Journal of Oleo Science Copyright ©2017 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess16240 J. Oleo Sci. 66, (8) 815-831 (2017)

REVIEW

Production of Valuable Lipophilic Compounds by Using Three Types of Interface Bioprocesses: Solid–Liquid Interface Bioreactor, Liquid–Liquid Interface Bioreactor, and Extractive Liquid-Surface Immobilization System Shinobu Oda1, 2＊ 1 2

Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho, Hakusan, Ishikawa 924-0838, JAPAN Integrated Technology Research Center of Medical Science and Engineering, 3-1 Yatsukaho, Hakusan, Ishikawa 924-0838, JAPAN

Abstract: Bioconversions such as enzymatic and microbial transformations are attractive alternatives to organic synthesis because of practical advantages such as resource conservation, energy efficiency, and environmentally harmonic properties. In addition, the production of secondary metabolites through microbial fermentation is also useful for manufacturing pharmaceuticals, agricultural chemicals, and aroma compounds. For microbial production of useful chemicals, the authors have developed three unique interfacial bioprocesses: a solid–liquid interface bioreactor (S/L-IBR), a liquid–liquid interface bioreactor (L/L-IBR), and an extractive liquid-surface immobilization (Ext-LSI) system. The S/L-IBR comprises a hydrophobic organic solvent (upper phase), a microbial film (middle phase), and a hydrophilic gel such as an agar plate (lower phase); the L/L-IBR and the Ext-LSI consist of a hydrophobic organic solvent (upper phase), a fungal mat with ballooned microspheres (middle phase), and a liquid medium (lower phase). All three systems have unique and practically important characteristics such as utilization of living cells, high concentration of lipophilic substrates/products in an organic phase, no requirement for aeration and agitation, efficient supply of oxygen, easy recovery of product, high regio- and stereoselectivity, and wide versatility. This paper reviews the principle, construction, characteristics, and application of these interfacial systems for producing lipophilic compounds such as useful aroma compounds, citronellol-related compounds, β-caryophyllene oxide, and 6-penty-α-pyrone. Key words: interface bioreactor, liquid-surface immobilization, fungal bioconversion, terpenoid conversion, pyrone fermentation 1 Introduction Biocatalysts such as enzymes, as well as whole cells of microorganisms, are known to catalyze many regio- and stereoselective reactions such as hydrolysis, esterification, and oxidoreduction1, 2）. Microbial transformation is therefore widely applied in various industries, including pharmaceutical, cosmetic, food, and petrochemical industries, as a solution to critical problems such as environmental pollution and resource and energy conservation. However, two grave obstacles, namely, water-insolubility and biocidal ac-

tivity of substrates and/or products, limit the industrial applications of these biocatalysts. To overcome the first obstacle, many approaches such as addition of surfactants3）or water-miscible4）and immiscible organic solvents5）, complexation with cyclodextrins6）, use of fungal dry mycelia7）, and immobilization of whole cells in organic solvents8）have been reported. In addition, solventtolerant bacteria such as Pseudomonas9）, Bacillus10）, Acinetobacter 11）, and Staphylococcus 12）, as well as some yeasts13）have been isolated and characterized. These sol-

＊

Correspondence to: Shinobu Oda, Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho, Hakusan, Ishikawa 924-0838, JAPAN E-mail: [email protected] Accepted April 27, 2017 (received for review December 19, 2016)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online

http://www.jstage.jst.go.jp/browse/jos/  http://mc.manusriptcentral.com/jjocs 815

