Liquid-surface immobilization system and liquid–liquid interface bioreactor: Application to fungal hydrolysis

Process Biochemistry 42 (2007) 1553–1560 www.elsevier.com/locate/procbio

Liquid-surface immobilization system and liquid–liquid interface bioreactor: Application to fungal hydrolysis Shinobu Oda *, Kunio Isshiki Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan Received 15 May 2007; received in revised form 28 August 2007; accepted 4 September 2007

Abstract Novel fungal cultivation and bioconversion systems are proposed. Spores and mycelia of a fungus suspended in a liquid medium were effectively floated on a liquid surface by the aid of a ballooned microsphere (MS). Many fungi such as Aspergillus and Penicillium formed a thick and physically strong fungus-MS mat on the liquid surface followed by stationary cultivation (LSI). The fungus-MS mat of Absidia coerulea IFO 4423 was overlaid by a solution of 2-ethylhexyl acetate (1) in n-decane (liquid–liquid interface bioreactor, L-L IBR). The strain could efficiently catalyze the hydrolysis of 1 to 2-ethyl-1-hexanol (2). The accumulation of 2 in the L-L IBR was significantly higher than those in emulsion and organic-aqueous two-liquid-phase systems and a formerly reported interface bioreactor (solid–liquid interface bioreactor, S-L IBR). Furthermore, lipase production in the LSI system was also higher than that in a submerged cultivation system. # 2007 Elsevier Ltd. All rights reserved. Keywords: Liquid-surface cultivation; Interface bioreactor; Immobilization; Microsphere; Lipase; Hydrolysis

1. Introduction Although many fungi produce various useful enzymes and secondary metabolites, the industrial applications are limited by some disadvantages in the present cultivation techniques, such as submerged, solid-state, and liquid-surface cultivation systems. In the submerged cultivation, it is generally difficult to control the fungal morphology that is the most important factor for the industrial application [1,2]. Moreover, it is also well known that the submerged cultivation has some serious disadvantages, such as the appearance of catabolite repression [3,4], the degradation of heterologous proteins by inherent proteases [3,5], and the damage of fungal mycelia by strong agitation [2,6]. On the other hand, the solid-state cultivation that is a traditional cultivation procedure in food industry has some advantages, such as higher fermentation productivity, endconcentration, and stability of product. It has also lower catabolite repression and energy demand compared with the submerged cultivation [7]. These advantages have, therefore, led to many applications of the solid-state cultivation to the production of enzymes [4,5], spores [8,9], pigments [10] and

* Corresponding author. Fax: +81 538 21 1135. E-mail address: [email protected] (S. Oda). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.09.002

biologically active secondary metabolites [11,12] being reported. However, the solid-state cultivation has also some disadvantages, such as poor heat dissipation and slow diffusion of nutrients, products, water and oxygen in a packed bed [7,13]. For the purpose of overcoming these disadvantages, some inert supports such as polyurethane foam [4,14], Amberlite [12], polystyrene [15], polypropylene [16], and nylon sponges [17] have been often been used. Although the liquid-surface cultivation using a microorganism floated on the surface of a liquid medium has been used to the production of citric acid [18] and enzymes [19], the system has also some disadvantages, such as the necessity of a long span cultivation, the instability of a fungal mat, and its narrow application. Recently, two unique cultivation systems, membrane-surface liquid cultivation (MSLC) and agar plate–organic solvent interfacial cultivation, were proposed. In the MSLC, although a fungus sufficiently grows on the surface of a porous polysulfone membrane set on the surface of a liquid medium to produce some enzymes [20,21] and kojic acid [22], the difficulty of up-scaling of the system and the blockage of micropores in the polysulfone membrane causes serious problems. On the other hand, in the agar plate–organic solvent interfacial cultivation, some fungi such as Aspergillus, Aureobasidium, and Penicillium can actively grow on an

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interface between a low toxic hydrocarbon (n-paraffin and nheptylbenzene) and a nutrient agar plate [23]. This system was developed to an interface bioreactor (solid–liquid interface bioreactor, S-L IBR), which is a microbial transformation device on an interface between a hydrophilic carrier and a hydrophobic organic solvent, and applied to various microbial transformations and biodesulfurization [24–28]. Although the bioreactor exhibits the superior toxicity alleviation effect toward hydrophobic substrates and products, some disadvantages, such as the difficulty of up-scaling of the bioreactor and the control of contents of water and nutrients, and the accumulation of toxic hydrophilic by-products such as organic acids in the agar plate, have been observed. Thus, the construction of a novel fungal cultivation system that can solve the abovementioned disadvantages in each traditional cultivation system is desired. In this report, we proposed a novel fungal cultivation system using a unique polymeric micro-material, a ballooned microsphere (MS). The cultivation system was tentatively named liquid-surface immobilization (LSI) system. Furthermore, a novel fungal bioconversion system named liquid–liquid interface bioreactor (L-L IBR) was also proposed. The L-L IBR was applied to the organic-aqueous interfacial hydrolysis of 2ethylhexyl acetate (1) with Absidia coerulea IFO 4423. The LL IBR exhibited the superior productivity of 2-ethyl-1-hexanol (2) compared with the emulsion and organic-aqueous twoliquid-phase systems, and the S-L IBR. The accumulation of 2 in the L-L IBR reached to over 100 mg ml 1 in an organic phase in spite of the strong inhibitory effects of 1 and 2 (Fig. 1). It was considered that the higher productivity of lipase and the alleviation effect of the inhibitory action of 1 and 2 in the L-L IBR contributed to the higher productivity of 2. 2. Experimental 2.1. Microorganisms, media, and chemicals All strains were stocked fungi in our laboratory containing ATCC (American Type Culture Collection), NRRL (Agricultural Research Service Culture Collection), and NBRC (NITE Biological Resource Center; former IFO) type

