Separation of salidroside from the fermentation broth of engineered Escherichia coli using macroporous adsorbent resins

Xiaocui Sun,Xue Liu,Guang-Rong Zhao,2,*

1 Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education),School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350,China

2 Georgia Tech Shenzhen Institute,Tianjin University,Shenzhen 518071,China

Keywords:Adsorption Desorption Fermentation Macroporous resin Salidroside

ABSTRACT Salidroside (8-O-β-D-glucoside of tyrosol),a plant-derived natural product,is used for treatment of hypoxia,fatigue and aging diseases.The availability of salidroside is restricted since it is extracted from 3-5 years old Rhodiola roots,which grow very slowly in the cold region of northern hemisphere of Earth.Our laboratory has constructed an engineered Escherichia coli and established a fermentation process to produce salidroside from glucose.In this article,nine macroporous resins from polarity to non-polarity,including NKA-9,S-8,AB-8,SP825,D101,LSA-8,LX-12,LX-18 and LX-68 resins,were tested to separate salidroside from fermentation broth.After static and dynamic experiments,the weakly polar SP825 resin had a better separation efficiency among nine resins.The adsorption kinetic and isotherm of salidroside on the SP825 resin were determined,and the pseudo-second-order kinetic model and Langmuir model could be fitted well.The effects of the pH on adsorption and ethanol concentration on desorption were investigated,and an optimal separation process was established.The adsorption for salidroside in the SP825 resin column was conducted with loading 150 ml at pH 7,and desorpted by washing 50 ml of 80% ethanol solution.Under the best process conditions,the purity and yield of salidroside in the final product were 91.6%and 74.0%,respectively.The results showed that the macroporous SP825 resin would be feasible and effective to prepare salidroside and has promising application in the downstream process of microbial fermentation.

1.Introduction

Salidroside (8-O-β-D-glucoside of tyrosol) is the main active ingredient of the traditional medicinal herbRhodiola,and has various pharmacological activities,including protective effects on liver,nerves and blood vessels of stress-induced impairments and disorders [1–4].In addition,salidroside has minimal cellular toxicity,and almost non-toxic to the liver and kidney [5].Salidroside would be useful to treat hypoxia and ischemia,fatigue and aging diseases [6].

At present,salidroside is mainly prepared from the root of 3–5 years wildRhodiolausing the traditional water or/and ethanol extraction,and salidroside with 3%–5% purity is obtained in the crude product.The microwave-assistance,supercritical carbon dioxide,molecularly imprinted polymers,ionic liquids,and deep eutectic solvents could be employed to enrich salidroside extraction fromRhodiolaroots [7–12].However,the products extracted by these methods still requires further purification and refining.Salidroside with high purity(over 90%)could be obtained by pooling the eluted salidroside peak fraction on high-speed countercurrent chromatography with two-phase solvents,which employed poisonous organic chemicals suchn-butanol and ethyl acetate used as mobile phases [13,14].Macroporous resins have strong adsorption capacity,high stability and easy regeneration[15] and are often used for the separation of natural active compounds such as polyphenols and flavonoids [16,17].Macroporous resins AB-8,AB-83,DA-201,D101,D301,NK-2,HP20,S-8,SP70,SP700,SP825 and XAD7HP have been tested to separate salidroside fromRhodiolaextract [18–21].Among those resins screened,after two adsorption and desorption runs on the HPD-200 resin,the salidroside product had 92%purity and 48%yield[19].Similarly,three separation cycles on the SP825 resin column increased 93% purity of salidroside and the yield of 80%[20].Although 94%purity of salidroside could be prepared by combining the HP20 resin with silica gel chromatography using a mixture of chloroform and methanol as eluents,the yield was disappointingly low [22].

TheRhodiolagrows at the high altitude of 2,500–5,500 meter above sea level and amount of salidroside in crude roots is low[1,23].The large-scale preparation of salidroside fromRhodiolaroot is inefficient,which limits the applications in medicines.Using synthetic biology,biosynthesis of the plant-derived natural products including taxol precursor taxadiene,artemisinic acid and cannabinoids have been achieved in engineered microbes [24–27].Salidroside-overproducingEscherichia coliandSaccharomyces cerevisiaestrains have been constructed [28–31],and our strain produced ten-fold higher amount of salidroside than that in plants[28].However,there is no report on the separation of salidroside from microbial fermentation.Exploring a new method to prepare salidroside from the fermentation broth with high yield and purity is of significance and necessity.