S. Oda

vent-tolerant microorganisms are expected to be genetically superior sources of biocatalysts for non- or micro-aqueous bioconversion 9）and producers of solvent-stable enzymes10）. However, the efficacy of additives such as surfactants and water-miscible and immiscible solvents is limited by their toxicity towards many microorganisms14）. Addition of high concentrations of these additives and organic solvents can cause serious damages to microbial cells, including decomposition of cell membrane 15）, inhibition of nutrient uptake16, 17）, inhibition of membrane permeability17, 18）, inhibition of oxygen uptake19, 20）, inhibition of metabolism19, 20）, inhibition of DNA synthesis20）, shrinking of cytoplasm21）, inhibition of respiration 22）, denaturation of intercellular enzymes17）, inhibition of cell division23）, acidification of cells23）, and release of intercellular components such as K＋, proteins, and DNA21）. Therefore, the surfactant or organic solvent must be carefully selected to minimize cell damage. Complexation of water-insoluble substrates with cyclodextrins is another method to overcome the challenge of water-insolubility. However, removal of cyclodextrins from a broth is troublesome and costly, because chromatographic procedures are necessary in many cases. Furthermore, the use of free and immobilized cells in an organic phase is strictly restricted to coenzyme-independent bioconversions such as esterification7）and hydrolysis24）. Although some coenzyme-dependent reactions such as reduction25）and biodegradation26）with lyophilized and/or immobilized microbial cells have been reported in an organic phase, the organic solvent promptly inactivated the regeneration of coenzymes in the microbial cells27）. The second challenge is the biocidal activity of substrates and/or products. Organic–aqueous two-liquid phase systems are known to be effective in reducing the toxicity of substrates and/or products5）. Two-liquid phase systems have been used in many microbial transformations such as

dehydrogenation28）, reduction29）, epoxidation30）, and hydroxylation31）. It was also reported that two-liquid phase systems are effective for side-chain cleavage of steroids5） and biodegradation of harmful substances 32）. Organic– aqueous two-liquid phase systems, which are classified into dispersion and dispersion-free systems, are known to be effective for alleviating solvent toxicity. However, they have some disadvantages, including phase toxicity, transfer of nutrients from the aqueous phase into the organic phase, and emulsion formation17）. Depending on the agitation rate, phase toxicity can cause disruption of the cell membrane. Therefore, more practical and efficient microbial transformation devices need to be developed. In this paper, two unique interfacial biotransformation systems, S/L-IBR and L/L-IBR, and an interfacial fermentation system, Ext-LSI system, are introduced and reviewed.

2 Solid–liquid interface bioreactor（S/L-IBR） 2.1 Principle and characteristics of S/L-IBR S/L-IBRs are interfacial microbial transformation systems that allow microbial cells to grow on an interface between a hydrophilic carrier and a hydrophobic organic solvent 33, 34） . Nutrient agar plates are often used as the hy（Fig. 1） drophilic carrier, and middle chain n-alkanes such as ndecane and n-dodecane or middle chain alkyl ethers such as di-n-hexyl ether and isoamyl ether are used as the hydrophobic organic solvent. These organic solvents can dissolve more oxygen than an aqueous phase. Thus, during cultivation with S/L-IBR, the culture is generally allowed to stand. Interestingly, the S/L-IBR can drastically reduce the toxicity of lipophilic compounds such as toluene and cholesterol, as the organic phase acts as a reservoir and a diluent of the toxic organic compounds35）. Thus, the concentration of the substrate and the accumulation of the

Fig. 1　The principle of S/L-IBR. A microbial film formed on an interface between an agar plate and a hydrophobic organic solvent efficiently catalyzes various microbial transformations of lipophilic substrates. The concentration of substrate and the accumulation of product can be drastically enhanced in accordance with the toxicity alleviation effect on the solid–liquid interface. 816

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

product are drastically increased in the S/L-IBR system. The S/L-IBR has been applied in many microbial transformations. Almost all bacteria, actinomycetes, yeasts, and fungi can actively grow on the carrier/solvent interface and efficiently catalyze various microbial reactions such as hydrolysis, esterification（Fig. 2）, oxidation（Fig. 3）, reduc. tion, and biodegradation （Fig. 4） The S/L-IBR has many characteristics of practical importance, such as high concentrations of substrates and products in the organic phase, solubilization of water-insoluble substrates and products, efficient supply of oxygen, easy recovery of products, and wide application（Fig. 1）. The protocol for using S/L-IBR is generally easy, as aeration, agitation, and solvent-extraction are unnecessary. The S/L-IBR also has some practical disadvantages. For example, harmful polar by-products such as organic acids may accumulate in the culture. In addition, it is difficult to control the pH in a carrier, and nutrient supplementation is difficult. These problems stem from the fact that the aqueous phase in a carrier cannot be modified （Fig. 1）.

