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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 fermentor by means of a pH control strategy under extensive power consumption in a fed-batch culture. Although an increase 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-products of contamination during production. There are some reports 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 bioreactor, 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: [email protected] 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 mechanical 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 production 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 30°C overnight on a rotary shaker

274

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) concentration, 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 30°C 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 harvested 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 concentration 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 culture conditions, a water-air system was employed for gen- eral measurement of power input for both types of fermentor. In the case of the 5-1 jar fermentor, the value of PJVwas a summation of the power consumption between the agitation 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.

where,

(1)

(2)

and

P ( ) 2 3 0.45

A

v agitation

=a45 F

[ I

provided that

p = %pn3D5 0 gc

(4)

(5)

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 cultures by means of DO control. Prolonged culture of S410 resulted in high viscosity of the broth, and this caused difficulties in the maintenance of aerobic conditions during the production of a-PL. Although increasing the agitation rate

216 KAHARETAL. BIOSCI.BIOENG.,

Jar f ermentor

ABR 700

600

500

400

300

200

100

0

100 200 300 400 500 600 700 800 9001000

Agitation rate (rpm)

a

0 1 2 3 4 5

Aeration rate (wm)

b

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 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.

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.

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

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 fermentor.

We also evaluated the leakage of INA-related substances into the culture broths harvested from both types of bioreactor. 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-related 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.

Comparison of fed-batch culture in a jar fermentor and ABR under different levels of power input To increase the production of E-PL, we also attempted to reproduce the fed-batch data from the 5-l jar fermentor in a laboratory-scale ABR. In this case, fed-batch cultures of E-PL in

VOL. 93,2002 E-POLYLYSINE PRODUCTION IN AIRLIFT BIOREACTOR 277

0 12 24 36 48 60 72 0 12 24 36 48 60 72

Time (h) Time(h)

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

b

from 0.5 to 2.5 wm.

a jar fermentor were carried out under two separate conditions 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.

As shown in Fig. 5, more than 40 gll of a-PL was produced at a high P$V (700 rpm) as previously reported. This model cannot be scaled up to a production-scale fermentor

0.4 c A a

0 ’ I 0 12 24 36 48 60 72

Time (h)

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 circle) as shown in Fig. 3.

due to the high P$V required. Thus, we attempted to produce E-PL under feasible conditions in a production plant at a low P V (400 rpm) and about 30 g/l of E-PL was produced. k the contrary, nearly 30 gll of E-PL, which was similar to the production level in the jar fermentor under a low P,JK was produced in the ABR with a P$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.

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 conventional SIRS, such as jar fermentors. We, therefore, investigated the leakage of the INA-related substances into the harvested culture broth from each system with regard to changes in the morphology of the cells during the production of a-PL.

Figure 6a shows that INA-related substances in the broth from the jar fermentor under a high PJV were present at 1.8 g/l at 168 h. However, in the ABR, the level was only 0.52 g/l, 2-fold less than that in the jar fermentor under a high P$K It was clear, however, after the glucose feeding started, that the leakage of INA-related substances per cell weight showed a decline in the ABR. Use of the optimal glucose feeding rate to improve the cell growth for enhanced E-PL production may result in high leakage of INA-related substances in the fed-batch jar fermentor (Fig. 6b). Decreasing the value of P.JV in the jar fermentor may possibly be employed to decrease this leakage. However, although the

278 KAHARETAL. J. BIOSCI. BIOENG.,

41 , , , 3

2.5 2

1.5 1

0.5 0

50 40 30

20

10

0

b

0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168

Time (h) Time (h) Time (h)

FIG. 5. Time course of fed-batch production of a-PL in the jar fermentor under high P$Vat 700 rpm (a), low P 4’

Vat 400 rpm (b) and compared with that in the ABR at the maximal aeration rate of 2.5 wm (c). The symbols used are the same as those in Fig. . In the jar fermentor, the initial agitation rate was 300 rpm and the aeration rate was fixed at 2.5 wm. In the ABR, the aeration rate was initially set at 0.5 wm and then increased to 2.5 wm according to the DO value.

value of P$V in the jar fermentor was set at a low level with an agitation rate of 400 rpm, it was still 2.2 fold higher than that in the ABR. In conclusion, the leakage of INA-related substances into the culture broth was the lowest in the ABR.