culture strains. All strains were stocked on modified Sabouraud agar plates consisted of 40.0 g of glucose, 10.0 g of Bacto peptone, 5 mg of FeSO4
7H2O, 20 mg of MnSO4
5H2O, 10 mg of CaCl2, and 15.0 g of agar in 1.0 l of deionized water (pH 6.0) at 4 8C. The medium was used for all tests with or without agar except the experiments for the effects of initial medium pH and glucose content. Seed cultivation was performed with F1 medium, consisted of 20.0 g of potato starch, 10.0 g of glucose, 20.0 g of soy protein, 1.0 g of KH2PO4, and 0.5 g of MgSO4
7H2O in 1.0 l of deionized water (pH 6.0). The ballooned microspheres (MSs), MFL-80GCA (CaCO3-coated type) and MFL-80GTA (talc-coated type), were gifted by Matsumoto Yushi-Seiyaku Co., Ltd. (Osaka). Both MSs were stable in hydrophobic organic solvents such as styrene. Mean diameter and density of both MSs were 20 mm and 0.2, respectively. All other chemicals were commercially available.

2.2. Floating test of fungi with or without MS Twenty milliliters of the modified Sabouraud medium and 70 mg of MFL80GTA were sufficiently mixed in a glass vial (volume, 50 ml; diameter, 3 cm). Then, 200 ml of a 3-day broth of each strain was added to the mixture, homogeneously mixed, and stand at 25 8C for 3 days. Rhizopus formosaensis IFO 4720 and R. oryzae ATCC 10404 were inoculated as a spore suspension. The liquid-surface cultivation without the MS was set as the reference to each strain. The evaluation of the fungal mat formation was exhibited as an occupation area on a liquid surface.

2.3. Screening of ester 1-hydrolyzing fungi Ester 1-hydrolyzing fungi were screened with the S-L IBR [24–28]. Ten milliliters of the agar medium was prepared in a glass vial whose volume and diameter were 50 ml and 3 cm, respectively. Three pieces (approximately 2 mm  2 mm) of each fungal mat were inoculated on the surface of the agar plate with a long toothpick, and precultivation was done at 25 8C by allowing the plate to stand for 2 days. After the precultivation, 2 ml of a 50% solution of 1 in n-decane was added onto the surface of fungal mats. Incubation was continued at 25 8C by allowing the plate to stand for 5 days. After the incubation, an organic phase in the vessel was directly analyzed with gas chromatography; the column (0.25 mm i.d.  30 m) contained Equity-5 (Supelco Co., Ltd., Bellefonte, PA), the column temperature was 100 8C, the injector temperature was 150 8C, the detector temperature was 155 8C, the carrier gas was He (20.5 mm s 1), and the split ratio was 1:50. The retention times of 2 and 1 were 10.21 and 15.59 min, respectively. The optical resolution of produced 2 in order to estimate the enantioselectivity of the hydrolysis of 1 was performed with gas chromatography; the column (0.25 mm i.d.  30 m) contained b-DEXTM325 (Supelco Co., Ltd.), the column temperature was raised from 80 to 110 8C at the rate of 1.0 8C min 1, the injector temperature was 150 8C, the detector temperature was 155 8C, the carrier gas was He

Fig. 1. Principles for LSI and L-L IBR systems. 1, 2-Ethylhexyl acetate; 2, 2-ethyl-1-hexanol. In the LSI system, a fungus immobilized in a fungus-MS mat floating on the surface of a liquid medium can efficiently produce enzymes and metabolites by using nutrients and water in a liquid medium. Oxygen is spontaneously supplied from the atmosphere. In the L-L IBR system, a fungus immobilized in the fungus-MS mat efficiently catalyzes the microbial transformations of lipophilic substrates (1) to lipophilic substrates (2).

S. Oda, K. Isshiki / Process Biochemistry 42 (2007) 1553–1560 (25.7 cm s 1), and the split ratio was 1:100. Alcohol 1 was detected as two peaks whose retention times were 18.57 and 19.00 min.