2.2 Application of S/L-IBR to hydrolysis, esterification, and transacetylation Enzymatic or microbial hydrolysis36）, esterification37）, and transesterification38）have been frequently used for the optical resolution of various enantiomers. The S/L-IBR has been used for the hydrolysis of 2-ethylhexyl acetate into 2-ethyl-1-hexanol by Candida cylindracea ATCC 14830, which was growing on an interface between a synthetic polymer gel （polyvinyl alcohol–glutaraldehyde/alginate–Ca2＋） and neat 2-ethylhexyl acetate. Although the enantioselectivity of the reaction was very low, 280 g/L of 2-ethyl39） . Additionally, 1-hexanol was obtained in 10 days（Fig. 2A） the enantiofacially selective hydrolysis of 2-benzylcyclohexanone enol ester into（R）-2-benzylcyclohexanone by Pichia farinosa IAM 4682 was also achieved with the S/ L-IBR. The enantiomeric excess （ee）and the molar yield of （R）-2-benzylcyclohexanone reached 80％ and 50％, re40） . spectively（Fig. 2B） For the esterification of cholesterol by octanoic acid （Fig. 2C）33）, a unique microbial transformation system for producing acetate esters, the double coupling system, was de-

Fig. 2　Application of S/L-IBR to hydrolysis, esterification and transacetylation.（A）Hydrolysis of 2-ethylhexyl acetate to 2-ethyl-1-hexanol by Candida cylindracea ATCC 14830, （B）enantioface-selective hydrolysis of 2-benzylcyclohexanone enol ester to（R） -2-benzylcyclohexanone by Pichia farinosa IAM 4682,（C） esterification of cholesterol with octanoic acid by lipase-producing bacteria,（D） enantioselective transacetylation of（RS） -citronellol with acetyl-CoA produced via glucose metabolism by Pichia kluyvery IFO 1165. 817

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

Fig. 3　Application of S/L-IBR to oxidation.（A） Regioselective oxidation of methylcyclohexanols to 2-methylcyclohexanone by Rhodococcus equi IFO 3730,（B）oxidation of 1-decanol to decanoic acid by Issatchenkia scutulata var. scutulata IFO 10070,（C）enantioselective oxidation of（RS）-citronellol to（ S）-citronellic acid by Hansenula saturnus IFO 0809,（D）enantioselective oxidation of（RS）-citronellol to（R）-citronellal by Rhodococcus equi IFO 3730. veloped. In this system, yeasts such as Hansenula and Pichia could produce various aliphatic and aromatic acetate esters through transacetylation of the corresponding primary alcohols by acetyl coenzyme A（acetyl-CoA）, which was produced via the metabolism of glucose41, 42）and free fatty acids43, 44）. This reaction is catalyzed by alcohol acetyltransferase（AATFase）, which is known to catalyze the acetylation of isoamyl alcohol by acetyl-CoA in sake production in Saccharomyces45）. The authors have firstly applied the enzyme to organic synthesis41）. The AATFase of Pichia kluyveri IFO 1165 could catalyze the enantioselective acetylation of（RS）-citronellol by acetyl-CoA to produce （S） -citronellyl acetate and （R）-citronellol at an E value of 40（Fig. 2D）46）. Optically active citronellol and its related terpenoids are useful for the syntheses of various insect pheromones47）. However, attempts to perform optical resolution of （RS）-citronellol by either esterification using lipase48）and microbial hydrolysis49）have not been successful. Thus, the double coupling system is very useful for the optical resolution of racemic citronellol, a primary alcohol with a remote chiral center and a reac. Despite the high biotoxicity, tive function （hydroxy group） it was possible to set up the initial concentration of（RS） -citronellol in n-decane as the organic phase to 30％50）.

