Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/ascecg
Recovery of L‑Arginine from Model Solutions and Fermentation Broth Using Zeolite‑Y Adsorbent Abrar Faisal,†,∥ Mattias Holmlund,‡ Mireille Ginesy,§ Allan Holmgren,*,† Josefine Enman,§ Jonas Hedlund,† and Mattias Grahn† †
Chemical Technology, Luleå University of Technology, SE 971 87 Luleå, Sweden Department of Forest Genetics and Plant Physiology, Swedish University of Agriculture Sciences, SE 901 83 Umeå Sweden § Biochemical Process Engineering, Luleå University of Technology, SE 971 87 Luleå Sweden ∥ Department of Chemical Engineering, COMSATS University Islamabad, Lahore Campus, Lahore 54000, Pakistan ACS Sustainable Chem. Eng. Downloaded from pubs.acs.org by OCCIDENTAL COLG on 04/24/19. For personal use only.
‡
ABSTRACT: Arginine was produced via fermentation of sugars using the engineered microorganism Escherichia coli. Zeolite-Y adsorbents in the form of powder and extrudates were used to recover arginine from both a real fermentation broth and aqueous model solutions. An adsorption isotherm was determined using model solutions and zeolite-Y powder. The saturation loading was determined to be 0.2 g/g using the Sips model. Arginine adsorbed from a real fermentation broth using either zeolite-Y powder or extrudates both showed a maximum loading of 0.15 g/ g at pH 11. This adsorbed loading is very close to the corresponding value obtained from the model solution showing that under the experimental conditions the presence of additional components in the broth did not have a significant effect on the adsorption of arginine. Furthermore, a breakthrough curve was determined for extrudates using a 1 wt % arginine model solution. The selectivity for arginine over ammonia and alanine from the real fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder, and 1.0, and 4.1, respectively, for extrudates. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent has been reported. KEYWORDS: Arginine, FAU, Fermentation broth, Escherichia coli, Adsorption, Selectivity, Breakthrough
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(glucose + sucrose).6 Growing environmental awareness has raised the demand for feedstock that is not only environmentally friendly but also does not compete with food production, such as lignocellulosic biomass. Lignocellulosic materials typically contain about 25% to 50% of hemicellulose, a fraction rich in xylose and arabinose. However, C. glutamicum and C. crenatum strains cannot metabolize five-carbon sugars. Escherichia coli (E. coli), on the other hand, is naturally able to use a wide variety of substrates, including pentoses, like xylose and arabinose; hence, efforts were made to engineer this microorganism for arginine production.16 In addition, E. coli is a robust organism for industrial processes, with a well characterized metabolism, and fast growth rate in low cost media, which makes it a good candidate for the production of arginine via fermentation. An arginine concentration of up to 19.3 g/L using an E. coli strain was reported.17 Chromatographic techniques,18 as well as organic ion exchange resins, are used today to recover amino acids from aqueous solutions.19 At the same time, adsorption has been identified as an efficient separation technique to recover
INTRODUCTION L-Arginine (or simply arginine) is a semi-essential amino acid that has vast applications in the pharmaceutical, food, and cosmetic industries.1,2 Moreover, it has been shown that arginine can efficiently replace inorganic nitrogen as a nitrogen source in plant fertilizers.3,4 This would be a more environmentally friendly approach toward more sustainable fertilizers. Different routes can be used for the production of amino acids, e.g., chemical or enzymatic synthesis, extraction from primary natural products, and microbiological fermentation. All these production methods, of course, require the use of energyefficient separation techniques.5 Scientists have been working to find a suitable strain for the production of arginine via fermentation of sugars since the early 1970s. Relatively high levels of arginine can be obtained by fermentation using Corynebacterium glutamicum or Corynebacterium crenatum strains.6,7 These strains were used to produce arginine in concentrations ranging from 1.2 to 34.8 g/L in the 1970s and 1980s.8−11 In 2009 and 2011, different groups reported high concentrations of arginine (between 45 and 52 g/L) using engineered C. gluamicum and C. crenatum strains.12−15 In a more recent study, Park et al. used an engineered C. glutamicum strain to produce 92.5 g/L of arginine during fed-batch fermentation with a yield of 0.4 g-arginine/g-sugar © XXXX American Chemical Society
Received: February 15, 2019 Revised: April 12, 2019 Published: April 15, 2019 A
