Production of ε-polylysine in an airlift bioreactor (ABR)

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<ul><li><p>JOURNAL OF BIOWENCE AND BIOENGINEERING Vol. 93, No. 3,274-280.2002 </p><p>Production of &amp;-Polylysine in an Airlift Bioreactor (ABR) PRIHARDI KAHAR, KENGO KOBAYASHI; TOSHIHARU IWATA,3 JUN HI-,3 </p><p>MAMI KOJIMA: AND MITSUYASU 0KABE2* </p><p>United Graduate School of Agricultural Science, Gifir Universiq, 1-I Yartagido, Gifu 501-1193, Japan, Laboratory of Biotechnology, Faculv of Agriculture, Shizuoka University, 836 Ohya, Shizuoka </p><p>422-8529, Japan,2 and Yokohama Research Cent@, Chisso Cooperation, 5-l Okawa, Kanazawa-ku, Yokohama 236-8605, Japan </p><p>Received 10 September 200lIAccepted.3 December 2001 </p><p>[Key words: E-poly+lysine, Streptomyces albulus, airlift bioreactor, power input] </p><p>As we reported in a previous paper (l), E-PL was success- fully produced at a level of more than 40 8/l in a 5-1 jar fer- mentor by means of a pH control strategy under extensive power consumption in a fed-batch culture. Although an in- crease in the production of E-PL was achieved, however, significant power consumption more than 8.0 kW/m3 was required per unit volume, which was impossible to scale up to a production-scale fermentor. Furthermore, the recovery of E-PL obtained from the culture in the jar fermentor under a high level of power consumption and also the purification yield were very low probably due to the leakage of intra- cellular nucleic acid (INA)-related substances as by-prod- ucts of contamination during production. There are some re- ports that have outlined the requirements in homo-polymer production when contamination does not occur (Joppien, R., Ph. D. Thesis, University of Hannover, Germany, 1992), and some discussion about the effect of INA-related substances on the recovery and purification of polymer products (2,3). We assumed that the increase in the INA-related substances leakage might cause difficulties in downstream processing and product recovery. </p><p>For aerobic bioprocessing, the stirred-tank reactor (STR), such as a jar fermentor, is the most popular type of biore- actor, Most of the reported Streptomyces cultivations were carried out in STRs (4-8). However, the shear stresses aris- ing in these bioreactors can cause undesired effects on mycelial morphology, product formation, and product yields (9). Many workers assume that is important to reduce shear </p><p>* Corresponding author. e-mail: phone/fax: +81-(0)54-238-4883 </p><p>stress to achieve optimal production. To this end, tower-type reactors, such as bubble columns, have been employed, and among them, airlift bioreactors (ABRs) have been the most widely studied (10,ll). Since ABRs do not require mechan- ical agitation and do not have mechanical parts, the shear stress is considerably less than that in STRs. Thus, the ap- plication of ABRs to many microbial productions has at- tracted much interest. Recently, further investigations have been directed towards the potential applicability and various advantages of ABRs when compared with conventional STRS. </p><p>However, few reports have discussed the advantages of ABRs from the economy viewpoint, particularly compari- sons between the power consumed per volume in both types of bioreactors and also downstream processing optimiza- tion. </p><p>In this study, the possibility of the energy-saving produc- tion of E-PL using Streptomyces albulus strain no. 4 10 in an ABR was evaluated, and compared with the production of E-PL in a jar fermentor. We also investigated the use of ABRs to reduce the production cost during the downstream processing of E-PL. </p><p>MATERIALS AND METHODS </p><p>Microorganism and culture medium S. albulus strain no. 410 (S410; Yokohama Research Center, Chisso Co. Ltd., Yoko- hama) was used throughout this study. The strain was maintained and cultured in