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The effects of initial medium pH, glucose content, medium volume, substrate concentration, organic phase volume, and the shaking of the bioreactor on the hydrolysis of 1 were examined by using the S-L IBR. Two hundred ml of a 2-day broth was inoculated on the surface of the agar plate (10 ml) prepared in a glass vial (volume, 50 ml; diameter, 3 cm). The medium pH and glucose content were varied from 5.5 to 8.0 and 1 to 10% (w/v), respectively. The medium volume and the organic phase volume were adjusted to 10, 15 or 20 ml and 3, 6 or 9 ml in the vial, respectively. Substrate concentration was varied from 10 to 90% (v/v) in n-decane or 100%. The hydrolytic reaction was carried out at 25 8C with or without shaking (100 rpm).

liquid medium prepared in the vessel (medium volume 50 ml; diameter, 7 cm). Following precultivation at 25 8C with agitation at 300 rpm for 3 days, 10 ml of neat 1 (emulsion system) or 20 ml of a 50% solution of 1 in n-decane (two-liquidphase system) was added to each broth, and incubation was continued for 10 days. As for the S-L IBR, 1 ml of a 3-day broth was inoculated on the surface of the agar plate prepared in the vessel and precultivation was done at 25 8C by allowing the vessel to stand for 3 days. Following the precultivation, 20 ml of a 50% solution of 1 in n-decane was added onto the surface of a fungal mat. Incubation was continued without shaking for 10 days. As for the L-L IBR, 20 ml of a 3-day broth was inoculated to 50 ml of the liquid medium containing 1 g of MFL-GCA. Following stationary precultivation for 3 days, 20 ml of a 50% solution of 1 in ndecane was added onto the surface of a fungus-MS mat, and incubation was continued without shaking for 10 days. A liquid medium in the L-L IBR was periodically exchanged by 50 ml of a fresh medium per 2 or 3 days. Thus, the addition of supplementary nutrients and the removal of harmful by-products such as acetic acid were effectively achieved in the L-L IBR.

2.5. Comparison of 1-hydrolytic activities among emulsion and organic-aqueous two-liquid-phase systems, S-L and L-L IBRs

2.6. Comparison of lipase production between submerged cultivation and LSI system

Comparison of 1-hydrolytic activities among four cultivation systems was done via two steps. First, as for the emulsion and two-liquid-phase systems, 1 ml of a 3-day broth was inoculated into 50 ml of the liquid medium prepared in a 250 ml-flask. Following the precultivation at 25 8C with shaking (220 rpm) for 3 days, 10 ml of neat 1 (emulsion system) or 20 ml of a 50% solution of 1 in n-decane (two-liquid-phase system) was added to each broth, and incubation was continued for 8 days. As for the S-L IBR, 200 ml of a 3-day broth was inoculated to the surface of the agar plate prepared in a glass vial (volume, 50 ml; diameter, 3 cm) and precultivation was performed at 25 8C by allowing the vial to stand for 3 days. Following the precultivation, 2 ml of a 50% solution of 1 in n-decane was added onto the surface of a fungal mat. Incubation was carried out with shaking (100 rpm) for 8 days. As for the L-L IBR, 200 ml of a 3-day broth was inoculated to 10 ml of the liquid medium containing 400 mg of MFL-80GCA. Following precultivation for 3 days, 3 ml of a 50% solution of 1 in n-decane was added onto the fungus-MS mat, and incubation was done at 25 8C with shaking (100 rpm) for 8 days. The accumulation of 2 in the organic phase was directly determined by gas chromatography on the fourth and eighth days of incubation in all systems. Secondly, the comparison of 1-hydrolytic activities in four cultivation systems was done with polypropylene vessels as shown in Fig. 2. As for the emulsion and two-liquid-phase systems, 2 ml of a 3-day broth was inoculated to 50 ml of the

As for the submerged cultivation, 2 ml of a 2-day broth was inoculated into 50 ml of the liquid medium prepared in a 250 ml-flask, and incubation was done at 25 8C with shaking (220 rpm) for 7 days. After the incubation, 8 ml of a supernatant gained by centrifugation of the broth was added to 2 ml of 1, and the hydrolytic reaction of 1 was carried out at 25 8C with shaking (220 rpm) for 4 h. As for the LSI system, 2 ml of a 2-day broth was inoculated to 50 ml of the liquid medium containing 1 g of MFL-80GCA. Incubation was performed at 25 8C with agitation (60 rpm) of an aqueous phase with a magnetic stirrer after 3-day stationary cultivation for 4 days. After the incubation, an aqueous phase was vigorously homogenized (18,000 rpm, 1 min) together with a fungus-MS mat, and a supernatant obtained by filtration with a cotton filter. The product 2 was determined with gas chromatography.

2.4. Characterization of the hydrolysis of 1 with Absidia coerulea IFO 4423

3. Results 3.1. Floating test of fungi with or without MS It is well known that some fungi can spontaneously float on the surface of a liquid medium to form a homologous fungal

Fig. 2. Schematic diagrams of the devices used in emulsion, two-liquid-phase, S-L IBR and L-L IBR systems. (A) Deep glass Petri dish; (B) liquid medium; (C) magnet; (D) organic phase; (E) fungal mat; (F) agar medium; (G) fungus-MS mat; (H) inlet; (I) outlet. In the emulsion and two-liquid-phase systems, an aqueous phase is intensely agitated with a magnetic stirrer. A hydrophobic organic solvent (n-decane) was added into the aqueous phase in the two-liquid-phase system. In the L-L IBR system, a liquid medium was periodically exchanged by a fresh medium via an inlet and an outlet line per 2 or 3 days.