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 20 mg of FeSO4·7H2O, 12 mg of MnSO4·H2O, 0.5 mg of thiamine·HCl, 0.4 g of antifoam, and 20 mg of tetracycline. Reactor and medium were sterilized at 121 °C for 10 min. Then, 14%−15% v/v ammonia solution was used to adjust and automatically maintain the pH at 7 throughout the fermentation. The fermentation was conducted at 37 °C, and the dissolved oxygen levels were kept above 30% with aeration and agitation. The fermentation was stopped after 40 h, when all the glucose was consumed. Cells were removed from the fermentation broth by two consecutive 10 min centrifugations at 4 °C and 10000 rpm. The supernatant was then filtered through 0.2 μm filters. Zeolite Ion Exchange. Zeolite-Y powder from Zeolyst International was received in sodium cation form. However, Krohn and Tsapatsis reported the highest adsorption for a zeolite-Y sample with Na/Al = 0.2 with the rest of the sites being protonated. The powder received was therefore ion exchanged using the procedure described in the work by Krohn and Tsapatsis.34 In short, the zeolite powder was mixed with twice its weight of a 1 M ammonium nitrate solution and stirred for 12 h. The slurries were then filtered and dried at 90 °C. This procedure was carried out twice. The ion-exchanged zeolite was then rinsed with four times its weight of deionized water and dried at 90 °C. The zeolite was thereafter calcined for 5 h at 500 °C at a heating rate of 1 °C/min. The Na/Al ratios of the samples before and after ion exchange were determined by inductively coupled plasmasector field mass spectroscopy (ICP-SFMS, ALS Analytica). Characterization. The ion-exchanged zeolite-Y powder and asreceived zeolite-Y extrudates were analyzed with X-ray diffractometry (XRD), using a Panalytical Empyrean diffractometer equipped with a Cu LFF HR X-ray tube, a graphite monochromator, and a PIXcel3D detector. Scanning electron microscopy (SEM) images were recorded using a FEI Magellan 400 field emission SEM without coating the samples. Batch Adsorption Experiments. A single component adsorption isotherm was determined for the zeolite-Y powder using model solutions, whereas both zeolite-Y powder and extrudates were used to recover arginine from both model solutions and a real fermentation broth. The fermentation broth had a native pH of 7.7. Due to limited availability of the fermentation broth, adsorption experiments were repeated twice at pH 11 but only once at pH 7.7. Liquid phase batch adsorption experiments using model solutions and real fermentation broths were carried out in closed 50 mL Erlenmeyer glass flasks. Approximately 40 mL of solution and 1 g of adsorbent were added to the flasks. The solution was thereafter allowed to equilibrate with the adsorbent for 48 h on a shaking table at room temperature. Samples were centrifuged at ca. 10,600g for 10 min at 4 °C and appropriately diluted with water. The supernatants were then filtered using a 0.2 μm syringe filter, and the arginine concentration was determined. The arginine, alanine, and ammonia concentrations in the samples were measured using the UPLC Amino Acid Analysis System Solution from Waters. In brief, after a precolumn derivatization with 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AccQ·Fluor UltraReagent), the amino acids were separated on a bridged ethyl hybrid C18 column (AccQ·Tag UltraColumn, 2.1 × 100 mm, 1.7 μm, Waters) and detected by a UV detector at 260 nm (ACQUITY UPLC System, Waters). The mobile phase consisted of AccQ·Tag Ultra Eluents A and B. To determine the loading of the adsorbate in the zeolite, a mass balance was used
biochemicals from dilute fermentation broths and aqueous solutions as compared to pervaporation, distillation, gas stripping, and freeze crystallization.20,21 The recovery of amino acids using various inorganic adsorbents, e.g., Al2O3, TiO2, SiO2, ferrihydrite, and kaolinite, has been reported earlier.22−31 In addition, a few studies also focused on adsorption of amino acids on different zeolites, e.g., zeolite ZSM-5, ZSM-11, zeolite-Y, and zeolite-beta. Titus et al. studied the adsorption of phenylalanine and tyrosine on NaZSM-5.32 Krohn and Tsapatsis investigated the adsorption of arginine and phenylalanine on zeolite-X, -Y, and -beta.33,34 In addition, Munsch et al. and Mesu et al. also reported adsorption of phenylalanine, glutamic, lysine, leucine, and histidine on ZSM-5, ZSM-11, zeolite-beta, and zeolite-Y.5,35 These studies indicate that zeolite adsorbents can potentially be used for arginine recovery from fermentation broths. However, recovery of arginine with these absorbents has rarely been discussed, and to the best of our knowledge, only Krohn and Tsapatsis have investigated the adsorption of arginine on zeolite-X, -Y, and -beta. The authors reported that zeolite-Y (Si/Al = 8, Na/Al ratio 0.2) can be efficiently used to adsorb arginine from aqueous solutions.33,34 It was found that the adsorption of arginine on zeolite-Y increases as the pH of the solution approaches the pH at which arginine is at its isoelectric point. Arginine has two amino functional groups that can gain positive charge and has the highest isoelectric point (pI = 10.76) of all common amino acids because of these two amino functional groups. The authors reported arginine loading from batch adsorption experiments using model aqueous solutions. They also varied the properties of the zeolites in terms of the Si/Al ratio and Na/H ratio at the cationic sites and evaluated these different samples during adsorption of a mixture of phenylalanine and arginine. Moreover, a model was derived to rationalize the observed experimental findings. The selection of material used in the present work is based on the findings reported by Krohn and Tsapatsis. However, no adsorption or breakthrough data for real fermentation broths were reported. In this study, zeolite-Y adsorbents in the form of powder and extrudate were used to recover arginine from model solutions as well as from a real fermentation broth. A single component adsorption isotherm for arginine in zeolite-Y was also determined for the first time, and the Sips adsorption model was fitted to the data. A column breakthrough experiment was also performed in order to gain a first insight into the dynamics of the adsorption.
