al. 2018; Molino et al. 2018a; Álvarez et al. 2020). Besides extraction from microalgae, scCO 2 extraction of astaxanthin has been also achieved from yeast (Lim et al. 2002; Harith et al. 2020), shrimp (Ahmadkelayeh et al. 2022), Gram-negative bacteria (Chougle et al. 2016), and oilseed (Xie et al. 2019). Renowned for the large-scale production of amino acids (Wendisch 2020), the Gram-positive soil bacterium Corynebacterium glutamicum has been proven to be a promising alternative to the aforementioned organisms for astaxanthin production. Harnessing its native carotenoid metabolic pathway, astaxanthin biosynthesis was enabled and improved in several studies, resulting in a titer of 103 mg/L astaxanthin in fed-batch fermentation (Henke et al. 2016; Henke and Wendisch 2019; Göttl et al. 2024). Recently, an extraction process based on ethanol was developed, resulting in a 94% recovery of astaxanthin (Seeger et al. 2023). An in vitro assay revealed a superior antioxidant activity of the obtained natural extract (astaxanthin oleoresin) compared to synthetic astaxanthin and a similar activity to microalgae-derived astaxanthin (Seeger et al. 2023). This study establishes a scCO₂-based extraction process for astaxanthin from C. glutamicum , aiming to provide an environmentally friendly and sustainable alternative that minimizes reliance on toxic organic solvents, thereby supporting advancements in the bio-economy. 2. Material and methods 2.1 Chemicals All chemicals were purchased from Carl Roth (Karlsruhe, Germany) or Sigma-Aldrich (St. Louis, MO, US). Organic solvents for extraction and analysis were HPLC grade. Carbon dioxide (99.9995% ( v / v )) was purchased from Messer (Bad Soden am Taunus, Germany). 2.2 Cultivation and harvesting of Corynebacterium glutamicum The astaxanthin producing strain Corynebacterium glutamicum ASTA* was cultivated as described in Henke and Wendisch (2019). After 48 h of cultivation, the cells were harvested by centrifugation at 10,000 x g for 20 min and were oven-dried at 50°C. The dried biomass contained 0.35 mg/g astaxanthin (quantification described in section 2.5). 2.3 Experimental apparatus A high-pressure variable-volume view cell (HPVVV; p max = 700 bar, T max = 180°C; New Ways of Analytics, Lörrach, Germany) was used for extraction. The required pressure was produced by adjusting the volume using a manual hydraulic press M(O) 189 (Maximator, Zorge, Germany). The system was homogenized by a stirrer. The temperature was measured inside the view cell and was regulated by a heating jacket. If applicable, ethanol was added through a 1/8″ port. A 260D syringe pump (Teledyne ISCO, Lincoln, NE, US) ( p max = 560 bar) was used for dosing compressed CO 2 into the HPVVV. 2.4 Experimental procedure 2.4.1 scCO 2 extraction without cosolvent The dried biomass (see section 2.2) was weighted, packed into a paper tea bag and positioned into the HPVVV. After sealing the HPVVV, CO 2 was loaded reaching a mass fraction of ≥ 0.99 compared to the biomass. Temperature and pressure were adjusted accordingly (see supplementary Table S1 for all tested conditions). If not stated differently, the extraction was terminated after 0.5 h by releasing the scCO 2 from the HPVVV. The biomass was taken out of the tea bag and subsequently processed for (residual) astaxanthin analysis (see supplementary Figure S1 for schematic experimental procedure). 2.4.2 scCO2 extraction with cosolvent Experimental procedure and loading of the cell with biomass was performed as described above. The cosolvent (ethanol) was added after the HPVVV front sapphire had been sealed. The CO 2 amount was introduced as in the previous case and temperature and pressure were adjusted accordingly (see supplementary Table S1 for all tested conditions). 2.5 Quantification of astaxanthin content by HPLC To determine the astaxanthin content of the biomass, a defined amount was extracted with 1 mL of a 7:3 ( v / v ) mixture of methanol:acetone at 1000 rpm and 60°C for 0.5 h using the ThermoMixer C (Eppendorf, Hamburg, Germany). After centrifugation for 10 min at 20,000 x g, the supernatant was analyzed via HPLC. The Agilent 1200 series (Agilent Technologies, Santa Clara, CA, US) equipped with a reversed-phase precolumn (LiChrospher 100 RP18 EC-5, 40 × 4 mm) (CS-Chromatographie, Langerwehe, Germany), a reversed-phase main column (LiChrospher 100 RP18 EC-5, 125 × 4 mm) (CS-Chromatographie, Langerwehe, Germany), and a diode array detector (DAD) was used for analysis. Methanol:water (9:1) (A) and methanol (B) were used as mobile phases. The injection volume was 50 µL, and a gradient at a flow rate of 1.5 mL min –1 was used as the following: 0 min B: 0%, 10 min B: 100%, and 32.5 min B: 100%. Carotenoids were quantified by recording the absorption at λ = 470 nm. Astaxanthin (Sigma-Aldrich, St. Louis, MO, US), adonirubin (CaroteNature, Münsingen, Switzerland), canthaxanthin (VWR, Darmstadt, Germany), echinenone (Sigma-Aldrich, St. Louis, MO, US), β-carotene (Sigma-Aldrich, St. Louis, MO, US), and lycopene (ExtraSynthese, Genay, France) were used to generate standard calibration curves. Exemplary HPLC chromatograms are shown in the supplementary Figure S2 . The extraction yield was calculated according to Eq. ( 1 ) with the total extracted amount of carotenoid calculated by comparing the initial carotenoid content of the biomass with the carotenoid content after the scCO 2 extraction (Eq. 2 ) $$\:\varvec{E}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:\varvec{y}\varvec{i}\varvec{e}\varvec{l}\varvec{d}\:\left[\varvec{\%}\right]=\left(\frac{\varvec{E}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{e}\varvec{d}\:\varvec{c}\varvec{a}\varvec{r}\varvec{o}\varvec{t}\varvec{e}\varvec{n}\varvec{o}\varvec{i}\varvec{d}\:\left[\varvec{m}\varvec{g}\:{\varvec{g}}^{-1}\right]}{{\varvec{C}\varvec{a}\varvec{r}\varvec{o}\varvec{t}\varvec{e}\varvec{n}\varvec{o}\varvec{i}\varvec{d}}_{\varvec{b}\varvec{e}\varvec{f}\varvec{o}\varvec{r}\varvec{e}\:\varvec{e}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}}\left[\varvec{m}\varvec{g}\:{\varvec{g}}^{-1}\right]}\:\times\:100\right)$$ 1 With $$\:\varvec{E}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{e}\varvec{d}\:\varvec{c}\varvec{a}\varvec{r}\varvec{o}\varvec{t}\varvec{e}\varvec{n}\varvec{o}\varvec{i}\varvec{d}=\:{\varvec{C}\varvec{a}\varvec{r}\varvec{o}\varvec{t}\varvec{e}\varvec{n}\varvec{o}\varvec{i}\varvec{d}}_{\varvec{b}\varvec{e}\varvec{f}\varvec{o}\varvec{r}\varvec{e}\:\varvec{e}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}}\left[\varvec{m}\varvec{g}\:{\varvec{g}}^{-1}\right]-\:{\varvec{C}\varvec{a}\varvec{r}\varvec{o}\varvec{t}\varvec{e}\varvec{n}\varvec{o}\varvec{i}\varvec{d}}_{\varvec{a}\varvec{f}\varvec{t}\varvec{e}\varvec{r}\:\varvec{e}\varvec{x}\varvec{t}\varvec{r}\varvec{a}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}}\left[\varvec{m}\varvec{g}\:{\varvec{g}}^{-1}\right]$$ 2 3. Results For the establishment of a scCO 2 -based extraction process of astaxanthin from corynebacterial biomass, three parameters were considered and optimized. (I) the impact of ethanol as a cosolvent, (II) the impact of extraction temperature, and lastly, the impact of (III) extraction time under optimized process conditions. 3.1 Impact of cosolvent addition Available literature data on the scCO 2 extraction of astaxanthin from microalgal biomass revealed the requirement of using a cosolvent for extraction (Machmudah et al. 2006; Nobre et al. 2006; Reyes et al. 2014). Furthermore, extraction temperature and pressure surpassing 50°C and 500 bar were needed for efficient extraction in several studies (Di Sanzo et al. 2018; Molino et al. 2018a; Álvarez et al. 2020). Based on that, the effect of ethanol as a cosolvent on the extraction of astaxanthin was investigated at 550 bar and 55°C for 0.5 h. Experiments were conducted as described in the materials and methods section. Without cosolvent 6.6% of the astaxanthin was extracted from the biomass. The extraction yield increased to 11.5% and 42.7% upon the addition of 4% ( w / w ) and 9% ( w / w ) ethanol, respectively (Fig. 1 ). Due to the strong positive effect of the cosolvent, 9% ( w / w ) ethanol was used for all following experiments. 