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Fig. 3. Photographs of various fungal and fungus-MS mats formed on the surface of a liquid medium. Left vial, the LSI system; right vial, the traditional liquidsurface cultivation system. Cultivation was performed at 25 8C by allowing the vials to stand for 3 days.

mat (liquid-surface cultivation). However, the fungal mat formation usually needs a long time and the formed fungal mat is unstable. First, we examined the floating ability of fungi with MFL-80GTA. As shown in Fig. 3 and Table 1, many fungi could form thick fungus-MS mats during the stationary cultivation only for 3 days. Aerial hyphae and spores were formed on the surface of mats of various fungi. On the other hand, many fungi could not cover a liquid-surface during the liquid-surface cultivation for 3 days. The fungus-MS mats formed in the LSI system were physically strong (Fig. 4A) and could be formed on a wider liquid-surface (30 cm  40 cm) in a stainless steel

tray (Fig. 4B). Thus, it was deduced that the LSI system was superior to the traditional liquid-surface cultivation for the formation of fungal mats. 3.2. Screening of 1-hydrolyzing fungi with S-L IBR In order to apply the L-L IBR to microbial transformation, the hydrolysis of ester 1 to alcohol 2 was examined (Fig. 1). First, we screened 1-hydrolyzing fungi with the S-L IBR among approximately 400 stocked fungi. As shown in Table 2, although the enantioselectivity of the hydrolysis of 1 were very

Table 1 Fungal mat formation on the surface of a liquid medium with or without microspherea Strain

LSI

Absidia blakesleeana ATCC 10148 Acremonium persicinum CBS 169.65 Armillaria tabescens IFO 9616 Aspergillus flavus ATCC 9643 Aspergillus niger NRRL 3122 Aspergillus ochraceum NRRL 405 Aspergillus oryzae IAM 2630 Aspergillus parasiticus IAM 2664 Bahusakala olivaceonigra CBS 499.66 Beauveria bassiana ATCC 7159 Cephalosporium mycophilum NL 24 Chaetomium globosum ATCC 6205 Cladosporium sphaerospermum IFO 4460 Corynespora cassicola IFO 6724 Cunninghamella sp. F 1490 Curvularia maculans PPT-4-2 Mortierella isabellina ATCC 42613 Neurospora crassa ATCC 14692 Penicillium chrysogenum IAM 7326 Penicillium citrinum IAM 7048

+++ ++ + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

a

Free

++ + + + ++ +++ + + + – +++ – + +++ + +

Strain

LSI

Free

Emericella unguis ATCC 13431 Emericellopsis minima IFO 8384 Flammulina velutipes IFO 30905 Glomerella fusarioides NBRC 8831 Hypomyces subiculosus IFO 6892 Penicillium echinulatum IAM 7208 Penicillium pinophilum IAM 7148 Peziza vesiculosa IFO 30324 Phanerochaete chrysosporium ATCC 34541 Phanerochaete sordida ATCC 90872 Phoma exigua ATCC 14728 Pleurotus pulmonarius IFO 31345 Pseudohydnum gelatinosum IFO 6984 Rhizopus formosaensis IFO 4720 Rhizopus oryzae ATCC 10404 Sesquicillopsis rosariensis DSM 8108 Spiroidium fuscum IFO 5479 Thanatephorus cucumeris IFO 5254 Trametes gibbosa IFO 4946 Trichoderma virens ATCC 9645

+++ +++ + +++ +++ +++ +++ +++ +++ ++ ++ + ++ +++ +++ +++ ++ +++ + +++

++

The degree of occupation area of a fungal mat during 3-day stationary cultivation: +++ (100%) > ++ > + >

(0%).

+ + ++ +

+

++

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Fig. 4. Some characteristics of the LSI system. The fungus-MS mat of various strains such as Pycnosporium sp. ATCC 12231 did not collapse by standing off the vial (A). Moreover, the fungus-MS mat could be easily enlarged by 30 cm  40 cm in a stainless-steel tray (B). Chaetomium globosum ATCC 6205 actively produced a secondary metabolite, a red pigment, in the LSI system (C).

low, many strains could efficiently hydrolyze 1 (concentration, 50% in n-decane) to 2. Penicillium fuscoflavum IAM 7196 and Fusarium oxysporum f. cubens FI 0738 could enantioselectively hydrolyze 1 in some degree, but the accumulations of 2 were very low. The highest accumulation of 2 was obtained in the case of using Absidia coerulea IFO 4423. 3.3. Characteristics of the hydrolysis of 1 with Absidia coerulea IFO 4423 in S-L IBR In order to characterize the hydrolysis of 1, we examined the effect of initial medium pH, agar medium volume, substrate concentration, organic layer volume, and the shaking of the reactor on the hydrolysis of 1 with the S-L IBR. The initial medium pH did not significantly affect 1-hydrolytic activity at the range from 5.5 to 8.5. The accumulation of 2 ranged from 34.2 to 40.2 mg ml 1 for 7 days, and the production rate ranged from 233 to 235 mg ml 1 h 1.