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EXPERIMENTAL SECTION
Materials. Zeolite-Y powder CBV100 with a Si/Al ratio of 2.6 was obtained from Zeolyst International, Netherlands. Zeolite-Y extrudates with a Si/Al ratio of >2.5 (vendor specification) were purchased from ACS Materials, USA. The average diameter and length of the extrudate was 2 and 5 mm, respectively. The extrudate was used in original form. Arginine (99%), ammonium nitrate (99%), sodium hydroxide (99.9%), and HCl (37%) were purchased from Merck Chemicals. All aqueous solutions used in this study were prepared from Milli-Q water. Fermentation Broth. The strain E. coli SJB009 previously engineered to overproduce arginine was used.16 The seed culture was grown at 37 °C and 200 rpm in a 500 mL shake flask containing 100 mL of the medium described above and supplemented with 2 g of CaCO3. After 12 h, cells were aseptically inoculated into the sterile bioreactor. The fermentation was performed in batch mode in a 1 L bioreactor (Biobundle 1 L, Applikon Biotechnology). The medium was composed of (per liter): 60 g of glucose, 15 g of corn steep liquor,
qi =
(Ci ,0 − Ci) × V0 mz ,0
(1)
In this equation, qi is the mass of adsorbate per unit mass of adsorbent, Ci,0 is the initial concentration of adsorbate in the solution, Ci is the concentration of the adsorbate in the solution at equilibrium, V0 is the volume of solution brought in contact with adsorbent, and mz,0 is the initial mass of the adsorbent. In addition, the Sips adsorption model, which may be loosely described as a combination of the Langmuir and Freundlich isotherms B
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering for predicting heterogeneous adsorption systems, was fitted to the experimental data of the isotherm.36 q = qsat
bC n 1 + bC n
recorded at room temperature (∼23 °C) using the double side forward−backward acquisition mode. A total number of 256 scans were co-added and signal averaged at an optical resolution of 4 cm−1 using a platinium attenuated total reflection (ATR) accessory equipped with a one-reflection diamond element. The samples were kept within an O-ring covered with a glass plate under mechanical pressure, admitting spectra to be recorded under vacuum. The resultant interferogram was Fourier transformed using the Mertz phase correction mode, a Blackman-Harris 3-term apodization function, and a zero filling factor of 2.
(2)
In this equation, qsat is the saturation loading, C is the equilibrium concentration of the adsorbate in solution, b is the Langmuir affinity parameter, and n is the Freundlich exponent (heterogeneity factor). This factor accounts for heterogeneity of the adsorption sites (i.e., that not all adsorption sites are equivalent), and it is the extra parameter in the Sips model as compared with the Langmuir model. Hence, the more the Freundlich exponent deviates from one, the greater the heterogeneity of the system.36 Breakthrough Experiments. Breakthrough experiments were carried out using a stainless steel column with an inner diameter of 10 mm and a length of 300 mm. Before the experiments, the zeolite-Y extrudates were calcined in a furnace for 5 h with heating and cooling rates of 1 °C/min. The column was loaded with 10 g of zeolite-Y extrudate, and the remaining part of the column was filled with glass beads to minimize the dead volume in the column. Breakthrough experiments were carried out using a model solution comprising 1 wt % of arginine in water at pH 11. The solution was pumped at a flow rate of 1 mL/min to the bottom of the vertical column using a pulse injection pump. The temperature of the column was maintained at 30 °C. The effluent at the top of the column was collected in vials and filtered using a 0.2 μm filter prior to analysis. The arginine concentration was determined using the same method as described above. The loading of adsorbed arginine was estimated using a mass balance.37
i ντ y ε Cint , i qi = jjj i − 1zzz kL { 1 − ε ρp
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RESULTS AND DISCUSSION Characterization. Figure 1 shows the X-ray diffraction patterns recorded for the zeolite-Y powder and extrudate.