3.2 Impact of extraction temperature In the next step, different extraction temperatures were screened at two different pressures (Fig. 2 ). Applying 550 bar, the extraction yield profile showed a clear optimum at 68°C with 67.5 ± 3.7% of astaxanthin being successfully extracted. In contrast, varying the temperature at 650 bar showed a decreased extraction yield with increasing temperature. For 650 bar, the maximal extraction yield of 61.5% was achieved at 50°C. Since high pressure did not improve the extraction, 450 bar and 500 bar were tested at 68°C resulting in a decreased extraction yield. 3.3 Effect of extraction time As it is well known that extraction time is crucial when considering scCO 2 extraction from biomass, the extraction time was extended up to 14 h to allow for prolonged penetration of the cellular membranes with scCO 2 . The effect of an extended process time was analyzed for both conditions, with and without the addition of ethanol as cosolvent (Fig. 3 ). Without the cosolvent, the extraction yield increased from 16.6% after 0.5 h to 56.3% after 14 h of process time. A similar effect was observed for the condition with cosolvent. The extended extraction time increased the extraction yield from 67.5 ± 3.7–93.3%. 3.4 Effect of extraction protocol on carotenoid composition The biomass examined in this study contained astaxanthin as the main product with 40.7% ( w / w ) of the total carotenoid content. The remaining 59.3% ( w / w ) of the cellular carotenoids were composed of different carotenoid intermediates of the astaxanthin biosynthetic pathway (see Figure S2 ). It was thus investigated, which other carotenoids, despite astaxanthin, are preferably extracted and thus presented in the obtained extract. The extraction yields of the different carotenoids with and without the use of ethanol as cosolvent were assessed (Fig. 4 ). Without the cosolvent, preferentially lycopene and β-carotene were extracted compared to the oxy-functionalized carotenoids astaxanthin, adonirubin, canthaxanthin and echinenone. This pattern switched upon adding the cosolvent, resulting in a better extractability of the xanthophylls. In both cases, the total extracted amounts of astaxanthin, adonirubin and canthaxanthin were comparable. 4. Discussion Among the different methods available for astaxanthin extraction, scCO 2 extraction is probably the most studied one as this is the method of choice for large scale extraction of astaxanthin from microalgae (Rodríguez-Sifuentes et al. 2020). Although scCO 2 extraction has also been investigated for some alternative astaxanthin sources, the available data for the extraction from bacterial sources is limited. However, bacterial processes for astaxanthin production are emerging (Park et al. 2018; Hasunuma et al. 2019; Diao et al. 2020), consequently, requiring appropriate extraction methods for product recovery. In this study, different process parameters of batchwise scCO 2 extraction were screened to optimize the extraction yield of astaxanthin from corynebacterial biomass. The addition of 9% ( w / w ) ethanol increased the extraction yield from initial 6.6–42.7%. This finding is in good agreement with several studies that found the addition of ethanol as a cosolvent to be beneficial for the astaxanthin recovery from microalgae (Valderrama et al. 2003; Machmudah et al. 2006; Nobre et al. 2006; Pan et al. 2012; Reyes et al. 2014). This improvement is caused by two reasons. First, scCO 2 is a highly apolar solvent (Kang et al. 2024). By addition of ethanol, the solvent mixture becomes more polar, thus favoring the solubility of astaxanthin. The inclusion of a polar cosolvent is particularly relevant for corynebacterial astaxanthin, surpassing its importance in the case of astaxanthin derived from microalgae. Unlike astaxanthin from microalgae, which is esterified with apolar fatty acids, the astaxanthin investigated in this study exists in its free form (Kumar et al. 2022), exhibiting a higher polarity. The relation between solute and solvent polarity becomes apparent in Fig. 1 . Without a cosolvent, the carotenes lycopene and β-carotene, consisting of just pure hydrocarbon, showed a better extraction yield than the xanthophylls (astaxanthin, adonirubin, canthaxanthin, and echinenone), which possess varying degrees of oxy-functionalization. These differences in solubility were also observed and discussed by de la Fuente et al. (2006). Upon modifying the polarity with ethanol, the extraction yield of astaxanthin and the xanthophylls increased (Fig. 4 ). A similar observation was reported by Montero et al. (2005), who also assigned the improved extraction of different xanthophylls to the increased polarity. The second reason for an improvement extraction using a cosolvent might be based on the swelling of the matrix, which in turn increases the contact area with the scCO 2 (Moore and Taylor 1996; Lim et al. 2002; Nobre et al. 2006). The amount of ethanol was limited to 9% ( w/w ), as high concentrations of the cosolvent were previously shown to reduce/negatively affect the density of the scCO 2 as well as the selectivity of the extraction (Machmudah et al. 2006). Next, the extraction temperature was optimized at 550 bar and 650 bar. At lower temperatures, higher pressure was favorable for the extraction yield. This can be explained by the higher density of the scCO 2 with increasing pressure, thus leading to a higher solvent power (Knez et al. 2014; Wang et al. 2021). The extraction yield at 650 bar decreased with increasing temperature, due to the decreased density at higher temperatures. However, at 550 bar, the extraction yield increased with increasing temperature, reaching a maximum of 67.5 ± 3.7% at 68°C. The observed optimum is in the same range as the optimum determined by Machmudah et al. (2006) (69.9°C, 550 bar) and by Molino et al. (2018a) (65°C, 550 bar). It appears that the extraction yield is more dependent on the temperature, and thus on the vapor pressure of the solute, than on the scCO 2 density, which is consistent with some observations from the literature (de la Fuente et al. 2006; Álvarez et al. 2020). The effect of temperature becomes also apparent when comparing the extraction yields at different temperatures at 550 bar without a cosolvent. The extraction yield increased from 6.6% at 55°C to 18.7% at 68°C (Fig. 2 ). As the extraction yield decreased with decreasing pressure, the optimal balance between fluid density and solute vapor pressure was at 68°C and 550 bar. A maximum mass fraction of 4*10 − 7 (without cosolvent) and 2*10 − 6 ± 5*10 − 7 (with cosolvent) has been achieved with the aforementioned optimum. This is up to two magnitudes lower than the values reported by de la Fuente et al. (2006) (2*10 − 6 , 300 bar, 40°C) and Youn et al. (2007) (7*10 − 4 , 300 bar, 60°C). Therefore, the limiting factor for the total amount of astaxanthin extracted from biomass is not the solubility, as the equilibrium is still far away from being reached. Extraction processes can be divided into a solubility- and diffusion-controlled mass transfer period (Knez et al. 2010). As the astaxanthin solubility was not reached even after 14 h (Fig. 3 ), it is plausible that the extraction is diffusion-controlled/limited. Shortening the diffusion path through e.g. reduced particle size or by cell disruption, can prospectively reduce the extraction time (Knez et al. 2010). Although cell disruption upon scCO 2 treatment has been observed for fungi and bacteria (Hossain et al. 2015; Primožič et al. 2019), this effect was already shown for diffusion-controlled extraction processed where cell disruption improved the astaxanthin extraction (Valderrama et al. 2003; Nobre et al. 2006), or smaller particle size improved the extraction of oil from seeds (Del Valle and Uquiche 2002). Extraction processes showing more than 85% astaxanthin recovery are regarded as the industrial benchmark (Álvarez et al. 2020). By applying different strategies and process conditions, recoveries up to 98.6% were