3.4. Comparison of 1-hydrolytic activities among emulsion and two-liquid-phase systems, S-L and L-L IBRs

Table 2 Screening of 1-hydrolyzing fungi by using S-L IBR Strain

Produced 2 mg ml

Absidia blakesleeana ATCC 10148 Absidia coerulea IFO 4423 Aspergillus parasiticus IAM 2665 Fusarium oxysporum f. cubens FI 0738 Penicillium fuscoflavum IAM 7196 Phoma cucurbitacearum A-7 Rhizopus formosaensis IFO 4720 Rhizopus japonicus IFO 4758 Rhizopus oryzae NBRC 5781 Rhizopus reflexus IAM 6253 Rhizopus sp. KS-206 Syncephalastrum racemosum IFO 4814 Tolypodadium niveum ATCC 34921

42.4 51.1 6.9 8.8 10.8 39.8 35.6 35.4 33.3 37.7 41.8 41.5 36.9

On the other hand, the agar medium volume affected 1hydrolytic activity. The production rates of 2 were 37.5, 45.8, and 59.6 mg ml 1 h 1 at 10, 15, and 20 ml of agar medium, respectively. The substrate concentration also affected the hydrolysis of 1. The higher the concentration of 1, the greater the accumulation of 2 as shown in Fig. 5. Thus, the inhibitory actions of 1 and 2 were effectively alleviated in the S-L IBR. Interestingly, the increase of an organic layer volume (50% 1 in n-decane) led to the increase of production rate of 2. The 2production rates per surface area of a fungal mat were 90.8, 112.4, and 126.8 mg cm 2 h 1 in 3, 6, and 9 ml of the organic layer volume, respectively. Thus, the larger the volume of organic layer, the higher the accumulation of 2. The shaking (100 rpm) of the S-L IBR also increased the production rate of 2. In a shaken S-L IBR, the production rate of 2 was 1.4 times higher than that in the stationary system (331 vs. 235 mg ml 1 h 1).

a

1

ee (%) 3.7b 5.8 60.6 55.0 54.9 5.3 5.3 6.3 5.4 2.7 0.1 2.8 6.0

Three pieces (approximately 2 mm  2 mm) of each fungal mat were inoculated on the surface of the Sabouraud agar plate (volume, 10 ml; surface area, 7.1 cm2) with a long toothpick, and precultivation was done at 25 8C by allowing the plate to stand for 2 days. After the precultivation, 2 ml of a 50% solution of 1 in n-decane was added onto the surface of a fungal mat. Incubation was continued at 25 8C by allowing the plate to stand for 5 days. a Concentration of 1 is exhibited as that in an organic phase. b Absolute configuration of 2 is unknown.

As shown in Fig. 5, the S-L IBR exhibited the superior inhibition alleviation effects on fungi in the same way as bacteria, yeasts, and actinomycetes [23–28]. Next, we compared the 1-hydrolytic activity in the L-L IBR with those in the emulsion and two-liquid-phase systems, and the S-L IBR. As shown in Fig. 6, it was observed that the strong inhibition by 1 was observed in the emulsion system. Although the hydrolysis of 1 did not significantly proceed in the emulsion system, 1 was efficiently hydrolyzed and much 2 was accumulated in an organic phase (50% solution of 1 in ndecane) in the two-liquid-phase system. It was assumed that the organic phase in the two-liquid-phase system played as a reservoir of inhibitory 1 and 2. As for the S-L IBR, the inhibition alleviation effect was higher than that in the two-liquid-phase system, although the production rate of 2 decreased in 8 days because of the strong inhibitory effect of 2. On the other hand, the L-L IBR afforded the highest production rate and accumulation of 2. The production rate and the final accumulation of 2 in the L-L IBR were 1.8 and 1.6 times higher than those in the S-L IBR, and the accumulation of 2 in the L-L IBR reached over 100 mg ml 1 by

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shown in Fig. 2. A liquid medium in the L-L IBR was periodically exchanged per 2 or 3 days. As shown in Fig. 7, both in the case of 20 and 100% concentrations of 1, the L-L IBR exhibited the highest 1-hydrolytic activities. Especially, for the hydrolysis of neat 1, whereby, the final accumulation of 2 in the L-L IBR was more than two-fold of that in the S-L IBR. Thus, the removal of inhibitory by-products such as acetic acid was effective for the improved performance of the hydrolysis of 1. 3.5. Comparison of lipase production between submerged cultivation and LSI system Fig. 5. Effect of substrate concentration on the hydrolysis of 1. Three milliliters of a 10:90 (v/v) solution of ester 1 in n-decane or neat 1 was added onto the surface of a 3-day-old fungal mat of Absidia coerulea IFO 4423. The hydrolysis of 1 to 2 was performed at 25 8C without shaking.

