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Separation of gibberellic acid (GA3) by n g D 084, A3) uili 402 ribu s sol aqueous solution on S-8 resin were determined at capacity of GA3 was measured as 4.56mggÿ1 wet resin 0% desor bed d to n li on l reas poro NOTATION C0 Original GA3 concentration in solution (gdmÿ3) Ci Instantaneous GA3 concentration in V Ef¯uent volume (cm3) X Xs X1 INT Gib horm imp yield The production of GA3 typically combines aerobic 2 extracted by a solvent. Finally, GA3 crystals are obtained by removing the solvent. However, some drawbacks exist in this process.2 (1) Low gibberellin yield. Low yield is attributed to the ®lm evaporator used to concentrate the fermentation is 0% s is ion an the the of in- rgy by . A concentrating method that does not need heating is Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:695±700 (2000) (Received 29 November 1999; revised version received 27 March 20 fermentation with solvent extraction. First, the gibberellic mold ingests the nutrient base, such as bran, cane sugar and salt, and egests the metabolized product-GA3 after assimulation. Next, the fermenta- tion liquor is concentrated by vaporization and then urgently needed to improve the conventional process. In recent years, some researchers have conducted studies on separation of GA3 by adsorption.3±12 GA3 is easily adsorbed by active carbon or XAD adsorption resins and eluted by organic solvents such as ethyl 00; accepted 3 April 2000) *Correspondence to: Zhigang Tang, Chemical Engineering Department, Tsinghua University, Beijing 100084, People’s Republic of China # 2 GA3 concentration in eluate or ef¯uent (gdmÿ3) Equilibrium GA3 concentration in solid state (mg/gÿ1 (wet resin)) Equilibrium GA3 concentration in liquid state (gdmÿ3) RODUCTION berellic acid (GA3) is an important plant growth one which can accelerate stalk and leaf growth, rove seed shooting and increase fructi®cation .1 liquor, the residence time in the heating region longer than 10h and the GA3 loss is great than 2 (wt%). The GA3 yield for the conventional proces only about 75%. (2) High energy consumption. Since the concentrat ratio during the concentration process is great th 7±8:1 and the evaporation heat of water is large, amount of heating steam needed to concentrate fermentation liquor is 2±3t (3±5kgcmÿ2) per kg GA3. Moreover, since high vacuum must be ma tained during the concentration process, the ene consumption for the vacuum is also high. Therefore, concentrating the fermentation liquor evaporation is the `bottle-neck' in GA3 production solution (gdmÿ3) t Time (h) hydrolyzation of GA3 when the aqueous solution is heated above 50 ° C. Even with a high vacuum falling- 000 So Keywords: gibberellic acid (GA3); adsorption; macro an adsorption and desorption cycle. # 2000 Society of Chemical Industry ous solution. The GA3 concentration was in was adsorbed by S-8 resin from the fermentati aque c of the ammonium sulfate in the fermentat dynamic adsorption capacity of GA3 was increase effect io ption performance. When GA3 was adsor S-8 resin was eluted with several eluents with 8 macroporous adsorptio Zhigang Tang,* Rongqi Zhou and Zhantin Chemical Engineering Department, Tsinghua University, Beijing 100 Abstract: Adsorption rates of gibberellic acid (G results indicating that the adsorption attained eq adsorption isotherms of GA3 on AB-8, X-5, S-8, D 20 ° C. S-8 resin had the largest solid/liquid dist adsorbent for separation of GA3 from the aqueou ciety of Chemical Industry. J Chem Technol Biotechnol 02 resins uan P R China on S-8 and X-5 resins were measured with the brium for adsorption times longer than 4h. The 0, D3520, D4006 and NKA-9 were determined at tion coef®cient of 10.5 and was selected as the ution. The breakthrough curves of GA3 from the different ¯ow rates. The dynamic adsorption at a ¯ow rate of 0.5cm3minÿ1. GA3 adsorbed on (v%) acetone aqueous