the medium described previously (1). </p><p>Culture methods For seed culture, a loopful of S410 was inoculated into a SOO-ml Erlemneyer flask containing 100 ml of M3G medium and precultured at 30C overnight on a rotary shaker </p><p>274 </p></li><li><p>VOL. 93,2002 E-POL~YS~ PRODUCTION IN AIRLIFT BIOREACTOR 275 </p><p>(220 rpm). For production, a 5-l ABR and 5-Z jar fermentor (type MDLSOO; </p><p>B. E. Marubishi Co. Ltd., Tokyo) were used. In the ABR, 200 ml of seed culture was inoculated into 1.8 I of M3G medium, and then cultured for 48-96 h. The aeration rate during cultivation was con- trolled based on a set value of dissolved oxygen (DO) concentra- tion, which was detected by a DO electrode (Toa Electronics Ltd., Tokyo). The pH change during cultivation was detected by a pH electrode (Toa Electronics) attached to a PID controller (MDLdC; B. E. Marubishi). The DO was kept at around 30% by varying the aeration rate from 0.5 to 2.5 wm. To maintain the pH at an appro- priate level, 2 N NaOH solution was automatically added to the culture broth. After 2-d of cultivation, foam appeared, leading to unstable culture conditions. Hence, autoclaved KM-70 (ShinEtsu Chemical Co. Ltd., Tokyo) was automatically added as an anti- foaming agent. The culture temperature was 30C and the initial pH was 6.8. Fed-batch culture was started based on the culture conditions as described previously (1). The jar fermentor was op- erated in the same manner as the ABR. </p><p>Experimental set-up of laboratory-scale ABR A schematic diagram of the experimental equipment is shown in Fig. 1. The fermentation was carried out in a modified ABR, which was 185 mm in diameter and 632 mm high. The bioreactor contained one- glass draft tubes in the center, which were 365 mm high, and 70 and 85 mm in diameter, respectively. The bioreactor, which was surrounded by a water jacket for temperature control, was made of glass. The air sparger was a multi porous plate (5 urn diameter per pore) located at the bottom of the bioreactor and between the draft tubes. Without the draf? tubes, the ABR became a simple bubble column. The DO and pH sensors were positioned at the top of the bioreactor. The foam probe was located 15 cm from the top of the upper broth surface. All the sensors and probes were interfaced with a control unit, an IBM PC/AT equipped with a PC-Lab&amp;d AD/DA Card (PCL-812PG, Advantech, Tokyo). </p><p>Recovery and purification of E-PL The culture broth har- vested from either jar fermentor or ABR was mixed with Topco Perlite no. 34 and then filtered. The resulting clear filtrate was ad- </p><p>Q m 19 </p><p>FIG 1. Schematic diagram of the ABR used throughout this study. Experimental apparatus: 1, antifoam reservoir; 2, alkaline reservoir; 3, pump; 4, pack-controller; 5, water jacket; 6, pH sensor; 7, dissolved oxygen probe: 8, foam sensor: 9, dispersed bubble; 10, draft tube; 11, sa&amp;in~ line; li, mesh screen; .13, stainless steel sparger; 14, air fil- ter; 15, flow meter; 16, air compressor; 17, CO, analyzer; 18, exhaust air line; 19, IBM PC/AT computer. </p><p>sorbed on an ion-exchange chromatography column of Amberlite IRC 5OH (pH 7.6) and eluted with 0.4 N HCl. Active fractions were combined and treated with active carbon, followed by con- centration under reduced pressure. E-PL was then precipitated from the resulting concentrate with addition of a solution of ace- tone and methanol (2 : 1, vol.). </p><p>Analytical methods The concentrations of a-PL, cells and glucose were measured as described previously (1). The analysis of ammonium concentration was carried out using a commercial enzyme analysis kit (Boehringer Mannheim code no. 542946). </p><p>Leakage of EVA-related substances from mycelia in the culture broth throughout the operation of both reactors was assessed by changes in the INA concentration according to the method of Schneider (12). </p><p>For