reached from microalgae (Valderrama et al. 2003; Nobre et al. 2006; Di Sanzo et al. 2018; Molino et al. 2018a) (Table 1 ). An astaxanthin recovery of 90% was also achieved for disrupted Phaffia rhodozyma by Lim et al. (2002). All these studies used semi-continuous extraction compared to batch extraction applied in this work. Due to the extraction yields and selectivity determined in this work, it is plausible that applying a multi-stage countercurrent extraction (increasing the driving force for extraction for depleted biomass by bringing it into contact with fresh CO 2 ) can also significantly enhance the overall extraction yield. Table 1 Comparison of scCO 2 extraction processes for astaxanthin extraction. Extraction yields correspond to the reference extraction method used in the respective study. Astaxanthin source Extraction yield Extraction conditions Process mode Reference C. glutamicum ASTA* 93% 68 °C, 550 bar, 9% ( w / w ) ethanol as cosolvent Batch This study H. pluvialis > 97% 60°C, 300 bar, 9.4% ( w / w ) ethanol as cosolvent Semi-continuous (Valderrama et al. 2003) H. pluvialis > 90% 60°C, 300 bar, 10% ( v / v ) ethanol as cosolvent Semi-continuous (Nobre et al. 2006) H. pluvialis 99% 50°C, 550 bar Semi-continuous (Di Sanzo et al. 2018) H. pluvialis 92% 65°C, 550 bar, 12.5% ( v / v ) ethanol as cosolvent Semi-continuous (Molino et al. 2018a) H. pluvialis 95% 50°C, 500 bar Semi-continuous (Álvarez et al. 2020) P. rhodozyma 90% 40°C, 500 bar, up to 5 % ( v / v ) ethanol as cosolvent Semi-continuous (Lim et al. 2002) Paracoccus sp. NBRC 101723 304% 40°C, 350 bar, 20% ( v / w ) ethanol as cosolvent Semi-continuous (Chougle et al. 2016) 5. Conclusion In this study, scCO 2 was employed to extract astaxanthin from the industrially relevant microorganism C. glutamicum . Three key findings emerged: (I) the addition of ethanol as a cosolvent was essential to achieve a high yield; (II) temperature influenced the extraction yield more than pressure; and (III) diffusion was identified as the controlling mechanism. Under optimized conditions (9% ( w / w ) ethanol, 68°C, 550 bar, 14 h), a yield of 93.3% was achieved. These results highlight the potential of C. glutamicum biomass as a valuable source for natural products like carotenoids, broadening its industrial applications. Declarations Authors’ contributions : Conceptualization, J.S., C.B. and N.A.H.; investigation, J.S. and C.B..; resources, C.B. and V.F.W.; writing—original draft preparation, J.S., M.Z., V.F.W., C.B. and N.A.H.; writing—review and editing, J.S., M.Z., V.F.W., C.B. and N.A.H.; supervision, N.A.H.; project administration, V.F.W and N.A.H.; funding acquisition, N.A.H. All authors have read and agreed to the published version of the manuscript. Acknowledgement: Competing interests : The authors declare no conflict of interest. Funding : This research was funded by the German Federal Ministry of Education and Research (BMBF) project KaroTec (grant number: 03VP09460). We acknowledge the financial support of the German Research Foundation (DFG) and the Open Access Publication Fund of Bielefeld University for the article processing charge. The funding bodies had no role in the design of the study or the collection, analysis, or interpretation of data, or in writing the manuscript. 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Rev Aquac 10:738–773 Machado FRS, Trevisol TC, Boschetto DL, et al (2016) Technological process for cell disruption, extraction and encapsulation of astaxanthin from Haematococcus pluvialis . J Biotechnol 218:108–114. https://doi.org/10.1016/J.JBIOTEC.2015.12.004 Machmudah S, Shotipruk A, Goto M, et al (2006) Extraction of astaxanthin from Haematococcus pluvialis using supercritical CO2 and ethanol as entrainer. Ind Eng Chem Res 45:3652–3657. https://doi.org/10.1021/IE051357K Molino A, Mehariya S, Iovine A, et al (2018a) Extraction of Astaxanthin and Lutein from Microalga Haematococcus pluvialis in the Red Phase Using CO2 Supercritical Fluid Extraction Technology with Ethanol as Co-Solvent. Marine Drugs 2018, Vol 16, Page 432 16:432. https://doi.org/10.3390/MD16110432 Molino A, Rimauro J, Casella P, et al (2018b) Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using generally recognized as safe solvents and accelerated extraction. 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Sci Rep 8:1–10. https://doi.org/10.1038/s41598-018-23854-w Pan JL, Wang HM, Chen CY, Chang JS (2012) Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier. Eng Life Sci 12:638–647. https://doi.org/10.1002/elsc.201100157 Park SY, Binkley RM, Kim WJ, et al (2018) Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metab Eng 49:105–115. https://doi.org/10.1016/J.YMBEN.2018.08.002 Picot-Allain C, Mahomoodally MF, Ak G, Zengin G (2021) Conventional versus green extraction techniques — a comparative perspective. Curr Opin Food Sci 40:144–156 Pitacco W, Samorì C, Pezzolesi L, et al (2022) Extraction of astaxanthin from Haematococcus pluvialis with hydrophobic deep eutectic solvents based on oleic acid. Food Chem 379:132156. https://doi.org/10.1016/j.foodchem.2022.132156 Praveenkumar R, Lee J, Vijayan D, et al (2020) Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure Homogenization for Astaxanthin Recovery. Applied Sciences 2020, Vol 10, Page 513 10:513. https://doi.org/10.3390/APP10020513 Primožič M, Čolnik M, Knez Ž, Leitgeb M (2019) Advantages and disadvantages of using SC CO2 for enzyme release from halophilic fungi. J Supercrit Fluids 143:286–293. https://doi.org/10.1016/J.SUPFLU.2018.09.001 Reyes FA, Mendiola JA, Ibañez E, Del Valle JM (2014) Astaxanthin extraction from Haematococcus pluvialis using CO2-expanded ethanol. J Supercrit Fluids 92:75–83. https://doi.org/10.1016/J.SUPFLU.2014.05.013 Rodríguez-Sifuentes L, Marszalek JE, Hernández-Carbajal G, Chuck-Hernández C (2020) Importance of Downstream Processing of Natural Astaxanthin for Pharmaceutical Application. Frontiers in Chemical Engineering 2:601483. https://doi.org/10.3389/FCENG.2020.601483 Sarada R, Vidhyavathi R, Usha D, Ravishankar GA (2006) An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis . J Agric Food Chem 54:7585–7588. https://doi.org/10.1021/JF060737T Sathasivam R, Ki JS (2018) A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Mar Drugs 16:26 Seeger J, Wendisch VF, Henke NA (2023) Extraction and Purification of Highly Active Astaxanthin from Corynebacterium glutamicum Fermentation Broth. Mar Drugs 21:530. https://doi.org/10.3390/MD21100530 Stachowiak B, Szulc P (2021) Astaxanthin for the food industry. Molecules 26 Valderrama JO, Perrut M, Majewski W (2003) Extraction of Astaxantine and phycocyanine from microalgae with supercritical carbon dioxide. In: Journal of Chemical and Engineering Data. American Chemical Society, pp 827–830 Wang W, Rao L, Wu X, et al (2021) Supercritical Carbon Dioxide Applications in Food Processing. Food Engineering Reviews 13:570–591. https://doi.org/10.1007/s12393-020-09270-9 Wendisch VF (2020) Metabolic engineering advances and prospects for amino acid production. Metab Eng 58:17–34 Wu T, Han B (2019) Supercritical Carbon Dioxide (CO2) as Green Solvent. In: Green Chemistry and Chemical Engineering. Springer, New York, NY, pp 173–197 Wu W, Lu M, Yu L (2011) A new environmentally friendly method for astaxanthin extraction from Xanthophyllomyces dendrorhous . European Food Research and Technology 232:463–467. https://doi.org/10.1007/S00217-010-1414-4 Xie L, Cahoon E, Zhang Y, Ciftci ON (2019) Extraction of astaxanthin from engineered Camelina sativa seed using ethanol-modified supercritical carbon dioxide. Journal of Supercritical Fluids 143:171–178. https://doi.org/10.1016/j.supflu.2018.08.013 Yabuzaki J (2017) Carotenoids Database: structures, chemical fingerprints and distribution among organisms. Database 2017:1–11. https://doi.org/10.1093/DATABASE/BAX004 Zhuang Y, Zhu MJ (2021) Recent developments in astaxanthin production from Phaffia rhodozyma and its applications. In: Global Perspectives on Astaxanthin: From Industrial Production to Food, Health, and Pharmaceutical Applications. Academic Press, pp 225–251 