In this study,our work is,for the first time,to develop an efficient method for the separation of salidroside from the fermentation broth of engineeredE.coliusing macroporous resins.Nine resins were screened and the LX-16,LX-36 and SP825 resins were further tested.The SP825 resin column was most suitable,and salidroside product with purity of 91.6% and yield of 74.0% was obtained under the optimal separation conditions.Our study provides an alternative approach for salidroside production from fermentation broth instead of collecting the wildRhodiola.

2.Materials and Methods

2.1.Chemicals and adsorbents

Methanol (HPLC grade),ethanol (analytical grade),n-butanol(analytical grade) and other reagents (analytical grade) were purchased from Kmart Chemical Technology (Tianjin,China).Salidroside standard was purchased from Solomon Biotechnology(Tianjin,China).

Nine macroporous resins were used in the experiments.The NKA,D101,AB-8 and S-8 resins were purchased from Nankai University Chemical Plants (Tianjin,China),while the LSA-8,LX-12,LX-18 and LX-68 resins were purchased from Xi’an Lanxiao Technology (Xi’an,China),and SP825 resin was purchased from Beijing Greenherbs Science and Technology (Beijing,China).The physical properties of nine resins were shown in Table 1.The macroporous resins were soaked in 95% ethanol solution for 24 h,and then washed thoroughly by distilled water for later use.The macroporous resins were dried to a constant weight at 80°C,and the water content of the resin was measured.

Table 1 Physical properties of the tested macroporous resins

2.2.Pretreatment of fermentation broth

EngineeredE.colistrain was cultivated at 37°C and 220 r?min-1in LB medium (pH 7.0),which contained 10 g?L-1NaCl,peptone 10 g?L-1,yeast extract 5 g?L-1and 50 mg?L-1streptomycin.When the optical density reached at 1.0,the cells were collected by centrifugation at 5000 r?min-1for 10 min and transferred into fermentation M9Y medium (pH 7.0),which contained 6.8 g?L-1Na2HPO4,3.0 g?L-1KH2PO4,1.0 g?L-1NH4Cl,0.5 g?L-1NaCl,10.0 g?L-1glucose,1.0 g?L-1yeast extract,50 mg?L-1streptomycin and 1 mmol?L-1of isopropyl β-D-thiogalactoside(IPTG).The fermentation was carried out in 250 ml shake flask with 50 ml M9Y medium at 30°C and 220 r?min-1.During fermentation,glucose was fed at 12 h intervals from 30 h to 78 h with the final concentration of 4.0 g?L-1for salidroside production.After the fermentation was completed for 102 h,the broth was centrifuged at 8000 r?min-1for 10 min,and the supernatant was collected.Absolute ethanol was added into the supernatant to make 80% ethanol at the final concentration,and placed in a 4°C refrigerator for 12 h to precipitate.After removing precipitants by centrifugation,the solution was concentrated by the rotary evaporation of ethanol and water,and then extracted with water-saturatedn-butanol three times.Finally,then-butanol layer was evaporated to dryness to obtain crude salidroside,which was dissolved in water as a pretreated sample of 6.0 g?L-1salidroside and decolorized using a LX-12 macroporous resin column.

2.3.Static adsorption and desorption tests

The appropriate macroporous resins were screened through experiments.The water-containing macroporous resins(dry mass:0.5 g)were added in 10 ml of the pretreated sample of a flask,and was shaken at 25°C and 160 r?min-1for 5 h in a thermostatic shaker.When the adsorption equilibrium of salidroside was reached,the adsorbed resin was rinsed with distilled water and then desorbed with 10 ml of 50% ethanol solution.The concentrations of salidroside in the solution after adsorption and desorption were determined by HPLC,and the adsorption and desorption capacities and ratios of the resin were calculated using the following equations:

whereqe(mg?g-1dry resin)is the adsorption capacity of the resin at the equilibrium of adsorption,AandDare adsorption ratio and desorption ratio,respectively.C0,Ce,andCd(mg?ml-1) are the concentrations of salidroside in the initial solution,and at the equilibriums of adsorption and desorption,respectively.ViandVd(ml) are the volume of the initial solution and the desorption solution,respectively.W(g) is the mass of dry resin.

2.4.Adsorption tests at different pH conditions

The LX-18,LX-68 and SP825 resins were chosen for better adsorption and desorption performance than others.To observe the effects of pH values on the adsorption performance of the resins,the resin and pretreated sample with different pH values were added to a flask.After shaken at 25 °C and 160 r?min-1for 5 h,the adsorption equilibrium was reached,and the sample was taken to determine the concentration of salidroside in the solution.

2.5.Desorption tests under different ethanol-water concentrations

To study the effects of different ethanol concentrations on resin desorption of salidroside,the resins which reached the adsorption equilibrium were desorbed with 10 ml of ethanol solution with different concentrations.After the desorption equilibrium was reached,the concentration of salidroside in the desorption solution was measured by HPLC.The desorption ratios at different ethanol concentrations were calculated.