solution having the best by S-8 resin from the fermentation liquor, the 12.02mggÿ1 wet resin in virtue of the salting-out quor. The yield of GA3 was above 90% after GA3 iquor at pH 2 and eluted by 80% (v%) acetone ed seven-fold from the fermentation liquor after us adsorption resin; separation 68±2575/2000/$30.00 695 layer of the solution and diluted to determine the GA3 concentration until the adsorption attained equili- Figure 1. Sketch of GA3 chemical structure. Figure 2. Experimental set up for static adsorption. Z Tang, R Zhou, Z Duan alcohol, methanol or acetone. This process does not require heating during concentration, so there is no GA3 loss due to high temperature and the energy consumption is reduced. However, the solid/liquid distribution coef®cients of GA3 on active carbon and XAD resins are relatively small. There has been much progress recently in the manufacture of domestic adsorption resins. Many types of novel macroporous adsorption resins have been developed, such as AB-8, X-5 and S-8, etc.13 Compared with classic ion exchange and adsorption resins, these resins have larger surface areas (always larger than 200m2gÿ1), are easily prepared (always derived from styrene) and are easily regenerated by normal organic solvents. These resins have been used successfully in some biological separations.14±16 AB-8, X-5 and other resins have large adsorption capacities and high recovery yield for the biological products shikonin, camptothecin and mogroside. However, before employing any of these adsorption resins, the insoluble residue in the feed must be removed to avoid contaminating the resins. Moreover, the feed concen- tration suitable for resin adsorption is always below 10gdmÿ3. Theoretically, adsorption is a function of structure, polarity, average pore diameter and surface area of the adsorbent. GA3 is not a large molecule(Fig 1) and is without strong polarity, so an adsorption resin with average pore diameter in the range of 100±300AÊ and medium or weak polarity, such as X-5, AB-5 and S-8 resins, should effectively separate GA3. The separa- tion results for these three resins were compared with Table 1. Properties of macroporous resins Name Grade matrix structure Polarity Pearl size (mm) Surfa (m D3520 Cross linked polystyrene Non 0.3±1.0 48 D4006 Cross linked polystyrene Non 0.3±1.0 40 D4020 Cross linked polystyrene Non 0.3±1.0 54 X-5 Cross linked polystyrene Weak 0.3±1.25 50 AB-8 Cross linked polystyrene Week 0.3±1.25 48 NKA-9 Cross linked polystyrene Polar 0.3±1.25 25 S-8 Cross linked polystyrene Medium 0.3±1.25 28 a Data taken from the speci®cations supplied by the Chemical Plant of Nankai Uni 696 brium. Since the sample volume was much smaller the adsorption of GA3 by non-polar resins D3520, D4006, D4020 and polar resin NKA-9 in this study. EXPERIMENTAL Materials GA3 was manufactured by No 18 Pharmaceutical Factory of Shanghai, P R China, with a purity above 96% (wt%). GA3 fermentation liquor was supplied by the same factory. The GA3 concentration in the fermentation liquor was in the range of 1.8±2.5gdmÿ3 and the pH of the fermentation liquor is in the range of 3.8±4.5. The macroporous adsorption resins AB-8, X-5, S-8, D4020, D3520, D4006 and NKA-9 were all pur- chased from the Chemical Plant of Nankai University, Tianjin, People's Republic of China. Some properties of the adsorption resins used in the work are listed in Table 1. All other reagents used were of analytical purity. Static adsorption experiments Determination of adsorption rate A speci®ed amount of GA3 was dissolved in deionized water to prepare GA3 aqueous solutions. Fiveg X-5 and 5g S-8 resins were put into an Erlenmeyer ¯ask in a constant temperature bath (Fig 2) and mixed with 150cm3 GA3 aqueous solution with a GA3 concen- tration of 0.5gdmÿ3. The mixture was stirred for 30min at 20 ° C, then allowed to settle. 