Supplementary Files GraphicalAbstract.png Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Editor assigned by journal 01 Apr, 2025 First submitted to journal 29 Mar, 2025 Editorial decision: Minor revision 21 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5798823","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437838565,"identity":"3922aee9-4441-4baa-a5c4-0baf38c4dc36","order_by":0,"name":"Jan Seeger","email":"","orcid":"","institution":"Bielefeld University: Universitat Bielefeld","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Seeger","suffix":""},{"id":437838566,"identity":"141a7b03-6703-4e45-9130-0f04c2cb8b21","order_by":1,"name":"Maximilian Zäh","email":"","orcid":"","institution":"TU Dortmund: Technische Universitat Dortmund","correspondingAuthor":false,"prefix":"","firstName":"Maximilian","middleName":"","lastName":"Zäh","suffix":""},{"id":437838567,"identity":"12cde472-b761-4067-a8d5-c22f2b16e911","order_by":2,"name":"Volker F. Wendisch","email":"","orcid":"","institution":"Bielefeld University: Universitat Bielefeld","correspondingAuthor":false,"prefix":"","firstName":"Volker","middleName":"F.","lastName":"Wendisch","suffix":""},{"id":437838568,"identity":"ccb7adfe-59a3-445c-9311-d79fb9267fc4","order_by":3,"name":"Christoph Brandenbusch","email":"","orcid":"","institution":"TU Dortmund: Technische Universitat Dortmund","correspondingAuthor":false,"prefix":"","firstName":"Christoph","middleName":"","lastName":"Brandenbusch","suffix":""},{"id":437838569,"identity":"d329f52f-e1fd-4295-be88-7327143475cf","order_by":4,"name":"Nadja Alina Henke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3OIQvCQBTA8TeErRysOpD5Fd4xcJj8KsrAZDibJpe0nN2PccksHJgmqw4MriytWMSw4B0YTLtFwfun43g/3gOw2X4zRIATAAGnTFF/TLuTHk31uwuBD3GDFDqQ2JOCveAWxkSe1wfWgO8tsJWM+ZxRDlU03m/nhVCHBbxuJ3haIBKQM5GTUXFXBK+GLZjXSBuQG5H7z6UmEyNRA5HaMsULdx19GPaNpGLRACUV2TkKDor3s4oZDkuOtF7JIWZJ+eBNGPq7RLQSlYvwdQkxjet69y5TNpvN9se9AfDASU3wBzUYAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1304-6310","institution":"Karlsruhe Institute of Technology: Karlsruher Institut fur Technologie","correspondingAuthor":true,"prefix":"","firstName":"Nadja","middleName":"Alina","lastName":"Henke","suffix":""}],"badges":[],"createdAt":"2025-01-09 19:20:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5798823/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5798823/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40643-025-00882-9","type":"published","date":"2025-05-26T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79882904,"identity":"92849b0d-4b2e-4d65-9cff-e924184e2628","added_by":"auto","created_at":"2025-04-04 05:01:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cosolvent on the astaxanthin extraction yield. \u003c/strong\u003eExtractions were performed at 550 bar and 55 °C for 0.5 h as single replicates. Ethanol was added as a cosolvent with indicated concentrations.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/cf3c7828fce1f2beb8701b59.png"},{"id":79882905,"identity":"86090312-7da7-451c-803f-689b1ad17b8c","added_by":"auto","created_at":"2025-04-04 05:01:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of temperature on astaxanthin extraction yield. \u003c/strong\u003eExtractions were performed for 0.5 h at 450 bar (purple), 500 bar (green),550 bar (blue) and 650 bar (orange) at the indicated temperatures. Single replicates were conducted except for the extraction at 68 °C and 550 bar (n = 3; mean ± sd).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/6a64ea7020609f33ec7d9483.png"},{"id":79884430,"identity":"bb992536-5ff2-4400-940b-e7e72d3c75df","added_by":"auto","created_at":"2025-04-04 05:35:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":152620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of process time on astaxanthin extraction yield. \u003c/strong\u003eExtractions were performed at 550 bar and 68 °C, with (red) and without (grey) ethanol as cosolvent for the indicated time. Single replicates were conducted except for the extraction at 0.5 h with cosolvent (n = 3; mean ± sd).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/105b8773f843725fc0063a05.png"},{"id":79884429,"identity":"ee6db546-56ba-4346-8d66-87f4bc5c7834","added_by":"auto","created_at":"2025-04-04 05:35:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtracted carotenoids. \u003c/strong\u003eExtractions were performed at 550 bar and 68 °C for 0.5 h without (grey) and with the addition of 9% (w/w) ethanol as a cosolvent (red; n = 3; mean ± sd). The respective carotenoid structures are shown; the arrows indicate the chronological order of the astaxanthin biosynthesis pathway starting from lycopene.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/9ef6d3ebbc23233a3d9e7a61.png"},{"id":83783226,"identity":"2ae66eba-3fa5-4b91-8c5d-e9c16b89c46f","added_by":"auto","created_at":"2025-06-02 16:11:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1476642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/aea1a655-9eaa-4c81-9999-3367d5765708.pdf"},{"id":79882910,"identity":"6cb3813e-312d-4dd8-a0f6-e5950e2818ac","added_by":"auto","created_at":"2025-04-04 05:01:54","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":298261,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/ac31a01b602c963946cbad7c.png"},{"id":79882912,"identity":"dd427285-23e8-404a-ab7e-402c80bf94ca","added_by":"auto","created_at":"2025-04-04 05:01:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":111941,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5798823/v1/4f57ec8052e35d1cf5351303.docx"}],"financialInterests":"","formattedTitle":"Supercritical carbon dioxide extraction of astaxanthin from Corynebacterium glutamicum","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlthough more than 1,100 carotenoids occur in nature (Yabuzaki 2017), only a few are industrially relevant (Sathasivam and Ki 2018). Among them is the red-colored marine carotenoid astaxanthin with a current market size of USD 2.83 bn and an expected compound annual growth rate of 17.1% until 2030 (Astaxanthin Market 2024). Besides its application as an animal feed additive (Lim et al. 2018), astaxanthin is being more and more used in the nutraceutical and cosmetics industries due to its various health promoting effects (Galasso et al. 2017; Barreiro and Barredo 2018; Stachowiak and Szulc 2021). However, only natural astaxanthin can be used for the latter, as the usage of synthetic astaxanthin is not considered for human consumption (Li et al. 2011). The most common microbial production systems for natural astaxanthin are the microalgae \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e (Rodr\u0026iacute;guez-Sifuentes et al. 2020; An et al. 2024), the red yeast \u003cem\u003eXanthophyllomyces dendrorhous\u003c/em\u003e (Zhuang and Zhu 2021), and the Gram-negative bacterium \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e (Hayashi et al. 2021). Based on its lipophilic nature, astaxanthin (and its esterified derivatives) are either incorporated into the cellular membrane (Kishimoto et al. 2016) or stored in intracellular lipid droplets (Ota et al. 2018). Due to the relatively rigid cell envelope of microalgae and yeasts, it is generally necessary to permeabilize or disrupt the cells before extraction (Rodr\u0026iacute;guez-Sifuentes et al. 2020). This can be achieved using physical methods like bead milling (Molino et al. 2018b; Irshad et al. 2019) or high-pressure homogenization (Praveenkumar et al. 2020), chemical methods such as acid treatment (Sarada et al. 2006; Wu et al. 2011), or biological methods like enzymatic lysis (Machado et al. 2016; Harith et al. 2020). Different extraction processes have been developed, comprising (pressurized) organic solvents, e.g., acetone, ethanol, and ethyl acetate (Molino et al. 2018b; Irshad et al. 2019; Praveenkumar et al. 2020), vegetable oils (Kang and Sim 2008), ionic liquids (Desai et al. 2016), and eutectic solvents (Pitacco et al. 2022).