2.6.Adsorption kinetics

The weighed resins(2.0 g)and 20 ml of pretreated sample were filled into a flask.0.2 ml of sample was taken at certain time intervals,and the concentration of salidroside was determined by HPLC.

2.7.Adsorption isotherms

The resins and pretreated sample with different concentrations of salidroside were added to a flask and shaken at 25 °C and 160 r?min-1for 5 h.When the adsorption equilibrium was reached,the concentration of salidroside in the solution was determined by HPLC.

2.8.Dynamic adsorption and desorption tests

The LX-18,LX-68 and SP825 resins were used to test the dynamic adsorption and desorption.Through static experiments,the adsorption and desorption processes of macroporous resins were optimized.Under the best conditions,dynamic adsorption and desorption experiments were performed in a glass column(16 mm×400 mm).The glass column was filled with 25 ml of wet resins at a height-to-diameter ratio of 8:1.The flow rate of the pretreated sample from the top to the bottom was controlled by a peristaltic pump to 50 ml?h-1,and the effluent solution was collected every 10 min at room temperature.After the adsorption was completed,the resin column was washed with 100 ml of distilled water.Then,ethanol solution was used to desorb the resin column at a flow rate of 50 ml?h-1,and the concentration of salidroside in the eluent was determined by HPLC.Finally,the eluent was spin-dried and lyophilized to prepare salidroside product.

2.9.HPLC analysis of salidroside

Salidroside in sample was analyzed by the HPLC system(HITACHI Primaide)which includes a UV detector,an autosampler and a pump,and a C18 column (4.6 mm×150 mm).The mobile phase was composed of 20% methanol,80% water and 0.1% formic acid.10 μl sample was injected with flow rate at 1 ml?min-1,and detected at 280 nm.All quantitative analysis was determined by 5-points standard curve,and the correlation coefficientR2was greater than 0.99.For investigation of adsorption kinetics,adsorption isotherms,and dynamic leakage and desorption curves,three independent experiments were conducted with almost the same results and one group of results were presented.For Screening the adsorbent resins,optimizing pH value and ethanol concentration in desorption solution,experiments were conducted in triplicates and data were shown as the mean±S.D.

3.Results and Discussion

3.1.Screening of the adsorbent resins

According to the properties of salidroside and the polarities,surface areas and average pore sizes of resins,nine polystyrene macroporous resins (Table 1) were initially selected for experiments in this study.Among them,the NKA-9 and S-8 resins have polarity;the AB-8 and SP825 resins show weakly polarity;the rest five resins (D101,LSA-8,LX-12,LX-18 and LX-68) are non-polar.

The adsorption capacities and desorption ratios of nine macroporous resins were shown in Table 2.The non-polar and weakly polar resins exhibited better adsorption capacities of salidroside than those of the polar resins,indicating that indicating that van der Waals hydrophobic force could be stronger between the polar salidroside and the non-polar and weakly polar resins,which was consistent with previous reports [19–21].The pore diameters and surface areas of resins also influence the adsorption [32,33],and the large pore diameters and small surface areas could weaken the adsorption of natural products.Compared with the weakly polar AD-8 resin,the SP825 resin had higher adsorption capacity probably because of its large surface areas.Analogously,among five non-polar resins,the smaller surface areas and larger pore diameters of the D101,LSA-8 and LX-12 resins might lead to the lower adsorption capacities than that of LX-18 and LX-68.Although the NKA-9 and D101 resins performed highest desorption ratios,their adsorption capacities were lower than other resins due to week adsorption affinity.Taken into account of the absorption capacity and desorption ratio,the LX-18,LX-68 and SP825 resins were superior and chosen for separation of salidroside in the following experiments.

Table 2 Adsorption capacities and desorption ratios of salidroside on different resins

3.2.Adsorption kinetics

The adsorption kinetics of salidroside on the LX-18,LX-68 and SP825 resins were shown in Fig.1.The adsorption tendencies of salidroside on three resins were almost the same and increased until reaching their maximum absorption capacities.The adsorption of salidroside increased rapidly in the first 5 min due to fast attachment of salidroside on the surface of resin,and then slowed down to the adsorption equilibrium at approximately 20 min,indicating that the LX-18,LX-68 and SP825 resins belonged to the fast adsorption resin type.

In order to better understand the adsorption mechanism of the LX-18,LX-68 and SP825 resins,three adsorption kinetic models were used to analyze the adsorption process [34,35].