1. Samples of 1cm3 were collected every hour from the upper clear ce area 3/gÿ1) Average pore diameter (AÊ ) Porosity (%) Pore volume (cm3gÿ1) 0±520 85±90 65±70 2.10±2.15 0±440 66±75 43±48 0.73±0.77 0±580 100±105 74±78 2.88±2.92 0±600 290±300 55±59 1.20±1.24 0±520 130±140 50±60 0.73±0.77 0±290 155±165 46±50 0.60±0.65 0±350 280±300 50±60 0.78±0.82 versity. J Chem Technol Biotechnol 75:695±700 (2000) than the solution volume during the experiments, the volumetric reduction due to the removal of samples can be ignored. The agitator rotation speed was changed from 100rpm to 300rpm to observe the effect of agitation. The starting concentration of GA3 in the aqueous solution was also changed from 0.4gdmÿ3 to 1.0g dmÿ3 to observe the effect of starting concentration. Determination of adsorption isotherms Fiveg S-8 resin was mixed with 50cm3 GA3 aqueous solution of different concentrations and the mixture were stirred for 30min at 20 ° C then allowed to settle. After 5h, samples were taken from the upper clear layer of the solution to determine the GA3 concentra- tion. The data were then plotted as the adsorption isotherm of GA3 on S-8 resin. organic solvent and deionized water. The desorption Adsorption and desorption experiments for GA3 Separation of gibberellic acid on resins effects of these eluents were compared with each other. The ¯ow rates during adsorption and desorption were 0.5cm3minÿ1 and 0.25cm3minÿ1, respectively. Figure 3. Experimental set-up for dynamic adsorption and de-sorption. The above experimental procedures were repeated for various resins to observe the adsorption isotherms of GA3 on the other resins. Dynamic experiments Determination of breakthrough curves GA3 aqueous solution with a GA3 concentration of 0.5gdmÿ3 was passed through an adsorption column packed with S-8 resin that had a large GA3 adsorption capacity. The adsorption column was 50mm in length and 10mm in diameter (Fig 3). Samples were periodically taken from the ef¯uent to determine the GA3 concentration and to calculate the dynamic adsorption capacity. Experiments were conducted at different ¯ow rates to study the in¯uence of ¯ow rate on the breakthrough curve. Determination of desorption curves GA3 was adsorbed dynamically on S-8 resin and then eluted by several eluents: ethanol, ethyl acetate, acetone, acetone aqueous solution prepared with J Chem Technol Biotechnol 75:695±700 (2000) fermentation liquor Static adsorption experiments Static adsorption experiments were conducted to determine the adsorption isotherms of GA3 on S-8 resin from fermentation liquor with a GA3 concentra- tion of 2.0gdmÿ3 over the pH range of 2 to 7. The method was similar to that described for the aqueous solutions (Fig 2). Dynamic adsorption and desorption experiments In a manner similar to that for the aqueous solution measurements, the dynamic adsorption measurements of the GA3 fermentation liquor were tested on S-8 resin at pH 2. Acetone aqueous solutions were used as the eluent to elute GA3 that had been adsorbed on the S-8 resin. Tests were conducted to measure the effects of eluent composition on the elution. The adsorption column was 50mm in length and 10mm in diameter. The GA3 concentration in the fermentation liquor was 2.0gdmÿ3 and the ¯ow rates during adsorption and desorption were 0.5cm3minÿ1 and 0.25cm3minÿ1, respectively. Recovery experiments An adsorption and desorption cycle of the GA3 fermentation liquor was carried out at pH 2 on an adsorption column 100mm in length and 10mm in diameter. The original GA3 concentration in the fermentation liquor was 2.0gdmÿ3, the adsorption ¯ow rate was 0.5cm3minÿ1 and the adsorption time was 120min. Then 80% (v%) acetone aqueous solu- tion was used to elute GA3 at a ¯ow rate of 0.25cm3 minÿ1. The eluate was collected to calculate the GA3 recovery yield. Analytical method The GA3 concentration in the aqueous solution was determined after dilution with 95% (wt%) ethanol aqueous solution by a Shimadzu UV-2501pc/2401PC