8 day. Thus, the L-L IBR exhibited a superior effect on the production of 2 compared with the emulsion and two-liquidphase systems, and the S-L IBR. It was expected that the L-L IBR was widely applicable to the fungal transformation of water-insoluble substrates. The continuous or periodical exchange of an aqueous phase by a fresh medium is an important advantage that there is not in the S-L IBR. In the S-L IBR, the exchange or modification of an aqueous phase in a gel is generally impossible. On the other hand, it was assumed that the harmful by-product such as acetic acid accumulated in aqueous phase in the L-L IBR could be effectively removed and thus enhancing the hydrolysis of 1. The 1-hydrolytic activity in a modified L-L IBR having an inlet and an outlet connected to an aqueous phase was compared with the conventional emulsion and two-liquid-phase systems, and the S-L IBR. The schematic diagram of each system is

Fig. 6. Comparison of 1-hydrolytic activities among emulsion, two-liquidphase, S-L IBR, and L-L IBR systems (first trial). In the emulsion and twoliquid-phase systems, 10 ml of ester 1 or 20 ml of a 50% (w/v) solution of 1 in ndecane was added to a 50 ml of a 3-day broth in a 250 ml-flask, respectively. In the S-L and L-L IBR systems, 2 ml of a 50% (w/v) solution of 1 in n-decane was added to the surface of a 3-day-old fungal mat (S-L IBR) and a fungus-MS mat (L-L IBR) in a glass vial (inner volume, 50 ml; diameter, 3 cm), respectively. The incubation was continued at 25 8C with shaking (220 rpm; emulsion and two-liquid-phase systems) or without shaking (S-L and L-L IBR systems).

Finally, we compared the lipase production in the LSI system with that in the submerged cultivation using 1 as a substrate. The accumulations of 2 in the submerged cultivation and the LSI system were 3.3 and 7.3 mg ml 1 for 4 h, respectively. The specific activities of lipase produced in the submerged cultivation and the LSI system were 26.5 and 58.5 mmol ml 1 min 1, respectively. Thus, the lipase productivity in the LSI system was over two-fold higher than that in the submerged cultivation. It was concluded that the LSI system achieved a superior 1-hydrolytic activity according to the excellent inhibition alleviation effect toward 1 and 2 and higher lipase productivity compared with the submerged cultivation. 4. Discussion The ballooned microspheres used in the LSI and L-L IBR systems are different from the filled types used in the immobilization of enzymes [29,30] and antibodies [31]. The unique characteristics, such as very low density and small particle size, enable fungal cells to floated on the surface of a liquid medium within 1 min. The floated and trapped fungal cells grow sufficiently and form a physically strong fungus-MS mat using water and nutrients in the liquid medium and oxygen in the atmosphere. The immobilized fungi differentiate and form much aerial hyphae and spores (Fig. 3 and Table 1). Although the differentiation of fungi in the LSI system resembles natural form same as traditional solid-state cultivation [32], the supply of water, nutrients and oxygen in the LSI system are higher than those in the solid-state cultivation. The LSI system has also some advantages compared with the liquidsurface and submerged cultivation, i.e., faster fungal mat formation, easier morphology control, efficient oxygen supply without agitation and aeration. It is well known that fungal morphology affects the productivity of enzymes and secondary metabolites [1,2,5,6]. For instance, while the filamentous form resulted in various unfavorable phenomena, such as the high viscosity of a broth and the decrease of dissolved oxygen. Moreover, vigorous agitation leads to a serious damage on fungal cells and higher energy consumption in the submerged cultivation. However, the LSI system consistently overcomes abovementioned disadvantages in the traditional cultivation systems; the submerged, solid-state, and liquid-surface cultivations. For the purpose of examination of the performance of the LL IBR that is a novel non-aqueous bioconversion system using

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Fig. 7. Comparison of 1-hydrolytic activities among emulsion, two-liquid-phase, S-L IBR, and L-L IBR systems (second trial). The schematic diagram of each system is shown in Fig. 2. In the emulsion and two-liquid-phase systems, 10 ml of neat 1 or 20 ml of a 50% (w/v) solution of 1 in n-decane was added to 50 ml of a 3day broth, respectively. In the S-L and L-L IBR systems, 20 ml of neat 1 or a 50% (w/v) solution of 1 in n-decane was added to the surface of a 3-day-old fungal mat (SL IBR) and a fungus-MS mat (L-L IBR), respectively. The hydrolytic reaction in the emulsion and two-liquid-phase systems was done at 25 8C with agitation with a magnetic stirrer (300 rpm). The hydrolysis in the S-L and L-L IBR systems was performed at 25 8C without agitation. In the L-L IBR system, an aqueous phase (50 ml) was periodically exchanged by 50 ml of a fresh medium per 2 or 3 days.