The pseudo-first-order equation:

Fig.1.Adsorption kinetics curves for salidroside on the LX-18,LX-68 and SP825 resins at 25 °C.

The pseudo-second-order equation:

The intraparticle diffusion equation:

whereqtis the adsorption capacity of salidroside on resins at timet(min)andqe(mg?g-1)is the adsorption capacity at adsorption equilibrium;Kt,K2andkpare the rate constants of pseudo-first-order,pseudo-second-order and intraparticle diffusion models,respectively;Cis a constant.

The adsorption kinetic parameters of the pseudo-first-order,pseudo-second-order and intragranular diffusion models for salidroside on the LX-18,LX-68 and SP825 resins were exhibited in Table 3.The correlation coefficients of the pseudo-first-order and pseudo-second-order models were higher than that of the intragranular diffusion model,which indicated that the boundary layer diffusion and the intragranular diffusion were not the main factors affecting the adsorption ratio of salidroside,in accordance with their fast adsorption features.For the LX-18 and LX-68 resins,the correlation coefficients of the pseudo-second-order model were higher than that of the pseudo-first-order model and the calculatedqevalues were closer to the experimental results.It indicated that the adsorption process of salidroside on the LX-18 and LX-68 resins could be described well by the pseudo-second-order kinetics model.As for the SP825 resin,although the correlation coefficient of the pseudo-first-order model was larger than the pseudosecond-order model,the calculatedqevalue of the pseudosecond-order model was consistent with the measured experimental result.It would be considered that the pseudo-second-order kinetics model was most suitable to describe the whole adsorption process of salidroside on the SP825 resin.

3.3.Adsorption isotherms

The adsorption isotherms of the LX-18,LX-68 and SP825 resins at adsorption equilibrium were measured at 25°C with salidroside solution of different concentrations,and shown in Fig.2.It is obvious that the adsorption capacities of salidroside on the LX-18,LX-68 and SP825 resins increased with increasing concentrations and reached saturation at 6.0 g?L-1of salidroside,which would be suitable initial concentration for resin adsorption.

The Langmuir and Freundich equations would describe the interaction between adsorbate and macroporous resin [19,36–38],and were used to fit the adsorption isotherms data of salidroside on the LX-18,LX-68 and SP825 resins.

Langmuir equation:

whereqmis the maximum adsorption capacity,qe(mg?g-1) is the adsorption capacity at adsorption equilibrium,Ceis the concentration of salidroside at the equilibrium of adsorption,andKLis the adsorption constant.

Freundlich equation:

whereKFis a constant related to adsorption capacity and 1/nis an empirical constant related to adsorption affinity.

The Langmuir equation mainly describes the adsorption process of the monomolecular layer adsorption where there is no interaction between the adsorbents and the adsorbate salidroside,while the Freundich equation is usually used in the multimolecular layer adsorption process.Table 4 provided the Langmuir and Freundlich parameters for adsorption of salidroside on the LX-18,LX-68 and SP825 resins.The higher correlation coefficientR2of Langmuir model than that of Freundich model suggested that the adsorption process of the LX-18,LX-68 and SP825 resins belonged to the monolayer adsorption,consistent with previous reports for adsorption of salidroside on other macrosporous resins [18,19,21].

Fig.2.Adsorption isotherms of salidroside on the LX-18,LX-68 and SP825 resins at 25 °C.

Table 3 Adsorption kinetics for salidroside on the LX-18,LX-68 and SP825 resins

Table 4 Langmuir and Freundlich parameters for adsorption of salidroside from the LX-18,LX-68 and SP825 resins

3.4.Optimization of the pH for adsorption and ethanol concentration for desorption

The effects of pH values on the adsorption of salidroside were investigated,and demonstrated in Fig.3.The adsorption ratios of salidroside on the LX-18,LX-68 and SP825 resins were over 95%from pH 4.0 to pH 9.0.When the pH value was lower than 5.0 or higher than 8.0,the adsorption ratios of salidroside seriously decreased.This might be that the affinity interaction between salidroside and resins was week and unstable under the strong acid and strong base conditions.The LX-18 and SP825 resins appeared the maximum adsorption ratios at pH 6 and at pH 7,respectively.The adsorption ratio on the LX-68 resin was stable from pH 5.0 to pH 8.0,it seems that the pH values less influenced the adsorption of salidroside on the LX-68 resin,compared with the LX-18 and SP825 resins.and In order to obtain the maximum desorption,the elution efficiencies of salidroside from the LX-18,LX-68 and SP825 resins with different concentrations of ethanol were studied.

Fig.3.Effects of the pH values of pretreated sample on adsorption ratios.