ultraviolet spectrophotometer at a wavelength of 233nm. The GA3 concentration in organic solutions such as acetone or ethyl acetate could not be analyzed directly because the samples from the organic phase could not be analyzed until the solvent was evapo- rated, the samples were dried at room temperature, then dissolved in 95% ethanol aqueous solution and analyzed by UV spectrometry. The samples from the fermentation liquor were extracted twice by ethyl acetate at a phase ratio of 2:1 (v/v), then the ethyl acetate extracts were combined and evaporated. The amount of GA3 was measured by UV spectrometry after being re-dissolved in 95% ethanol aqueous solution. To calibrate the system, the absorbencies at 233nm of several GA3 95% ethanol aqueous solutions with different known concentrations were measured to get a calibration curve. Then the absorbency of the unknown aqueous solution was measured at the same wavelength to determine the concentration from the calibration curve. 697 the fermentation liquor, the compositions of the fermentation liquor, the ef¯uent and the eluate were Figure 5. Adsorption isotherms of GA3 on some adsorption resins at 20 ° C: ‡, AB-8; ^, NKA-9; &, D3520;*, D4020;~ D4006;$, S-8; QX-5. Z Tang, R Zhou, Z Duan analyzed by HPLC. The chromatograph column was 4.6mm in diameter and 25cm in length and was packed with Supelcosil LC-18 (5mm). The mobile phase composition was A (methanol): B (deionized water)=70:30 (v/v) at a ¯ow rate of 1.0cm3minÿ1. RESULTS AND DISCUSSION Static Adsorption experimental results GA3 adsorption rates measured on S-8 and X-5 resins from the GA3 aqueous solution for a GA3 concentra- tion of 0.5gdmÿ3 are shown in Fig 4. The experi- mental results show that the adsorption capacity reached 95% of the saturation adsorption capacity after 4h. Therefore, the adsorption time was always longer than 4h during the static experiments. Since a few side-products or impurities in GA3 fermentation liquor may also absorb in UV light, they would affect the analytical precision of the UV spectrometry for determining GA3 concentration in fermentation liquor experiments. Therefore, a Hewlett Packard 1050 High Performance Liquid Chromato- graph (HPLC) was used with a detector (Hewlett Packard 1040M Ultraviolet Spectrophotometer) to improve the analytical precision. In experiments for Figure 4. Adsorption rate of GA3 on S-8 resin and X-5 resin at 20 ° C:*, on S-8 resin; *, on X-5 resin. The results also indicated that the agitation rate and the GA3 starting concentration in the feed had little effect for agitator speeds from 100rpm to 300rpm and starting concentrations from 0.4gdmÿ3 to 1.0gdmÿ3. The static adsorption isotherms on AB-8, X-5, D3520, D4020, D4006 and NKA-9 are shown in Fig 5. The abscissa represents the GA3 liquid phase concentration, and the ordinate represents the amount of GA3 adsorbed on the resin phase. As shown in Fig 5, the solid/liquid distribution coef®cients for GA3 on S-8 and AB-8 were larger than 10, the distribution coef®cient for GA3 on NKA-9 was the smallest (<1), and the distribution coef®cients on the other resins were between those two values. Here the solid/liquid distribution coef®cient is de®ned as: amount of GA3 adsorbed on the solid phase per unit mass of 698 resin/amount of GA3 in the solution per unit mass of solution. Although the polarity of the molecular skeleton is very small, the GA3 molecule has some moderate polarity which is derived from some polar radicals such as hydroxyl, carboxyl and carbonyl. Thus the adsorp- tion capacities of resins with moderate polarity, such as S-8 and AB-8, were larger, while the capacities of weak polarity resins, such as D3520 and D4020, and the strong polarity resin NKA-9, were much smaller. The S-8 resin, which had the largest adsorption capacity, was selected to conduct the following experiments. Dynamic experimental results Breakthrough curves measured