(3)
In this equation, ν is the interstitial velocity (cm/s) in the column, τi is the average breakthrough time for component i (s), L is the length of the adsorbent bed (cm), ε is the bed porosity, Cint,i is the concentration of component i at the inlet (g/mL), ρp is the adsorbent particle density (g/mL), and qi is the loading of component i in the zeolite adsorbent (g/g). Since the pressure and flow rate are constant during the breakthrough experiment, the mean residence time τi is given by38 τi =
∫0
Figure 1. XRD pattern for the ion-exchanged zeolite-Y powder, zeolite-Y extrudate, and database pattern for FAU zeolite (00-0430168).
Zeolite-Y powder was characterized after the ion exchange, and the extrudate was characterized as received. Reflections from the Faujasite (FAU) phase were indexed and compared with the database pattern. For the powder, no reflections originating from other crystalline phases were observed, confirming that the adsorbents are composed of pure FAU. However, the diffractogram for the extrudate showed some extra reflections that most probably originate from the binder material. Figure 2a and b shows the zeolite crystal size in zeolite-Y powder and extrudate, respectively. The crystals in the powder are quite large, with a length exceeding 1 μm, while the crystals in the extrudate are much smaller with diameters of about 0.2 μm. Table 1 shows the Si/Al and Na/Al ratios of zeolite powder and extrudate. It is important to mention here that the extrudate was not ion exchanged, as it already had the desired Na/Al ratio.14 Single Component Adsorption from Aqueous Solution. As mentioned in the Introduction, Krohn and Tsapatsis observed the highest adsorption of arginine in zeolite-Y at pH 11, i.e., close to the isoelectric point of arginine. Keeping this in mind, single component batch adsorption experiments were performed at pH 11. Figure 3 shows the adsorption isotherm of arginine at pH 11 and 25 °C for adsorption in H/Na−Y zeolite powder. The experimental data is indicated by points. The shape of the isotherm is of Type 1, typical for monolayer adsorption, and the maximum observed loading of arginine was
∞i j
y jj1 − xi , t zzzdt jjj z xi ,0 zz{ k
(4)
where xi,o and xi,t are the fractions of component i at time 0 and t, respectively. The adsorption selectivity was calculated as Si / j = (xi/xj)/(yi /yj )
(5)
where x is the mole fraction in the adsorbed phase, y is the mole fraction in the liquid phase at equilibrium, i represents arginine, and j represents any other component of interest. To assess if axial dispersion may have affected the breakthrough experiments, the dispersion number (D/uL) for fluid flowing in packed beds was determined,39 where D is the axial dispersion coefficient (cm2/s), u is the superficial velocity (cm/s), and L is the length of the packed bed (cm). This is done by first calculating the Reynolds number for the fluid in the bed. After calculating the Reynolds number, the corresponding chart relating the intensity of dispersion (Dε/udp) to the Reynolds number was consulted, see Levenspiel.39 The vessel dispersion numbers were thereafter determined by multiplying the intensity of dispersion obtained with the ratio between the characteristic diameter and the length of the adsorbent bed, e.g., dp/(εL) for the packed bed. FTIR Spectroscopy. Infrared spectra were recorded on a Bruker Vertex 80v vacuum FTIR spectrometer equipped with a DLaTGS detector and ceramic mid-infrared external source. All spectra were C
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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experimental data well as illustrated by the curve in the figure. The saturation loading of arginine was determined to be 0.20 g/g from the Sips model. In addition, the Sips adsorption parameter (b) and heterogeneity factor (n) were determined to be 0.19 L/g and 1.65, respectively. Farzaneh et al. reported the heterogeneity factor values ranging from 1.46 to 1.61 for butanol adsorption in silicalite-1 at different temperatures.40 Kumar et al. reported the value of Sips heterogeneity factor of 1.8 for the adsorption of dye in a novel cashew nut shell adsorbent.41 Vargas et al. reported the heterogeneity factor value of 3.2 for the adsorption of methylene blue on an activated carbon adsorbent.42 The heterogeneity factor value reported in this study is quite similar to the values reported by Farzaneh and Kumar; however, it is lower than the value reported by Vargas. Thus, the heterogeneity of the adsorption sites in the present work is modest compared to other previous reports. Krohn and Tsapatsis reported a slightly higher loading of 0.26 g/g for the H/Na−Y zeolite at pH 11.34 As the zeolite reaches saturation, these findings suggest that arginine shows a high affinity toward zeolite-Y at pH 11. Additional experiments were carried out to compare the adsorption in the powder with that in the beads containing a binder material. In these batch adsorption experiments, the initial concentration of the aqueous solution was chosen to be similar as in the fermentation broths, i.e., 11 g/L. For the powder sample, the obtained arginine loading was 0.16 g/g at the equilibration concentration of 6.9 g/L. This adsorbed loading is close to the expected, as judged from the adsorption isotherm (Figure 3). For the extrudate, an arginine loading of 0.10 g/g was obtained at an equilibrium concentration of 8.9 g/L. This is somewhat lower than the loading of about 0.16 g/ g that may have been expected from the isotherm. However, this lower loading may be explained by the presence of some binder material in the extrudate. In addition, this lower loading is in agreement with the difference in specific surface area between the powder and the extrudate (Table 1). Due to the presence of the binder, the surface area of the extrudate is lower than the surface area of the powder adsorbent, consequently giving less amount of zeolite adsorbed per gram of sample. Arginine Adsorption from Real Fermentation Broths. Table 2 shows the concentrations