Fig.4.Effects of the concentrations of ethanol solution on desorption ratios.

It has been reported that the concentrations of ethanol affected the elution efficiencies of salidroside from the macrosporous resins[20,21].Here,as shown in Fig.4,the significant effects of ethanol concentrations from 50% to 90% in the desorption of salidroside were also observed,and desorption ratios from the LX-18,LX-68 and SP825 resins varied from 60.43% to 97.41%.The desorption of salidroside from the SP825 and LX-18 resins showed more sensitive to ethanol concentrations than that of LX-68.The LX-68 resin showed highest desorption ratio with ethanol concentrations in the broad range from 50%to 80%.The similar desorption tendency from the LX-18 and SP825 resins was shown and the highest desorption ratios appeared with 70% and 80% ethanol solutions,respectively.

Fig.5.Dynamic leakage curves of salidroside on the LX-18,LX-68 and SP825 resins.

Fig.6.Dynamic desorption curves of salidroside on the LX-18,LX-68 and SP825 resins.

3.5.Dynamic breakthrough curves and dynamic desorption curves

For the purpose of calculating the quantity of resin and the loading volume of the pretreated sample,the dynamic leakage curve was investigated.The leakage point was defined when the eluent reached 10% of the initial concentration of salidroside[39,40].As shown in Fig.5,salidroside was completely adsorbed on the LX-68 resin in the first 175 ml and the leakage process increased rapidly after 200 ml,the leakage point was 212.5 ml when the initial salidroside concentration was 6.0 g?L-1.Similar tendencies of leakage process were shown for the SP825 and LX-18 resins,and the leakage points were 150 ml and 187.5 ml,respectively.

According to the volume of desorption solution and the salidroside concentration,the dynamic desorption curves of salidroside on the LX-18,LX-68 and SP825 resins were shown in Fig.6.After eluted by ethanol solution with the volume of 100 ml at a flow rate of 50 ml?h-1,the maximum concentrations of salidroside in the desorption solutions from the LX-18,LX-68,and SP825 resins appeared at 16.75 ml,and the LX-68 resin exhibited the highest concentration of salidroside than LX-18 and SP825 resins.Salidroside on the LX-18 resin had almost been eluted when the eluent volume reached 7.5 ml,while salidroside on the LX-68 and SP825 resins had almost been eluted at 50 ml.

3.6.Preparation of salidroside under optimal conditions

Through static and dynamic adsorption and desorption experiments,the optimal processes for the separation of salidroside by the LX-18,LX-68 and SP825 resin were determined.The salidroside in the LX-18 resin column was adsorpted with loading 187.5 ml at pH 6.0 and desorpted by washing 32.5 ml of 70% ethanol solution.The adsorption for salidroside in the LX-68 resin column was conducted with loading 212.5 ml at pH 8.0 and desorpted by washing 50 ml of 50%ethanol solution.The adsorption for salidroside in the SP825 resin column was conducted with loading 150 ml at pH 7.0 and desorpted by washing 50 ml of 80%ethanol solution.The purities of and the yields were calculated after separation.

As shown in Fig.7,most of the impurities in the prepared product had been removed.Since tyrosol and salidroside has the same benzene ring structure,minor tyrosol remained in the salidroside product.

Fig.7.The HPLC chromatograms of prepared products.Salidroside standard(a);Desorption solutions rich in salidroside from the LX-18 resin(b),from the SP825 resin(c)and from the LX-68 resin (d).

The salidroside product had highest purity (92.6%) with lowest yield (62.8%) prepared from the LX-68 resin column than those from the LX-18 and SP825 resins.Although preparation from the LX-18 resin column gave highest yield(75.9%),the lowest purity of salidroside (88.6%) was obtained.The SP825 resin column gave a comparable purity (91.6%) to the LX-68 resin column and higher yield (74.0%) than the LX-18 resin column.Thus,the SP825 resin would be suitable for separation of salidroside from microbial fermentation broth.

4.Conclusions

In this experiment,a method of separating salidroside from fermentation broth was established by macroporous resin.Among the nine macroporous resins,the weakly polar SP825 resin exhibited the best separation performance.The adsorption kinetic of salidroside on the SP825 resin fitted the pseudo second-order model well,and the isothermal adsorption process conformed to the Langmuir model and belonged to the monolayer adsorption process.The separation conditions were optimized using the SP825 resin column,91.6% purity of salidroside product was prepared with the overall yield of 74.0%.The results showed that macroporous resin SP825 was effective to separate salidroside from engineeredE.colifermentation broth,which provided an efficient route for mass production of salidroside.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by The Key-Area Research and Development Program of Guangdong Province (2020B0303070002),China.