at different ¯ow rates are shown in Fig 6. The original concentration of the GA3 in the feed was 0.5gdmÿ3, the adsorption column was 50mm in length and 10mm in diameter. The results indicate that low ¯ow rate facilitates adsorption. The dynamic adsorption capacities of GA3 on S-8 were 2.88mggÿ1 wet resin, 3.82mggÿ1 wet resin and 4.56mggÿ1 wet resin at ¯ow rates of 2.25cm3minÿ1, 0.8cm3minÿ1 and 0.5cm3minÿ1, re- spectively. Since the very low ¯ow rate resulted in very long experimental periods, a ¯ow rate of 0.5cm3minÿ1 was used in the following experiments. Figure 6. Breakthrough curves for GA3 on S-8 resin at different flow rates (20 ° C): *, 2.25cm3minÿ1; &, 0.8cm3minÿ1; ~, 0.5cm3minÿ1. J Chem Technol Biotechnol 75:695±700 (2000) and the aqueous solution, with the dynamic adsorp- ÿ1 Figure 7. Desorption curves of different solvents for GA3 adsorbed on S-8 at 20 ° C: ‡, ethanol;*, ethyl acetate; ~, acetone; *, 80% (v%) acetone aqueous solution. Figure 9. Breakthrough curves of GA3 from fermentation liquor on S-8 resin (pH=2, 20 ° C). Separation of gibberellic acid on resins After the adsorption, ethanol, ethyl acetate, acetone and 80% (v%) acetone aqueous were selected as eluents to elute the GA3 adsorbed on the S-8 resin. The experimental results at a desorption ¯ow rate of 0.25cm3minÿ1 are shown in Fig 7. The results show that the desorption strength was greatest for 80% acetone aqueous solution, followed in decreasing order by acetone, ethyl acetate, and ethanol. Adsorption and desorption experimental results for GA3 fermentation liquor The adsorption isotherms for GA3 from the fermenta- tion liquor onto S-8 resin at 20 ° C are shown in Fig 8. The ordinate represents the amount of GA3 on the resin (mg GA3gÿ1 resin). The results indicate that pH strongly in¯uences the adsorption isotherms. Because GA3 has an acid radical carboxyl, some reactions may occur at high pH. The experimental data points acquired at high pH (pH>6) were unsteady and the ®delity was poor. The experimental results show that an acidic environment facilitates the adsorption. Compared with the results in Fig 5, the GA3 distri- bution coef®cients between the resins and the fermen- tation liquor were higher than those between the resins Figure 8. Adsorption isotherms of GA3 adsorbed from fermentation liquor on S-8 resin at different pH values (20 ° C): *, pH=4; *, pH=2. J Chem Technol Biotechnol 75:695±700 (2000) tion capacity of GA3 increased to 12.02mgg wet resin (Fig 9) The increase was mainly due to the salting-out effect of ammonium sulfate in the fer- mentation liquor. GA3 fermentation liquor contains 2%±5% (wt%) ammonium sulfate which may decrease the activity of water in the fermentation liquor which increases the distribution coef®cients and adsorption capacity.13 Besides the ammonium sulfate, some nutrient base, such as bran, cane sugar, or some side-product in the fermentation liquor may also affect the GA3 distribu- tion behavior relative to the `pure' aqueous solution, causing the improved distribution coef®cient and adsorption capacity. The mechanism for GA3 adsorp- tion on S-8 resin will be investigated in the future. The experimental conditions were chosen so that the GA3 concentration in the fermentation liquor was 2.0gdmÿ3 and the pH was 2, the adsorption column was 50mm in length, 10mm in diameter and the adsorption ¯ow rate was 0.5cm3 minÿ1. Desorption was achieved using acetone aqueous solutions of different concentrations on a column 50mm in length and 10mm in diameter at 20 ° C. The results are shown in Fig 10. In the experiments, the Figure 10. Eluting GA3 from S-8 resin with acetone aqueous solutions (v%) at 20 ° C: ^, 90% acetone; *, 80% acetone;~, 70% acetone. 699 GA3 concentration of the fermentation liquor feed ÿ3 tion process, GA3 can be successfully separated (East) Patent 152,578 (1981). 10 Gerhard R, Matteis I, Dieter B and Rimmer A. Recovery of gibberell