of the main components in the real fermentation broth at pH 7.7, the natural pH of the broth, as well as the adsorbed loadings and their corresponding equilibrium concentrations in solution. In addition to arginine, ammonia and alanine were also present in smaller quantities in the broth. The adsorbed loadings of arginine in the powder and extrudate adsorbents were as low as 0.045 and 0.038 g/g, respectively, at this pH. The adsorption of ammonia and alanine in the zeolite were even lower, i.e., the zeolite is still selective toward arginine. The selectivity of arginine over ammonia and alanine was determined to be 2.6 and 1.7, respectively, for zeolite-Y powder at pH 7.7. The corresponding selectivities for the extrudate were 4.5 and 1.4, respectively, at the same pH value. The loading of arginine was approximately half of the loading achieved during single component and batch adsorption experiments using zeolite-Y powder at pH 11. The lower adsorption at pH 7.7 is likely the result of a less favorable speciation of arginine in combination with the presence of additional electrolytes in the real fermentation broths. For example, the fermentation broth has high concentrations of KH2PO4 (1 g/L) and MgSO4 (0.5
Figure 2. (a) SEM image of zeolite-Y crystals in powder form. (b) SEM image of zeolite-Y crystals in the extrudate.
Table 1. Zeolite Composition Sample
Si/Al
Na/ Al
Surface area (m2/g)
Zeolite-Y powder before ion exchange Zeolite-Y powder after ion exchange Zeolite-Y extrudate (as received)
2.6 2.6 >2.5
0.8 0.3 0.2
900 − 650
Figure 3. Adsorption isotherm for arginine in zeolite-Y powder at pH 11 and 25 °C. The curve represents the Sips model fitted to the experimental data.
determined to be 0.2 g/g for 22 g/L equilibrium concentration. The net volume change of the solution upon adsorption is regarded as insignificant. However, it seems like the isotherm has not leveled out at this concentration, and the saturation loading may be somewhat higher. The Sips adsorption model was fitted to the experimental data, and the model fits the D
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering
Table 2. Initial Concentrations and Adsorbed Amount of Arginine from Real Fermentation Broth at pH 7.7 and 25 °C Adsorbate
Initial concentration in fermentation broth (g/L)
Equilibrium concentration with powder (g/L)
Equilibrium concentration with extrudates (g/L)
Amount adsorbed in powder (g/g)
Amount adsorbed in extrudates (g/g)
Arginine Ammonia Alanine
11.5 1.29 0.44
10.3 1.23 0.40
10.5 1.25 0.41
0.045 0.002 0.001
0.038 0.001 0.001
Table 3. Initial concentration and adsorbed amount of arginine from real fermentation broth at pH 11 and 25 °C. The amounts adsorbed are average values from two experiments with standard deviations indicated Adsorbate
Initial concentration in fermentation broth (g/L)
Equilibrium concentration with powder (g/L)
Equilibrium concentration with extrudates (g/L)
Amount adsorbed in powder (g/g)
Amount adsorbed in extrudates (g/g)
Arginine Ammonia Alanine
11.46 1.29 0.44
7.60 0.92 0.41
9.13 1.03 0.41
0.154 ± 0.001 0.010 ± 0.002 0.001 ± 0.001
0.093 ± 0.001 0.010 ± 0.003 0.001 ± 0.002
g/L). The presence of these K+ and Mg2+ cations was possibility affecting the adsorption of arginine at this pH. Table 3 shows the corresponding results from the batch adsorption experiments after increasing the pH of the broth to 11. As expected, arginine shows a much higher affinity toward the adsorbent at this pH. In this case, the arginine loading was determined to be 0.154 g/g for the powder sample and 0.093 g/g for the extrudate at equilibrium concentrations of 7.6 and 9.13 g/L, respectively. The amount of ammonia adsorbed also increases as the pH was increased. However, increased pH did not affect the adsorption of alanine at the conditions studied. At pH 11, the selectivity of arginine over ammonia and alanine was determined to be 1.9 and 8.3, respectively, for the zeolite-Y powder. For the extrudates, the selectivity of arginine over ammonia and alanine was lower, viz., 1.0 and 4.1 at pH 11. With increasing pH, the arginine selectivity over ammonia was slightly reduced, whereas the selectivity of arginine over alanine was slightly increased. Column Breakthrough Experiments. Breakthrough experiments may give information on the uptake rate and the dispersion in the column, which are valuable parameters for the design of columns used in operation on an industrial scale. In this study, column breakthrough experiments were performed at 30 °C using the zeolite-Y extrudate as adsorbent and a model solution as feed with an arginine content of 1 wt %, which is close to the concentration of 10 g/L of arginine found in the fermentation broth for an initial assessment of the adsorbent. Figure 4 shows the breakthrough curve of arginine with the concentration of arginine in the effluent plotted against a collected volume of effluent. Breakthrough occurs after approximately 120 mL of collected effluent, and steady state was reached after 220 mL of collected effluent. The shape of the breakthrough is quite sharp initially, up to an arginine concentration of ca. 0.7 wt %, after which the curve broadens significantly. The broadening of the breakthrough front may be due to the dispersion in the column or a somewhat slow uptake of arginine during the adsorption process. To estimate the role of dispersion during adsorption, the column dispersion number was calculated to be 0.12. If the dispersion number is higher than 0.01, dispersion may affect the performance of the process.39 However, a more uniform broadening of the breakthrough front would have been expected if axial dispersion was the main cause. Instead, we suggest that mass transfer resistance is the main cause for the observed broadening of the breakthrough front. The aggregated crystals in combination with the presence of the binder may perhaps
Figure 4. Breakthrough curve for a 1 wt % arginine model solution at 25 °C and pH 11 using zeolite-Y extrudate. Error bars are based upon three independent experiments.