\u003c/p\u003e \u003cp\u003eAnother solvent-based method for the extraction of astaxanthin is the supercritical fluid extraction (SFE). By reaching the supercritical point, the properties of the gas and the liquid converge, leading to a state characterized by (low) gas-like surface tension, diffusivity and viscosity, and liquid-like density (Knez et al. 2014). Being non-toxic, non-flammable, chemically stable as well as readily affordable, carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) is considered as a green solvent (Wu and Han 2019) making it the most common used solvent for SFE in food processing (Picot-Allain et al. 2021). The rather low supercritical point (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 31.1\u0026deg;C, \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 73.8 bar) of CO\u003csub\u003e2\u003c/sub\u003e (Wu and Han 2019) enables an extraction under mild conditions, thus avoiding thermal or chemical degradation of the extract (Picot-Allain et al. 2021). The main factor affecting the solvent power of supercritical CO\u003csub\u003e2\u003c/sub\u003e (scCO\u003csub\u003e2\u003c/sub\u003e) is its density, which can be adjusted by pressure and temperature (Knez and L\u0026uuml;tge 2023). Increasing the pressure increases the density, favoring the solubility of the solute. Conversely, higher temperatures elevate the solute's vapor pressure while reducing the solvent's density. Therefore, optimization of the process conditions is required for an efficient extraction process (Knez et al. 2014; Wang et al. 2021). The polarity of the scCO\u003csub\u003e2\u003c/sub\u003e can be modified by cosolvents (polar modifier) like polar organic solvents or plant oils (Krichnavaruk et al. 2008; Wang et al. 2021). After the extraction, simple pressure reduction enables the residue-free removal of the CO\u003csub\u003e2\u003c/sub\u003e from the extract (Kang et al. 2024). By employing multiple separators and gradually reducing pressure (and thus solvent power of scCO\u003csub\u003e2\u003c/sub\u003e), different fractions of the extract can be collected (Knez and L\u0026uuml;tge 2023). Recovery of the gas after expansion allows its recycling without the need of solvent purification (Knez et al. 2014; Kang et al. 2024). Apart from being used for the extraction of essential oils, phenolic compounds, and alkaloids from natural sources (Wang et al. 2021), scCO\u003csub\u003e2\u003c/sub\u003e extraction has been applied and optimized for the extraction of astaxanthin from \u003cem\u003eH. pluvialis\u003c/em\u003e in numerous studies (Valderrama et al. 2003; Machmudah et al. 2006; Nobre et al. 2006; Krichnavaruk et al. 2008; Pan et al. 2012; Reyes et al. 2014; Di Sanzo et al. 2018; Molino et al. 2018a; \u0026Aacute;lvarez et al. 2020). Besides extraction from microalgae, scCO\u003csub\u003e2\u003c/sub\u003e extraction of astaxanthin has been also achieved from yeast (Lim et al. 2002; Harith et al. 2020), shrimp (Ahmadkelayeh et al. 2022), Gram-negative bacteria (Chougle et al. 2016), and oilseed (Xie et al. 2019).\u003c/p\u003e \u003cp\u003eRenowned for the large-scale production of amino acids (Wendisch 2020), the Gram-positive soil bacterium \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e has been proven to be a promising alternative to the aforementioned organisms for astaxanthin production. Harnessing its native carotenoid metabolic pathway, astaxanthin biosynthesis was enabled and improved in several studies, resulting in a titer of 103 mg/L astaxanthin in fed-batch fermentation (Henke et al. 2016; Henke and Wendisch 2019; G\u0026ouml;ttl et al. 2024). Recently, an extraction process based on ethanol was developed, resulting in a 94% recovery of astaxanthin (Seeger et al. 2023). An \u003cem\u003ein vitro\u003c/em\u003e assay revealed a superior antioxidant activity of the obtained natural extract (astaxanthin oleoresin) compared to synthetic astaxanthin and a similar activity to microalgae-derived astaxanthin (Seeger et al. 2023). This study establishes a scCO₂-based extraction process for astaxanthin from \u003cem\u003eC. glutamicum\u003c/em\u003e, aiming to provide an environmentally friendly and sustainable alternative that minimizes reliance on toxic organic solvents, thereby supporting advancements in the bio-economy.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003eAll chemicals were purchased from Carl Roth (Karlsruhe, Germany) or Sigma-Aldrich (St. Louis, MO, US). Organic solvents for extraction and analysis were HPLC grade. Carbon dioxide (99.9995% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e)) was purchased from Messer (Bad Soden am Taunus, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cultivation and harvesting of \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe astaxanthin producing strain \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e ASTA* was cultivated as described in Henke and Wendisch (2019). After 48 h of cultivation, the cells were harvested by centrifugation at 10,000 x g for 20 min and were oven-dried at 50\u0026deg;C. The dried biomass contained 0.35 mg/g astaxanthin (quantification described in section 2.5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental apparatus\u003c/h2\u003e \u003cp\u003eA high-pressure variable-volume view cell (HPVVV; \u003cem\u003ep\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e = 700 bar, \u003cem\u003eT\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e = 180\u0026deg;C; New Ways of Analytics, L\u0026ouml;rrach, Germany) was used for extraction. The required pressure was produced by adjusting the volume using a manual hydraulic press M(O) 189 (Maximator, Zorge, Germany). The system was homogenized by a stirrer. The temperature was measured inside the view cell and was regulated by a heating jacket. If applicable, ethanol was added through a 1/8\u0026Prime; port. A 260D syringe pump (Teledyne ISCO, Lincoln, NE, US) (\u003cem\u003ep\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e = 560 bar) was used for dosing compressed CO\u003csub\u003e2\u003c/sub\u003e into the HPVVV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental procedure\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 scCO\u003csub\u003e2\u003c/sub\u003e extraction without cosolvent\u003c/h2\u003e \u003cp\u003eThe dried biomass (see section 2.2) was weighted, packed into a paper tea bag and positioned into the HPVVV. After sealing the HPVVV, CO\u003csub\u003e2\u003c/sub\u003e was loaded reaching a mass fraction of \u0026ge;\u0026thinsp;0.99 compared to the biomass. Temperature and pressure were adjusted accordingly (see supplementary \u003cem\u003eTable S1\u003c/em\u003e for all tested conditions). If not stated differently, the extraction was terminated after 0.5 h by releasing the scCO\u003csub\u003e2\u003c/sub\u003e from the HPVVV. The biomass was taken out of the tea bag and subsequently processed for (residual) astaxanthin analysis (see supplementary \u003cem\u003eFigure S1\u003c/em\u003e for schematic experimental procedure).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 scCO2 extraction with cosolvent\u003c/h2\u003e \u003cp\u003eExperimental procedure and loading of the cell with biomass was performed as described above. The cosolvent (ethanol) was added after the HPVVV front sapphire had been sealed. The CO\u003csub\u003e2\u003c/sub\u003e amount was introduced as in the previous case and temperature and pressure were adjusted accordingly (see supplementary \u003cem\u003eTable S1\u003c/em\u003e for all tested conditions).