JOURNAL OF BIOWENCE AND BIOENGINEERING Vol. 93, No. 3,274-280.2002
Production of &-Polylysine in an Airlift Bioreactor (ABR) PRIHARDI KAHAR, KENGO KOBAYASHI; TOSHIHARU IWATA,3 JUN HI-,3
MAMI KOJIMA: AND MITSUYASU 0KABE2*
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
422-8529, Japan,2 and Yokohama Research Cent@, Chisso Cooperation, 5-l Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received 10 September 200lIAccepted.3 December 2001
[Key words: E-poly+lysine, Streptomyces albulus, airlift bioreactor, power input]
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.
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
* Corresponding author. e-mail: firstname.lastname@example.org phone/fax: +81-(0)54-238-4883
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.
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.
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.
MATERIALS AND METHODS
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).
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
VOL. 93,2002 E-POL~YS~ PRODUCTION IN AIRLIFT BIOREACTOR 275
(220 rpm). For production, a 5-l ABR and 5-Z jar fermentor (type MDLSOO;
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.
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&d AD/DA Card (PCL-812PG, Advantech, Tokyo).
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-
Q m 19
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&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.
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.).
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).
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).
For microscopic observation, a stereoscopic microscope (SZH- 10, Olympus Co., Tokyo) equipped with a monochrome CCD camera (XC-77CE, Sony, Tokyo) was used.
RESULTS AND DISCUSSION
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> 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 ( ) 2 3 0.45
p = %pn3D5 0 gc
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.
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
216 KAHARETAL. BIOSCI.BIOENG.,
Jar f ermentor
100 200 300 400 500 600 700 800 9001000
Agitation rate (rpm)
0 1 2 3 4 5
Aeration rate (wm)
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.
above the optimal level determined for aeration control might be one