slow the uptake to certain domains in the beads resulting in a broadening of the breakthrough front in the latter part of the curve. The arginine loading was determined from the breakthrough curve to be 0.088 g/g using eqs 3 and 4. The arginine loading was slightly lower than the expected loading of 0.1 g/g from batch adsorption experiments using the extrudate as adsorbent. However, breakthrough experiments usually show slightly lower loadings than batch adsorption experiments.43,44 Although the conditions of the breakthrough experiments were not optimized, the experiment clearly illustrates the applicability of the zeolite-Y extrudate for arginine separation. Consequently, one possible application could be to selectively remove arginine from the fermentor during operation by pumping a part of the broth (after removal of cells and other particulate matter) to an adsorption column and recycle the effluent from the column, which is lean in arginine but still contain nutrients. In summary, the results obtained show that zeolite-Y may be an option for recovery of arginine from fermentation broths. However, the adsorbent and operating conditions should be optimized, and an efficient eluation strategy should be developed. Infrared Absorption. Infrared spectroscopy was used with the aim to find a molecular reason to the experimental finding from this investigation that the adsorption of aqueous arginine onto the surface sites of zeolite-Y was larger at pH 11 (Table 3) than at pH 7.7 (Table 2), where the arginine molecule is E
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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(pKa1 = 2.1).45 The shift to a higher wavenumber at pH 8 is presumably due to the hydrogen-bonding interaction with the adjacent NH3+ moiety (pKa2 = 9.15)45 giving more of a double bond character to the asymmetric stretch. Simultaneously, the infrared band at 1361 cm−1 disappears, and a new band appears at 1353 cm−1, indicating the absorbance of these bands to, at least partly, originate from symmetric stretching vibration of the carboxylate group.46 Another clear difference between the arginine spectra in Figure 5 is the appearance of a strong infrared band at 1520 cm−1 (pH 8), which is not present in the spectrum of the amino acid at pH 11 (Figure 5, upper trace). This band appears because of the protonation of the NH2 function adjacent to the carboxylate group and is assigned to the symmetric bending and symmetric rocking vibration of the formed NH3+ moiety. Together, the bending and rocking vibrations contribute to about 75% of the potential energy distribution.46 Another positively charged amino group in the arginine structure may imply more extensive hydrogenbonding interaction with water molecules as indicated by the hydrogen-bonding region of the infrared spectrum in Figure 5 (lower trace). In this region, the infrared absorption increases between 3500 and 3300 cm−1, gets more structured between 3200 and 3000 cm−1, and shows a slight increase around 2650 cm−1. As suggested by Xian et al.,47 based on proton NMR and H−H NOESY experiments, the conformation of arginine changes from a partly folded state to an extended state in the pH range 9−12, i.e., by passing the isoelectric point. A folded structure, accomplished by rotation of C−C bonds, would imply that the distance between the guanidinium group and the carboxylate function decreases, while keeping the distance between the two positively charged groups as large as possible. The strong Coulombic attraction between the oppositely charged guanidinium and carboxylate moieties may be further stabilized by a water molecule in between the two charges and therefore exposed to bond polarization that results in a strong hydrogen bond.48 This would contribute to the stability of the conformation. Arginine in a partly folded state should have impact on its ability to pass through the narrow pores of zeolite-Y (7.4 Å× 7.4 Å)49 and may be one reason why arginine adsorbed more at pH 11 than at pH 7.7. Figure 6 shows infrared spectra of arginine adsorbed in ionexchanged zeolite-Y powder (Table 1) at pH 11 and pH 8. Adsorption at pH 11 (upper trace) results in a shift to lower