2.3 Application of S/L-IBR in oxidative reactions While alcohol dehydrogenase（ADH） is useful for producing aldehydes or ketones from alcohols51）, aldehyde dehydrogenase（ALDH）is useful for preparing carboxylic acids from aldehydes51）. The S/L-IBR was used for the ADH-dependent oxidation of methylcyclohexanols by Rhodococcus . 2-Methylcyclohexanone was regiequi IFO 3730（Fig. 3A） oselectively synthesized from 2-methylcyclohexanol with a high accumulation rate（11 g/L）, despite their high biotoxicities33）. The superior ability of S/L-IBR to alleviate toxicity was also observed during the oxidative synthesis of decanoic acid, an antiseptic, from 1-decanol by Issatchenkia scutulata var. scutulata IFO 10070（Fig. 3B）52）. Middle chain are known to have high molecular toxic1-alkanols（C6–C10） ity17）, and decanoic acid is more biotoxic against Gram-positive bacteria53）, yeast53, 54）, and fungi54）. In these reports, the minimal inhibitory concentration（MIC） of decanoic acid was found to be less than 1 mM （0.172 g/L）; however, with S/L-IBR, accumulation of high amounts of decanoic acid （32.5 g/L）was observed52）. While many yeasts such as Hansenula and Candida catalyze（S）-selective oxidation of citronellol to produce （S） -citronellic acid and （R） -citronellol （Fig. 3C） , some acti-

818

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

Fig. 4　Application of S/L-IBR to reduction and degradation.（A）Asymmetric reduction of 6-methyl-5-heptene-2-one to（R） -sulcatol by Picha farinosa IAM 4682,（B）asymmetric reduction of ethyl 2-oxo-4-phenylbutanoate to ethyl （R） -2-hydroxy-4-phenylbutanoate by Candida holmii KPY 12402,（C）asymmetric reduction of methyl 7-ketolithocholate to methyl ursodeoxycholte by Eubacterium aerofaciens JCM 7790,（D）stereoselective degradation of（RS）-ibuprofen to（S）-ibuprofen by Trichosporon cutaneum KPY 30802,（E）biodegradation of dibenzothiophene by Rhorococcus erythropolis ATCC 53968. nomycetes such as Rhodococcus equi catalyze（R）-selec-citronellal and （S） tive oxidation of citronellol to yield（R） 55, 56） . Using Geotrichum candidum as citronellol（Fig. 3D） a biocatalyst, 65 g/L of citronellic acid was produced in 13 days, despite the high biotoxicity of the substrate and product. Thus, by combining enantioselective transacetylation and oxidation by select microorganisms,（R）-citronellol,（R）-citronellal,（R）-citronellic acid,（S）-citronellol, and（S）-citronellic acid were synthesized with high values of ee and yield57）. 2.4 Application of S/L-IBR in reductive reactions The reductase enzyme of microorganisms is a versatile and useful enzyme for preparing chiral alcohols by asymmetric reduction58）. S/L-IBR has been used for reducing ketones, as microbial asymmetric reduction is important

for preparing chiral alcohols in industries. Sugai et al. had applied S/L-IBR in asymmetric reduction of 6-methyl5-heptene-2-one into（R）-sulcatol, a useful chiral synthon, 59） . The ee and conby Pichia farinosa IAM 4682（Fig. 4A） version yield of（R）-sulcatol were 90％ and 51％, respectively. Ethyl（R）-2-hydroxy-4-phenylbutanoate［（R）-EHPB］is an important intermediate in the synthesis of angiotensinconverting enzyme（ACE）inhibitors such as enalapril and Lisinopril 60）. S/L-IBR has been used for producing（ R）EHPB through microbial asymmetric reduction of a prochi（Fig. ral precursor, ethyl 2-oxo-4-phenylbutanoate（EOPB） 4B）61）. An yeast, Candida holmii KPY 12402, could（R） -preferentially reduce EOPB to（R）-EHPB in an n-decane layer. The overall yield, chemical purity, and ee of the（R） -EHPB were 58％, 99％, and 90％, respectively61）. 819