This result may mean that the ABR would be of practical use, to reduce the difficulties in E-PL recovery, particularly to obtain high purity of a-PL products. The basis of this proposition is the shear stress acting on the cells due to fluid dynamics forces. As clearly shown in Fig. 7, the shear stress

2 a

? 1.5 -

S

2 1 _

0.5 -

0.125

z b 0.1

01 I I I , I I I I 0 24 48 72 96 120 144 168

Time (h)

FIG 6. Time course of the INA concentration (a) and its values with respect to cell concentration (b) in broth harvested from the fed- batch cultures in both the jar fermentor and ABR. Symbols: open triangle down, jar fermentor at 700 rpm; open triangle up, jar fermentor at 400 rpm; closed circle, ABR at 2.5 wm.

promotes variation of the morphology of S410 cells during the fed-batch production of E-PL and it may limit cell growth through cell damage. Also shear stress effectively decreases the formation of mycelial clusters and produces filament-like cells, which can potentially increase the ap- parent viscosity of broth (14) despite high INA-related substance leakage.

Recovery yield of E-PL To clarify the advantages of using an ABR in a-PL production, we tried to recover and purify E-PL from the culture broth harvested from both the ABR and the jar fermentor under different P$V values. As shown in Table 1, the recovery yield of E-PL achieved in the jar fermentor after the production at high P$V (700rpm) was 68.4%. The recovery yield in the jar fermentor could be improved by decreasing the agitation rate and it was 7 1.3% at 400 rpm. Unfortunately, the accumulation of E-PL in the jar fermentor at 300 rpm was extremely low, and we could not purify the a-PL product probably due to product loss. If we want to increase the recovery yield of a-PL (68.4%) from the culture broth in the jar fermentor under a high P$K further downstream processing is required, such as the use

TABLE 1. Comparison of the purification rate of E-PL in the jar fermentor and ABR

Jar fermentor (rpm) ABR (vvm) \ ---I

3oc 400 700 2.5

Final concentration of E-PL (g/I) 1.3 31 40 30 *Recovery yield (%) ND 71.3 68.4 78.7 INA (glr) ND 1.12 1.87 0.52 Power input (W/m’) 600 1000 8000 350

ND, Not detected. (purified E-PL)x(purity)

* = (final concentration of E- PL) x(tina1 broth volume) x 100%

VOL. 93.2002 E-POLYLYSINF PRODUCTION IN AIRLIFT BIOREACTOR 279

t=96 h

a

b

FIG 7.

t=24 h k168 h

The morphology changes of S410 cells during fed-batch culture in a jar fermentor under an agitation rate of 700 rpm (a) and in an ABR ^_ _ ,.. under an aeration rate of 2.5 wm (b).

of a peptide-protein adsorption column, which leads to increased total production costs. On the contrary, the recovery yield of s-PL in the case of the ABR was 78.7%, higher than that in the jar fermentor.

The results obtained in this study are promising for the low-cost production of E-PL. Although the E-PL productiv- ity in the ABR is lower than that in the jar fermentor under high P$V (700 rpm, 8.0 kW/m3), it has advantages both in the ease of scale-up to production-scale (larger than 60 m’) and also in the low overall cost for the production of E-PL of high purity. Finally, further studies on scale-up, particularly the optimal design of a production-scale ABR, are consid- ered important to decrease the total production cost of E-PL.

J, : P * 1 *

P2 : fair: T ; PO :

Ps :

N, : P : n :

2;

v :

NOMENCLATURE

coefficient for transformation from J to kW, kW/J pressure in medium at the height of the air sparger, atm back pressure, atm volume of air per volume of broth per minute, min-’ gas constant, 8.3 14 J/mol/K temperature, K power consumed for agitation in an ungassed system, kW power consumed for agitation in a gassed system, kW aeration number density of liquid, kg/m3 agitation rate, rps conversion factor, kg m’/kW s3 impeller diameter, m volume of liquid, m3

ACKNOWLEDGMENTS

The authors are grateful to Dr. Enoch Y. Park, Faculty of Agri- culture, Shizuoka University, for his helpful advice regarding the measurement of the leakage of the INA-related substances. Also the authors wish to thank Mr. Noriaki Itoda, a graduate student of the Faculty of Agriculture, Shizuoka University, for his help regarding the calculation of the power input both in the jar fermentor and ABR.

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