the LSI system, the fungal hydrolysis of 1 was examined. As shown in Table 2, Absidia coerulea IFO 4423 was the strain as it afforded the highest 2-accumulation (51.1 mg/ml) in the S-L IBR, although the enantioselectivity was very low. Indeed, the optical resolution of aliphatic primary alcohols via enzymatic or microbial hydrolysis is generally difficult [33,34]. However, Absidia coerulea IFO 4423 could catalyze the hydrolysis of 100% of ester 1 to afford over 80 mg/ml of alcohol 2 during 8day incubation (Fig. 5). Thus, the S-L IBR exhibited a superior toxicity or inhibition alleviation effect on a hydrophobic substrate (ester 1). Furthermore, the L-L IBR exhibited more excellent 1hydrolytic activity compared with the emulsion and the twophase systems, and the S-L IBR as shown in Fig. 6. In the emulsion system, the hydrolytic reaction did not significantly proceed because of the strong inhibitory action of ester 1. It was assumed that the interfacial denaturation of lipase and/or the phase toxicity of 1 toward fungal cells appeared in the emulsion system [35,36]. In the two-liquid-phase system, the hydrolysis of ester 1 smoothly proceeded to afford 40 mg ml 1 of alcohol 2 for 8 days since n-decane might be play as a reservoir for 1 and/or 2. On the other hand, in both the interface bioreactors (S-L and L-L IBRs), the accumulation of 2 was higher than that in the two-liquid-phase system (Fig. 6). Especially, the L-L IBR exhibited excellent accumulation of 2 compared with the emulsion, twoliquid-phase, and S-L IBR systems. In the L-L IBR, the accumulation of 2 in an organic phase reached to over 100 mg ml 1 in spite of the strong inhibitory action of 1 and 2 as shown in the emulsion system. An aqueous phase (liquid medium) in the L-L IBR was periodically (per 2 or 3 days) exchanged to remove acetic acid produced via the hydrolysis of 1, and as a result, the

accumulation of 2 in the L-L IBR was at least 2 times higher than that in the S-L IBR (Fig. 7). It was assumed that the harmful acetic acid was effectively removed by the periodically exchange of the aqueous phase. Furthermore, the production of lipase in the LSI system was at least 2 times higher than that in the submerged culture. The specific activities of lipase produced in the submerged culture and the LSI system were 26.5 and 58.5 mmol ml 1 min 1, respectively. Therefore, it was concluded that the L-L IBR was a superior fungal transformation system for hydrophobic substrates since the system enabled the production of much biocatalyst in addition to the alleviation of the inhibitory actions of hydrophobic substrates and products. In addition to the efficient proceeding of fungal hydrolysis and lipase production, it was observed that some fungi such as Chaetomium globosum ATCC 6205 actively produced watersoluble pigments in the LSI system (Fig. 4C). On the other hand, the accumulation of lipophilic pigments in an organic phase was also observed in the L-L IBR with many fungi (data not shown). Thus, it is strongly expected that the LSI system is applicable to the efficient production of secondary metabolites such as lipophilic antibiotics. By the present, it is not yet clear whether catabolite repression and proteolytic digestion of enzymes produced are suppressed or not in the LSI and L-L IBR systems as shown in the solid-state cultivation. If these suppressive effects are present, the application and efficiency of the systems could drastically spread. We have continued studies for the application of the LSI and L-L IBR systems to the production of biologically active secondary metabolites and enzymes, and the fungal conversion of some synthetic substrates.

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Acknowledgments We express our sincere thanks to Director Nobuo Ichimaru, Matsumoto Yushi-Seiyaku, Co., Ltd., for the kind gift of microspheres. We are also grateful to Miss Aki Kobayashi and Mrs. Akiho Ohmura for their helpful technical assistance. References [1] Znidarsic P, Pavko A. The morphology of filamentous fungi in submerged cultivations as a bioprocess parameter. Food Technol Biotechnol 2001;39: 237–52. [2] Johansen CL, Coolen L, Hunik JH. Influence of morphology on product formation in Aspergillus awamori during submerged fermentations. Biotechnol Prog 1998;14:233–40. [3] Aguilar CN, Augur C, Favela-Torres E, Viniegra-Gonza´lez G. Induction and repression patterns of fungal tannase in solid-state and submerged cultures. Process Biochem 2001;36:565–70. [4] Diza-Godinez G, Soriano-Santos J, Augur C, Viniegra-Gonza´lez G. Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study. J Ind Microbiol Biotechnol 2001;26: 271–5. [5] Xu J, Wang L, Ridgway D, Gu T, Moo-Young M. Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth. Biotechnol Prog 2000;16:222–7. [6] Papagianni M, Nokes SE, Filer K. Submerged and solid-state fermentation by Aspergillus niger: effects of agitation and medium viscosity on phytase production, fungal morphology and inoculum performance. Food Technol Biotechnol 2001;39:319–26. [7] Ho¨lker U, Ho¨fer M, Lenz J. Biotechnological advantages of laboratoryscale solid-state fermentation with fungi. Appl Microbiol Biotechnol 2004;64:175–86. [8] Bapat PM, Kundu S, Wangikar PP. An optimized method for Aspergillus niger spore production on natural carrier substrates. Biotechnol Prog 2003;19:1683–8. [9] Viccini C, Mannich M, Capalbo DMF, Valdebenito-Sanhueza R, Mitchell DA. Spore production in solid-state fermentation of rice by Clonostachys rosea, a biopesticide for gray mold of strawberries. Process Biochem 2007;42:275–8. [10] Rosenblitt A, Agosin E, Delgado J, Pe´rez-Correa R. Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis. Biotechnol Prog 2000;16:152–62. [11] Adinarayana K, Prabhakar T, Srinivasulu V, Rao MA, Lakshmi J, Ellaiah P. Optimization of process parameters for cephalosporin C production under solid state fermentation from Acremonium chrysogenum. Process Biochem 2003;39:171–7. [12] Gelmi C, Pe´rez-Correa R, Agosin MGE. Solid substrate cultivation of Gibberella fujikuroi on an inert support. Process Biochem 2000;35:1227– 33. [13] Viccini G, Mitchell DA, Boit SD, Gern JC, de Rosa AS, Costa RM, Dalsenter FDH, von Meien OF, Krieger N. Analysis of growth kinetic profiles in solid-state fermentation. Food Technol Biotechnol 2001;39:1– 23. [14] Aranda C, Robledo A, Loera O, Contreras-Esquivel JC, Rodrı´guez R, Aguilar N. Fungal invertase expression in solid-state fermentation. Food Technol Biotechnol 2006;44:229–33. [15] Sabu A, Keerthi TR, Kumar SR, Chandrasekaran M. L-Glutaminase production by marine Beauveria sp. under solid state fermentation. Process Biochem 2000;35:705–10. [16] Couto SR, Ro¨tto¨ M, Domı´nguez A, Sanroma´n A. Strategies for improving ligninolytic enzyme activities in semi-solid-state bioreactors. Process Biochem 2001;36:995–9.