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Quantification of astaxanthin content by HPLC\u003c/h2\u003e \u003cp\u003eTo determine the astaxanthin content of the biomass, a defined amount was extracted with 1 mL of a 7:3 (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) mixture of methanol:acetone at 1000 rpm and 60\u0026deg;C for 0.5 h using the ThermoMixer C (Eppendorf, Hamburg, Germany). After centrifugation for 10 min at 20,000 x g, the supernatant was analyzed via HPLC. The Agilent 1200 series (Agilent Technologies, Santa Clara, CA, US) equipped with a reversed-phase precolumn (LiChrospher 100 RP18 EC-5, 40 \u0026times; 4 mm) (CS-Chromatographie, Langerwehe, Germany), a reversed-phase main column (LiChrospher 100 RP18 EC-5, 125 \u0026times; 4 mm) (CS-Chromatographie, Langerwehe, Germany), and a diode array detector (DAD) was used for analysis. Methanol:water (9:1) (A) and methanol (B) were used as mobile phases. The injection volume was 50 \u0026micro;L, and a gradient at a flow rate of 1.5 mL min\u003csup\u003e\u0026ndash;1\u003c/sup\u003e was used as the following: 0 min B: 0%, 10 min B: 100%, and 32.5 min B: 100%. Carotenoids were quantified by recording the absorption at λ\u0026thinsp;=\u0026thinsp;470 nm. Astaxanthin (Sigma-Aldrich, St. Louis, MO, US), adonirubin (CaroteNature, M\u0026uuml;nsingen, Switzerland), canthaxanthin (VWR, Darmstadt, Germany), echinenone (Sigma-Aldrich, St. Louis, MO, US), β-carotene (Sigma-Aldrich, St. Louis, MO, US), and lycopene (ExtraSynthese, Genay, France) were used to generate standard calibration curves. Exemplary HPLC chromatograms are shown in the supplementary \u003cem\u003eFigure S2\u003c/em\u003e. The extraction yield was calculated according to Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) with the total extracted amount of carotenoid calculated by comparing the initial carotenoid content of the biomass with the carotenoid content after the scCO\u003csub\u003e2\u003c/sub\u003e extraction (Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{E}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\varvec{y}\\varvec{i}\\varvec{e}\\varvec{l}\\varvec{d}\\:\\left[\\varvec{\\%}\\right]=\\left(\\frac{\\varvec{E}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{e}\\varvec{d}\\:\\varvec{c}\\varvec{a}\\varvec{r}\\varvec{o}\\varvec{t}\\varvec{e}\\varvec{n}\\varvec{o}\\varvec{i}\\varvec{d}\\:\\left[\\varvec{m}\\varvec{g}\\:{\\varvec{g}}^{-1}\\right]}{{\\varvec{C}\\varvec{a}\\varvec{r}\\varvec{o}\\varvec{t}\\varvec{e}\\varvec{n}\\varvec{o}\\varvec{i}\\varvec{d}}_{\\varvec{b}\\varvec{e}\\varvec{f}\\varvec{o}\\varvec{r}\\varvec{e}\\:\\varvec{e}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}}\\left[\\varvec{m}\\varvec{g}\\:{\\varvec{g}}^{-1}\\right]}\\:\\times\\:100\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWith\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{E}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{e}\\varvec{d}\\:\\varvec{c}\\varvec{a}\\varvec{r}\\varvec{o}\\varvec{t}\\varvec{e}\\varvec{n}\\varvec{o}\\varvec{i}\\varvec{d}=\\:{\\varvec{C}\\varvec{a}\\varvec{r}\\varvec{o}\\varvec{t}\\varvec{e}\\varvec{n}\\varvec{o}\\varvec{i}\\varvec{d}}_{\\varvec{b}\\varvec{e}\\varvec{f}\\varvec{o}\\varvec{r}\\varvec{e}\\:\\varvec{e}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}}\\left[\\varvec{m}\\varvec{g}\\:{\\varvec{g}}^{-1}\\right]-\\:{\\varvec{C}\\varvec{a}\\varvec{r}\\varvec{o}\\varvec{t}\\varvec{e}\\varvec{n}\\varvec{o}\\varvec{i}\\varvec{d}}_{\\varvec{a}\\varvec{f}\\varvec{t}\\varvec{e}\\varvec{r}\\:\\varvec{e}\\varvec{x}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}}\\left[\\varvec{m}\\varvec{g}\\:{\\varvec{g}}^{-1}\\right]$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eFor the establishment of a scCO\u003csub\u003e2\u003c/sub\u003e-based extraction process of astaxanthin from corynebacterial biomass, three parameters were considered and optimized. (I) the impact of ethanol as a cosolvent, (II) the impact of extraction temperature, and lastly, the impact of (III) extraction time under optimized process conditions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Impact of cosolvent addition\u003c/h2\u003e \u003cp\u003eAvailable literature data on the scCO\u003csub\u003e2\u003c/sub\u003e extraction of astaxanthin from microalgal biomass revealed the requirement of using a cosolvent for extraction (Machmudah et al. 2006; Nobre et al. 2006; Reyes et al. 2014). Furthermore, extraction temperature and pressure surpassing 50\u0026deg;C and 500 bar were needed for efficient extraction in several studies (Di Sanzo et al. 2018; Molino et al. 2018a; \u0026Aacute;lvarez et al. 2020). Based on that, the effect of ethanol as a cosolvent on the extraction of astaxanthin was investigated at 550 bar and 55\u0026deg;C for 0.5 h. Experiments were conducted as described in the materials and methods section. Without cosolvent 6.6% of the astaxanthin was extracted from the biomass. The extraction yield increased to 11.5% and 42.7% upon the addition of 4% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) and 9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Due to the strong positive effect of the cosolvent, 9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol was used for all following experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Impact of extraction temperature\u003c/h2\u003e \u003cp\u003eIn the next step, different extraction temperatures were screened at two different pressures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cem\u003e).\u003c/em\u003e Applying 550 bar, the extraction yield profile showed a clear optimum at 68\u0026deg;C with 67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% of astaxanthin being successfully extracted. In contrast, varying the temperature at 650 bar showed a decreased extraction yield with increasing temperature. For 650 bar, the maximal extraction yield of 61.5% was achieved at 50\u0026deg;C. Since high pressure did not improve the extraction, 450 bar and 500 bar were tested at 68\u0026deg;C resulting in a decreased extraction yield.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of extraction time\u003c/h2\u003e \u003cp\u003eAs it is well known that extraction time is crucial when considering scCO\u003csub\u003e2\u003c/sub\u003e extraction from biomass, the extraction time was extended up to 14 h to allow for prolonged penetration of the cellular membranes with scCO\u003csub\u003e2\u003c/sub\u003e. The effect of an extended process time was analyzed for both conditions, with and without the addition of ethanol as cosolvent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Without the cosolvent, the extraction yield increased from 16.6% after 0.5 h to 56.3% after 14 h of process time. A similar effect was observed for the condition with cosolvent. The extended extraction time increased the extraction yield from 67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u0026ndash;93.3%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of extraction protocol on carotenoid composition\u003c/h2\u003e \u003cp\u003eThe biomass examined in this study contained astaxanthin as the main product with 40.7% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) of the total carotenoid content. The remaining 59.3% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) of the cellular carotenoids were composed of different carotenoid intermediates of the astaxanthin biosynthetic pathway (see \u003cem\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/em\u003e). It was thus investigated, which other carotenoids, despite astaxanthin, are preferably extracted and thus presented in the obtained extract. The extraction yields of the different carotenoids with and without the use of ethanol as cosolvent were assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Without the cosolvent, preferentially lycopene and β-carotene were extracted compared to the oxy-functionalized carotenoids astaxanthin, adonirubin, canthaxanthin and echinenone. This pattern switched upon adding the cosolvent, resulting in a better extractability of the xanthophylls. In both cases, the total extracted amounts of astaxanthin, adonirubin and canthaxanthin were comparable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAmong the different methods available for astaxanthin extraction, scCO\u003csub\u003e2\u003c/sub\u003e extraction is probably the most studied one as this is the method of choice for large scale extraction of astaxanthin from microalgae (Rodr\u0026iacute;guez-Sifuentes et al. 2020). Although scCO\u003csub\u003e2\u003c/sub\u003e extraction has also been investigated for some alternative astaxanthin sources, the available data for the extraction from bacterial sources is limited. However, bacterial processes for astaxanthin production are emerging (Park et al. 2018; Hasunuma et al. 2019; Diao et al. 2020), consequently, requiring appropriate extraction methods for product recovery. In this study, different process parameters of batchwise scCO\u003csub\u003e2\u003c/sub\u003e extraction were screened to optimize the extraction yield of astaxanthin from corynebacterial biomass.