wavenumbers of the infrared bands assigned to the guanidinium moiety, as compared to the position of these bands in the spectrum of aqueous arginine at the same pH (Figure 5, upper trace). The peak absorbance appears now at 1625 cm−1 with a shoulder at 1658 cm−1, indicating that the guanidinium moiety experiences a new environment. Even more interesting is the disappearance of the vibrational mode of the carboxylate ion at 1558 cm−1 and in addition the simultaneous appearance of the infrared band at 1510 cm−1 assigned to the symmetric bending and symmetric rocking vibration of the NH3+ moiety. These findings show that the amine function on the C(α) carbon of arginine becomes protonated upon adsorption of arginine onto the porous zeolite-Y structure at pH 11. The protonation may be accomplished during an ion-exchange reaction where arginine adsorbs and a proton leaves its site. The infrared spectrum of adsorbed arginine at pH 8 (Figure 6, lower trace) exhibited all the spectral features that arginine dissolved in deionized water showed in this spectral range
positively charged. To that end, infrared spectra of aqueous arginine solutions were recorded, where more than 94% of the arginine molecules are either neutral or positively charged (pKa2 = 9.15).45 Infrared spectra of saturated aqueous Larginine solutions adjusted to pH 8 and 11 are shown in Figure 5. In the hydrogen-bonding region between 3700 and 2500
Figure 5. Infrared spectra of arginine at pH 11 (upper trace) and pH 8. (lower trace), where the spectrum of water as solvent was subtracted. Spectra are shifted along the absorbance scale for clarity.
cm−1, in addition to absorption due to H-bonded OH and NH groups, the expected infrared absorption from asymmetric and symmetric methylene vibrations appears on the right-hand side of the broad hydrogen-bonding feature, while methylene bending vibration appears at about 1472 and 1453 cm−1. However, the two different pH values give rise to quite different hydrogen-bonding line shapes. Decreasing pH from 11 to 8 results in an increase of the absorbance around 3360 cm−1, indicating an additional hydrogen-bonding possibility. At the lower pH value, the amine group adjacent to the carboxyl function is protonated, which could imply stronger interaction with the carboxylate function but also stronger interaction with water molecules due to their well-known proton acceptor ability. At lower wavenumbers, viz., between 1800 and 1200 cm−1, dramatic changes occur in the spectral appearance of arginine upon decreasing pH from 11 to 8. For this spectral range, Mahmod Ghomi et al. have reported a vibrational analysis of aqueous arginine solution using the density functional theory method (DFT) and the hybrid functional (B3LYP) employing the 6-31++G* basis set.46 Although they did not explicitly report the pH value of the aqueous arginine solution investigated, the report was helpful and facilitated the interpretation of certain spectral changes. At pH 11 (Figure 5, upper trace), the infrared absorption of arginine gives rise to two distinct bands at 1665 and 1632 cm−1. These bands are the result of overlapping bands from the bending vibration of NH2 adjacent to the carboxylate group and NH2 bending in the protonated guanidinium moiety (pKa3 = 13.5)45 involving C−N stretch of the resonance structure of that moiety. The line shape of this band changes when pH is lowered to eight with peak absorbance at 1626 cm−1 and shoulders at each side of the absorption band, viz., at 1667 and 1601 cm−1. The latter band originates from the strong infrared absorption band with peak absorbance at 1558 cm−1 (Figure 5, upper trace), which disappears in the spectrum at pH 8 and is assigned to the asymmetric stretch of the carboxylate group F
DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Allan Holmgren: 0000-0001-9794-8305 Mattias Grahn: 0000-0002-4755-5754 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Bio4Energy, a strategic research environment appointed by the Swedish government, for financial support. In addition, sincere thanks go to Dr. Ming Zhou for his help with XRD measurements and SEM imaging and Dr. Daniela Rusanova-Naydenova for her helpful discussions.