[17] Domı´nguez A, Rivela I, Couto SR, Sanroma´n MA. Design of a new rotating drum bioreactor for ligninolytic enzyme production enzyme production by Phanerochaete chrysosporium grown on an inert support. Process Biochem 2001;37:549–54. [18] El-Holi MA, Al-Delaimy KS. Citric acid production from whey with sugar and additives by Aspergillus niger. African J Biotechnol 2003;2:356–9. [19] Zyla K, Gogol D. In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation. J Agric Food Chem 2002;50:899–905. [20] Morita M, Shimamura H, Ishida N, Imamura K, Sakiyama T, Nakanishi K. Characteristics of a-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods. J Biosci Bioeng 2004;98:200–6. [21] Ogawa A, Yasuhara A, Tanaka T, Sakiyama T, Nakanishi K. Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704. J Ferment Bioeng 1995;80:35–40. [22] Ogawa A, Wakisaka Y, Tanaka T, Sakiyama T, Nakanishi K. Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484. J Ferment Bioeng 1995;80:41–5. [23] Oda S, Ohta H. Alleviation of toxicity of poisonous organic compounds on hydrophilic carrier/hydrophobic organic solvent interface. Biosci Biotech Biochem 1992;56:1515–7. [24] Oda S, Sugai T, Ohta H. Interface bioreactor: microbial transformation device on an interface between a hydrophilic carrier and a hydrophobic organic solvent. In: Vulfson EN, Halling PJ, Holland HL, editors. Enzyme in nonaqueous solvents. Totowa: Humana Press; 2001. p. 401–16. [25] Oda S, Ohta H. Microbial transformation device on interface between hydrophilic carriers and hydrophobic organic solvents. Biosci Biotech Biochem 1992;56:2041–5. [26] Oda S, Inada Y, Kobayashi A, Kato A, Matsudomi N, Ohta H. Coupling of metabolism and bioconversion: microbial esterification of citronellol with acetyl coenzyme A produced via metabolism of glucose in an interface bioreactor. Appl Environ Microbiol 1996;62:2216–20. [27] Oda S, Sugai T, Ohta H. Preparation of methyl ursodeoxycholate via microbial reduction of methyl 7-ketolithocholate in an anaerobic interface bioreactor. J Biosci Bioeng 2001;91:202–7. [28] Oda S, Ohta H. Biodesulfurization of dibenzothiophene with Rhodococcus erythropolis ATCC 53968 and its mutant in an interface bioreactor. J Biosci Bioeng 2002;94:474–7. [29] Aksoy S, Tumturk H, Harirci N. Stability of a-amylase immobilized on poly(methylmethacrylate-acrylic acid) microspheres. J Biotechnol 1998;60:37–46. [30] Li S, Hu J, Liu B. Use of chemically modified PMMA microspheres for enzyme immobilization. Biosystems 2004;77:25–32. [31] Van Erp R, Linders YE, van Sommeren AP, Gribnau TC. Characterization of monoclonal antibodies physically adsorbed onto polystyrene latex particles. J Immunol Methods 1992;152:191–9. [32] Sakurai Y, Misawa S, Shiota H. Growth respiratory activity of Aspergillus oryzae grown on solid state medium. Agric Biol Chem 1985;49: 745–50. [33] Majerie´ M, Sunjie´ V. Preparation of S-2-ethylhexyl-para-methoxycinnamate by lipase catalyzed sequential kinetic resolution. Tetrahedron: Asymmetry 1996;7:815–24. [34] Weissfloch ANE, Kazlauskas RJ. Enantiopreference of lipase from Pseudomonas cepacia toward primary alcohols. J Org Chem 1995;60: 6959–69. [35] Williams AC, Woodley JM, Ellis PA, Lilly MD. Denaturation and inhibition studies in a two-liquid phase biocatalytic reaction: the hypothesis of menthyl acetate by pig liver esterase. In: Laane C, Tramper J, Lilly MD, editors. Biocatalysis in organic media. Amsterdam: Elsevier; 1986. p. 399–404. [36] Bar R. Effect of interface mixing on a water-organic solvent two-liquid phase microbial system: ethanol fermentation. J Chem Tech Biotechnol 1988;43:49–62.