\u003c/p\u003e \u003cp\u003eThe addition of 9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol increased the extraction yield from initial 6.6\u0026ndash;42.7%. This finding is in good agreement with several studies that found the addition of ethanol as a cosolvent to be beneficial for the astaxanthin recovery from microalgae (Valderrama et al. 2003; Machmudah et al. 2006; Nobre et al. 2006; Pan et al. 2012; Reyes et al. 2014). This improvement is caused by two reasons. First, scCO\u003csub\u003e2\u003c/sub\u003e is a highly apolar solvent (Kang et al. 2024). By addition of ethanol, the solvent mixture becomes more polar, thus favoring the solubility of astaxanthin. The inclusion of a polar cosolvent is particularly relevant for corynebacterial astaxanthin, surpassing its importance in the case of astaxanthin derived from microalgae. Unlike astaxanthin from microalgae, which is esterified with apolar fatty acids, the astaxanthin investigated in this study exists in its free form (Kumar et al. 2022), exhibiting a higher polarity. The relation between solute and solvent polarity becomes apparent in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Without a cosolvent, the carotenes lycopene and β-carotene, consisting of just pure hydrocarbon, showed a better extraction yield than the xanthophylls (astaxanthin, adonirubin, canthaxanthin, and echinenone), which possess varying degrees of oxy-functionalization. These differences in solubility were also observed and discussed by de la Fuente et al. (2006). Upon modifying the polarity with ethanol, the extraction yield of astaxanthin and the xanthophylls increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A similar observation was reported by Montero et al. (2005), who also assigned the improved extraction of different xanthophylls to the increased polarity. The second reason for an improvement extraction using a cosolvent might be based on the swelling of the matrix, which in turn increases the contact area with the scCO\u003csub\u003e2\u003c/sub\u003e (Moore and Taylor 1996; Lim et al. 2002; Nobre et al. 2006). The amount of ethanol was limited to 9% (\u003cem\u003ew/w\u003c/em\u003e), as high concentrations of the cosolvent were previously shown to reduce/negatively affect the density of the scCO\u003csub\u003e2\u003c/sub\u003e as well as the selectivity of the extraction (Machmudah et al. 2006).\u003c/p\u003e \u003cp\u003eNext, the extraction temperature was optimized at 550 bar and 650 bar. At lower temperatures, higher pressure was favorable for the extraction yield. This can be explained by the higher density of the scCO\u003csub\u003e2\u003c/sub\u003e with increasing pressure, thus leading to a higher solvent power (Knez et al. 2014; Wang et al. 2021). The extraction yield at 650 bar decreased with increasing temperature, due to the decreased density at higher temperatures. However, at 550 bar, the extraction yield increased with increasing temperature, reaching a maximum of 67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% at 68\u0026deg;C. The observed optimum is in the same range as the optimum determined by Machmudah et al. (2006) (69.9\u0026deg;C, 550 bar) and by Molino et al. (2018a) (65\u0026deg;C, 550 bar). It appears that the extraction yield is more dependent on the temperature, and thus on the vapor pressure of the solute, than on the scCO\u003csub\u003e2\u003c/sub\u003e density, which is consistent with some observations from the literature (de la Fuente et al. 2006; \u0026Aacute;lvarez et al. 2020). The effect of temperature becomes also apparent when comparing the extraction yields at different temperatures at 550 bar without a cosolvent. The extraction yield increased from 6.6% at 55\u0026deg;C to 18.7% at 68\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As the extraction yield decreased with decreasing pressure, the optimal balance between fluid density and solute vapor pressure was at 68\u0026deg;C and 550 bar.\u003c/p\u003e \u003cp\u003eA maximum mass fraction of 4*10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e (without cosolvent) and 2*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e \u0026plusmn; 5*10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e (with cosolvent) has been achieved with the aforementioned optimum. This is up to two magnitudes lower than the values reported by de la Fuente et al. (2006) (2*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e, 300 bar, 40\u0026deg;C) and Youn et al. (2007) (7*10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, 300 bar, 60\u0026deg;C). Therefore, the limiting factor for the total amount of astaxanthin extracted from biomass is not the solubility, as the equilibrium is still far away from being reached.\u003c/p\u003e \u003cp\u003eExtraction processes can be divided into a solubility- and diffusion-controlled mass transfer period (Knez et al. 2010). As the astaxanthin solubility was not reached even after 14 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it is plausible that the extraction is diffusion-controlled/limited. Shortening the diffusion path through e.g. reduced particle size or by cell disruption, can prospectively reduce the extraction time (Knez et al. 2010). Although cell disruption upon scCO\u003csub\u003e2\u003c/sub\u003e treatment has been observed for fungi and bacteria (Hossain et al. 2015; Primožič et al. 2019), this effect was already shown for diffusion-controlled extraction processed where cell disruption improved the astaxanthin extraction (Valderrama et al. 2003; Nobre et al. 2006), or smaller particle size improved the extraction of oil from seeds (Del Valle and Uquiche 2002).\u003c/p\u003e \u003cp\u003eExtraction processes showing more than 85% astaxanthin recovery are regarded as the industrial benchmark (\u0026Aacute;lvarez et al. 2020). By applying different strategies and process conditions, recoveries up to 98.6% were reached from microalgae (Valderrama et al. 2003; Nobre et al. 2006; Di Sanzo et al. 2018; Molino et al. 2018a) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). An astaxanthin recovery of 90% was also achieved for disrupted \u003cem\u003ePhaffia rhodozyma\u003c/em\u003e by Lim et al. (2002). All these studies used semi-continuous extraction compared to batch extraction applied in this work. Due to the extraction yields and selectivity determined in this work, it is plausible that applying a multi-stage countercurrent extraction (increasing the driving force for extraction for depleted biomass by bringing it into contact with fresh CO\u003csub\u003e2\u003c/sub\u003e) can also significantly enhance the overall extraction yield.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eComparison of scCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eextraction processes for astaxanthin extraction.\u003c/b\u003e Extraction yields correspond to the reference extraction method used in the respective study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAstaxanthin source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtraction yield\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtraction conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProcess mode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. glutamicum\u003c/em\u003e\u003c/p\u003e \u003cp\u003eASTA*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68\u0026nbsp;\u0026deg;C,\u003c/p\u003e \u003cp\u003e550 bar,\u003c/p\u003e \u003cp\u003e9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBatch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH.\u0026nbsp;pluvialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u0026deg;C, \u003c/p\u003e \u003cp\u003e300 bar, \u003c/p\u003e \u003cp\u003e9.4% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Valderrama et al. 2003)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH.\u0026nbsp;pluvialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u0026deg;C, \u003c/p\u003e \u003cp\u003e300 bar, \u003c/p\u003e \u003cp\u003e10% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Nobre et al. 2006)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH.\u0026nbsp;pluvialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u0026deg;C, \u003c/p\u003e \u003cp\u003e550 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Di Sanzo et al. 2018)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH.\u0026nbsp;pluvialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65\u0026deg;C, \u003c/p\u003e \u003cp\u003e550 bar, \u003c/p\u003e \u003cp\u003e12.5% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Molino et al. 2018a)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH.\u0026nbsp;pluvialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u0026deg;C, 500 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(\u0026Aacute;lvarez et al. 2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. rhodozyma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026deg;C, \u003c/p\u003e \u003cp\u003e500 bar, \u003c/p\u003e \u003cp\u003eup to 5 % (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Lim et al. 2002)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParacoccus sp.\u003c/em\u003e NBRC 101723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e304%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026deg;C, \u003c/p\u003e \u003cp\u003e350 bar, \u003c/p\u003e \u003cp\u003e20% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol as cosolvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSemi-continuous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Chougle et al. 2016)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, scCO\u003csub\u003e2\u003c/sub\u003e was employed to extract astaxanthin from the industrially relevant microorganism \u003cem\u003eC. glutamicum\u003c/em\u003e. Three key findings emerged: (I) the addition of ethanol as a cosolvent was essential to achieve a high yield; (II) temperature influenced the extraction yield more than pressure; and (III) diffusion was identified as the controlling mechanism. Under optimized conditions (9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol, 68\u0026deg;C, 550 bar, 14 h), a yield of 93.3% was achieved. These results highlight the potential of \u003cem\u003eC. glutamicum\u003c/em\u003e biomass as a valuable source for natural products like carotenoids, broadening its industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e: Conceptualization, J.S., C.B. and N.A.H.; investigation, J.S. and C.B..; resources, C.B. and V.F.W.; writing\u0026mdash;original draft preparation, J.S., M.Z., V.F.W., C.B. and N.A.H.; writing\u0026mdash;review and editing, J.S., M.Z., V.F.W., C.B. and N.A.H.; supervision, N.A.H.; project administration, V.F.W and N.A.H.; funding acquisition, N.A.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was funded by the German Federal Ministry of Education and Research (BMBF) project KaroTec (grant number: 03VP09460). We acknowledge the financial support of the German Research Foundation (DFG) and the Open Access Publication Fund of Bielefeld University for the article processing charge. The funding bodies had no role in the design of the study or the collection, analysis, or interpretation of data, or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmadkelayeh S, Cheema SK, Hawboldt K (2022) Supercritical CO2 extraction of lipids and astaxanthin from Atlantic shrimp by-products with static co-solvents: Process optimization and mathematical modeling studies. 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Marine Drugs 2018, Vol 16, Page 432 16:432. https://doi.org/10.3390/MD16110432\u003c/li\u003e\n\u003cli\u003eMolino A, Rimauro J, Casella P, et al (2018b) Extraction of astaxanthin from microalga \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e in red phase by using generally recognized as safe solvents and accelerated extraction. J Biotechnol 283:51\u0026ndash;61. https://doi.org/10.1016/j.jbiotec.2018.07.010\u003c/li\u003e\n\u003cli\u003eMontero O, Mac\u0026igrave;as-S\u0026aacute;nchez MD, Lama CM, et al (2005) Supercritical CO2 extraction of \u0026beta;-carotene from a marine strain of the cyanobacterium \u003cem\u003eSynechococcus\u003c/em\u003e species. J Agric Food Chem 53:9701\u0026ndash;9707. https://doi.org/10.1021/jf051283n\u003c/li\u003e\n\u003cli\u003eMoore WN, Taylor LT (1996) Extraction and quantitation of digoxin and acetyldigoxin from the Digitalis lanata leaf via near-supercritical methanol-modified carbon dioxide. 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Database 2017:1\u0026ndash;11. https://doi.org/10.1093/DATABASE/BAX004\u003c/li\u003e\n\u003cli\u003eZhuang Y, Zhu MJ (2021) Recent developments in astaxanthin production from \u003cem\u003ePhaffia rhodozyma\u003c/em\u003e and its applications. In: Global Perspectives on Astaxanthin: From Industrial Production to Food, Health, and Pharmaceutical Applications. Academic Press, pp 225\u0026ndash;251\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Astaxanthin, supercritical carbon dioxide, extraction, Corynebacterium glutamicum","lastPublishedDoi":"10.21203/rs.3.rs-5798823/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5798823/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstaxanthin, a red carotenoid with potent antioxidant properties, holds significant value in the feed, cosmetics, and nutraceutical industries. While traditionally sourced from microalgae, \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e, a well-established industrial microorganism, has been engineered to serve as an efficient host for astaxanthin production. As astaxanthin integrates into the cellular membrane, effective extraction methods are essential to access this valuable compound. In this study, a sustainable batch extraction process using supercritical carbon dioxide (scCO₂) as a green solvent was developed. The effects of cosolvent concentration (0\u0026ndash;9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e)), temperature (50\u0026ndash;75\u0026deg;C), and pressure (450\u0026ndash;650 bar) were investigated with regard to the extraction yield. An optimized extraction was achieved with 9% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ew\u003c/em\u003e) ethanol as a cosolvent, at 68\u0026deg;C and 550 bar, allowing the extraction of 67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% of the cellular astaxanthin within 0.5 hours. Prolonging the extraction time further increased the recovery to 93.3%, which is comparable to processes that have been established for the extraction of astaxanthin from microalgae and yeast. This approach provides a scalable and environmentally friendly solution for industrial astaxanthin recovery.\u003c/p\u003e","manuscriptTitle":"Supercritical carbon dioxide extraction of astaxanthin from Corynebacterium glutamicum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 05:01:49","doi":"10.21203/rs.3.rs-5798823/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-03T09:01:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T08:36:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T11:57:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2025-03-29T05:51:01+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2025-02-21T07:45:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"024cd137-6f66-46b9-b1b9-66ca73afec9f","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:08:05+00:00","versionOfRecord":{"articleIdentity":"rs-5798823","link":"https://doi.org/10.1186/s40643-025-00882-9","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2025-05-26 15:57:02","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2025-04-04 05:01:49","video":"","vorDoi":"10.1186/s40643-025-00882-9","vorDoiUrl":"https://doi.org/10.1186/s40643-025-00882-9","workflowStages":[]},"version":"v1","identity":"rs-5798823","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5798823","identity":"rs-5798823","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}