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(1) Alvares, T. S.; Meirelles, C. M.; Bhambhani, Y. N.; Paschoalin, V. M.; Gomes, P. S. L-Arginine as a potential ergogenic aid in healthy subjects. Sports Med. 2011, 41 (3), 233−248. (2) Siani, A.; Pagano, E.; Iacone, R.; Iacoviello, L.; Scopacasa, F.; Strazzullo, P. Blood pressure and metabolic changes during dietary Larginine supplementation in humans. Am. J. Hypertens. 2000, 13 (5), 547−551. (3) Ö hlund, J.; Näsholm, T. Low Nitrogen Losses with a New Source of Nitrogen for Cultivation of Conifer Seedlings. Environ. Sci. Technol. 2002, 36 (22), 4854−4859. (4) Näsholm, T.; Ekblad, A.; Nordin, A.; Giesler, R.; Högberg, M.; Högberg, P. Boreal forest plants take up organic nitrogen. Nature 1998, 392, 914−916. (5) Munsch, S.; Hartmann, M.; Ernst, S. Adsorption and separation of amino acids from aqueous solutions on zeolites. Chem. Commun. 2001, 19, 1978−1979. (6) Park, S. H.; Kim, H. U.; Kim, T. Y.; Park, J. S.; Kim, S.; Lee, S. Y. Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commun. 2014, 5, 4618. (7) Xu, M.; Rao, Z.; Dou, W.; Yang, J.; Jin, J.; Xu, Z. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production. Amino Acids 2012, 43 (1), 255−266. (8) Kisumi, M.; Kato, J.; Sugiura, M.; Chibata, I. Production of lArginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis. Appl. Microbiol. 1971, 22 (6), 987−991. (9) Kubota, K.; Onoda, T.; Kamijo, H.; Yoshinaga, F.; Okumura, S. Microbial production of L-arginine. J. Gen. Appl. Microbiol. 1973, 19, 339−352. (10) Kato, J.; Kisumi, M.; Takagi, T.; Chibata, I. Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis. Appl. Environ. Microbiol. 1977, 34 (6), 689−694. (11) Yoshida, H.; Araki, K.; Nakayama, K. L-arginine Production by Arginine Analog-resistant Mutants of Microorganisms. Agric. Biol. Chem. 1981, 45 (4), 959−963. (12) Xu, M.; Rao, Z.; Yang, J.; Xia, H.; Dou, W.; Jin, J.; Xu, Z. Heterologous and homologous expression of the arginine biosynthetic argC ∼ H cluster from Corynebacterium crenatum for improvement of l-arginine production. J. Ind. Microbiol. Biotechnol. 2012, 39 (3), 495− 502. (13) Dou, W.; Xu, M.; Cai, D.; Zhang, X.; Rao, Z.; Xu, Z. Improvement of l-Arginine Production by Overexpression of a Bifunctional Ornithine Acetyltransferase in Corynebacterium crenatum. Appl. Biochem. Biotechnol. 2011, 165 (3−4), 845−855. (14) Xu, H.; Dou, W.; Xu, H.; Zhang, X.; Rao, Z.; Shi, Z.; Xu, Z. A two-stage oxygen supply strategy for enhanced l-arginine production by Corynebacterium crenatum based on metabolic fluxes analysis. Biochem. Eng. J. 2009, 43 (1), 41−51. (15) Ikeda, M.; Mitsuhashi, S.; Tanaka, K.; Hayashi, M. Reengineering of a Corynebacterium glutamicum L-Arginine and LCitrulline Producer. Appl. Environ. Microbiol. 2009, 75 (6), 1635− 1641.
Figure 6. Infrared spectra of adsorbed arginine in the spectral range 1800−1250 cm−1. Spectra were obtained by subtracting a spectrum of aqueous zeolite-Y treated with deionized water at pH 11, from a spectrum of aqueous zeolite-Y loaded with 30 g/L of arginine at pH 11 (upper trace) and the corresponding spectrum recorded at pH 8. (lower trace). Spectra are shifted along the absorbance scale for clarity.
(Figure 5, lower trace). The only clear difference is the peak absorbance position of the 1520 cm−1 band that shifted downward to 1513 cm−1 when arginine was adsorbed on zeolite-Y. According to these results, it is evident that before adsorption at pH 11, arginine adopts an unfolded conformation, while it adopts a folded conformation at pH 8. Furthermore, the folded conformation is accompanied by extensive hydrogen-bonding interaction (Figure 5). Together, these findings may explain the observed behavior with larger amounts adsorbed at pH 11 (Table 3) than at pH 7.7 (Table 2).
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CONCLUSIONS In the present work, the adsorption of arginine on zeolite-Y adsorbents in the form of powder and extrudate was studied. An adsorption isotherm was determined at 25 °C and pH 11. The highest adsorbed loading obtained was 0.2 g/g-zeolite at a concentration of 21 g-arginine/L. Batch adsorption experiments were also performed using real arginine fermentation broths. At the original pH of the broth, i.e., 7.7, arginine showed lower affinity toward the zeolite-Y adsorbent, whereas at pH 11 arginine loading was close to the loading achieved in single component batch adsorption experiments showing that at this pH the presence of additional compounds in the broth had a negligible influence on the amount of arginine adsorbed. Breakthrough experiments showed that the adsorbent efficiently took up arginine during dynamic conditions. The present work showed that adsorption on zeolite-Y can be a feasible way to recover arginine from aqueous solutions and fermentation broths; however, the adsorbent and operating conditions should be optimized.
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REFERENCES
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DOI: 10.1021/acssuschemeng.9b00918 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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