microscopic observation, a stereoscopic microscope (SZH- 10, Olympus Co., Tokyo) equipped with a monochrome CCD camera (XC-77CE, Sony, Tokyo) was used. </p><p>RESULTS AND DISCUSSION </p><p>Comparison of the power input for oxygen supply in a jar fermentor and an ABR To compare the power input used to supply oxygen in a 5-Z jar fermentor and a 5-1 ABR, power consumed per unit volume (p$v&gt; was calculated for each operational condition. Due to the various types of cul- ture conditions, a water-air system was employed for gen- eral measurement of power input for both types of fermen- tor. In the case of the 5-1 jar fermentor, the value of PJVwas a summation of the power consumption between the agita- tion and aeration with continuous gas injection, calculated per unit volume using Eq. 1 as described by Aiba et al. (13). On the other hand, in the case of the ABR, the PJV value was calculated using Eq. 2. </p><p>where, </p><p>(1) </p><p>(2) </p><p>and </p><p>P ( ) 2 3 0.45 </p><p>A </p><p>v agitation </p><p>=a45 F </p><p>[ I </p><p>provided that </p><p>p = %pn3D5 0 gc </p><p>(4) </p><p>(5) </p><p>The relationships between PJV and agitation rate for the jar fermentor and aeration rate for the ABR are shown in Fig. 2a and 2b, respectively. </p><p>To achieve high-level production of a-PL at around 40 g/l, it is important to maintain the aerobic condition of cul- tures by means of DO control. Prolonged culture of S410 resulted in high viscosity of the broth, and this caused diffi- culties in the maintenance of aerobic conditions during the production of a-PL. Although increasing the agitation rate </p></li><li><p>216 KAHARETAL. BIOSCI.BIOENG., </p><p>Jar f ermentor </p><p>ABR 700 </p><p>600 </p><p>500 </p><p>400 </p><p>300 </p><p>200 </p><p>100 </p><p>0 </p><p>100 200 300 400 500 600 700 800 9001000 </p><p>Agitation rate (rpm) </p><p>a </p><p>0 1 2 3 4 5 </p><p>Aeration rate (wm) </p><p>b </p><p>FIG. 2. Comparison of the power consumed per unit volume of oxygen supply in both the 5-Z jar fermentor (a) and the ABR (b) with connec- tion of operating variables (agitation rate of the former and aeration rate of the later). Conditions: p= 1200 kg/m3, ~=0.02 kg/m s. </p><p>above the optimal level determined for aeration control might be one of the ways to resolve the above-mentioned problem, increasing the agitation and aeration rates might increase the value of PBK As shown clearly in from Fig. 2a, about 8.0 kW/m3 of power is required to produce a-PL at around 40 g/l in the jar fermentor due to the maintenance of the DO concentration at around 30% by increasing the agitation rate to 700 rpm. Otherwise, production of E-PL at around 40 g/l could not be achieved due to a lack of oxygen. To date, there is no operational agitator motor available for industrial-scale STRs that can fulfill the requirement of power input at 8.0 kW/m3 or higher. Thus, we assumed that it would be impossible to reproduce the results obtained in a laboratory-scale jar fermentor in a STR larger than 60m3. We therefore investigated the production of E-PL using an ABR with a low-level of power input in place of a SIX. </p><p>Comparison of batch cultures in a jar fermentor and an ABR under a low level of power input To evaluate E-PL production, batch cultures both in a jar fermentor under a low P,JV (300 and 400 rpm) and in an ABR under the usual operational conditions (2.5 wm) were carried out. </p><p>In the case of the jar fermentor at 300 rpm, only 1.3 g/l of E-PL could be produced probably due to DO limitation after the cell growth became maximal (data not shown). How- ever, if the agitation rate was increased from 300 to 400 rpm such that aerobic conditions could be attained in the culture, the accumulation of E-PL was increased to 3 gll as shown in Fig. 3a. In the case of the ABR, fermentation characteristics such as the glucose consumption rate, the growth rate and </p><p>the change in pH during fermentation were almost the same as in the jar fermentor, and the same production level of E-PL was obtained, even though the culture time was 12 h longer than that using the jar fermentor. According to the calculation as shown in Fig. 2, it is interesting to note that only 0.35 kW/m3 of P,JV was required to produce E-PL in the ABR at the same production level as in the jar fermen- tor. </p><p>We also evaluated the leakage of INA-related substances into the culture broths harvested from both types of bio- reactor. As shown in Fig. 4a, the leakage of the MA-related substances in the ABR was less than 70% of that in the jar fermentor. Also the leakage of INA-related substances per cell weight tended to slightly decrease in the ABR (Fig. 4b). This result suggests that further improvement in cell growth with associated to high cell yield may not elevate leakage of INA-related substances in the ABR. Because the INA-re- lated substances in the broth may become contaminants in the downstream processing of E-PL, a production system with low INA-related substance leakage is desirable. In this study, an ABR was therefore selected as one alternative to reduce power consumption in the case of scaling-up and to decrease the leakage of INA-related substances, which may affect the downstream processing of e-PL. </p><p>Comparison of fed-batch culture in a jar fermentor and ABR under different levels of power input To in- crease the production of E-PL, we also attempted to repro- duce the fed-batch data from the 5-l jar fermentor in a labo- ratory-scale ABR. In this case, fed-batch cultures of E-PL in </p></li><li><p>VOL. 93,2002 E-POLYLYSINE PRODUCTION IN AIRLIFT BIOREACTOR 277 </p><p>0 12 24 36 48 60 72 0 12 24 36 48 60 72 </p><p>Time (h) Time(h) </p><p>FIG. 3. Comparison of fermentation time courses in batch cultures using the 5-I jar fermentor (a) and ABR (b). Symbols: closed circle, pH; closed diamond, dry cell weight concentration (g/Q open triangle, ammonium concentration (g/Z); open circle, residual glucose concentration (g/o; closed circle, a-PL concentration (g/Q Agitation rate was 400 rpm. Aeration rate in jar fermentor was fixed at 2.5 vvm, and in the ABR was varied </p><p>b </p><p>from 0.5 to 2.5 wm. </p><p>a jar fermentor were carried out under two separate con- ditions per unit volume; low PJV (maximum agitation rate, 400 rpm) and high PJV (maximum agitation rate, 700 rpm). The culture in the ABR was carried out under continuous aeration in the range from 0.5 to 2.5 wm. </p><p>As shown in Fig. 5, more than 40 gll of a-PL was pro- duced at a high P$V (700 rpm) as previously reported. This model cannot be scaled up to a production-scale fermentor </p><p>0.4 c A a </p><p>0 I 0 12 24 36 48 60 72 </p><p>Time (h) </p><p>FIG. 4. Comparison of the INA concentration (a) and its value with respect to cell concentration (b) in broth harvested from the batch cultures in both the jar fermentor (open triangle) and ABR (closed cir- cle) as shown in Fig. 3. </p><p>due to the high P$V required. Thus, we attempted to pro- duce E-PL under feasible conditions in a production plant at a low P V (400 rpm) and about 30 g/l of E-PL was pro- duced. k the contrary, nearly 30 gll of E-PL, which was similar to the production level in the jar fermentor under a low P,JK was produced in the ABR with a P$V value 35% less than that in the jar fermentor. This suggested that the ABR was suitable for the energy-saving production of a-PL. </p><p>INA-related substances and cell morphology changes during E-PL production Since the ABR does not require mechanical agitation and does not have moving parts, the shear stress generated is considerably less than that in con- ventional SIRS, such as jar fermentors. We, therefore, in- vestigated the leakage of the INA-related substances into the harvested culture broth from each system with regard to ch...</p></li></ul>