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

Furthermore, methyl ursodeoxyxcholate（Me-UDCA）, which is widely used as a cholesterol gallstone-dissolving agent 62）, was synthesized by asymmetrically reducing methyl 7-ketolithocholate（Me-7KLCA）with an anaerobic bacterium, Eubacterium aerofaciens JCM 7790（ Fig. 4C）63−65）. Anaerobic conditions in the S/L-IBR were regulated using a set of GasPakTM catalyst and an oxygen indicator. The productivity of Me-UDCA was enhanced by a combination of glucose and mannitol as the carbon and hydride sources and by using phosphate buffer as the aqueous phase in the gel63）. 2.5 Application of S/L-IBR in biodegradation S/L-IBR has been applied in the synthesis of （S） -ibuprofen, a pharmaceutically active anti-inflammatory drug66）, via enantioselective biodegradation of racemic ibuprofen 67） . by a yeast, Trichosporon cutaneum KPY 30802 （Fig. 4D） The addition of hydroquinone（10 mM）into a hydrophilic carrier effectively increased the ee and repressed excess degradation of（S） -ibuprofen （E value: 9.3） . S/L-IBR has also been used for the biodesulfurization of dibenzothiophene into 2-hydroxybiphenyl by Rhodococcus 68） . The development of erythropolis ATCC 53968 （Fig. 4E） a practical process for the biodesulfurization of organo-sulfur compounds such as dibenzothiophene in fossil fuels was considered necessary, as the sulfur dioxide formed during the combustion of fossil fuels caused acid rains69）. It was observed that a biofilm of the R. erythropolis ATCC 53968 strain peeled off from the surface of the agar plate to form an organic phase（n-dodecane or n-tetradecane）. To overcome this challenge, screening was performed for a UV mutant that could strongly adhere onto the carrier surface. The selected UV mutant （UM-021） could effectively catalyze the biodesulfurization, and the biofilm did not peel off from the carrier surface. The final removal rate of dibenzothiophene（2 mM）by the mutant was 95％, after still cultivation for 5 days68）. 2.6 Practical disadvantages of S/L-IBR As described in the previous sections, S/L-IBR generates high yield and ee of products, in addition to many other merits, as shown in Fig. 1. However, S/L-IBR also has some fatal disadvantages. These disadvantages result from the fact that the aqueous phase in the hydrophilic carrier of the S/L-IBR cannot be modified. For instance, harmful byproducts such as organic39, 63）and inorganic acids68）could accumulate in the hydrophilic carrier, the carrier pH could decrease, and the nutrients may get depleted46）. Therefore, the development of a new type of interface bioreactor with a modifiable aqueous phase is necessary. Thus, the next generation of interface bioreactors, the L/L-IBR has an aqueous phase in the hydrophilic carrier that can be modified or exchanged.

3 Liquid–liquid interface bioreactor（L/L-IBR） 3.1 Principle and characteristics of liquid-surface immobilization （LSI）systems Ballooned microspheres（MS）, used as fillers in composite materials such as paints and putty, have micro-scale diameters and very low density70）. MS were used as key materials for generating fungal mats on the surface of an . Fungal cells, mycelia, and aqueous phase（liquid medium） spores could float on liquid surfaces with a crowd of MS, as shown in Fig. 5A. Many fungi were found to form thick fungus–MS mats with many aerial hyphae and spores, after only 3 days of stationary cultivation（Figs. 5B, 5C）. This interfacial cultivation system for fungi was called the liquidsurface immobilization（LSI）system（Fig. 6A） . The fungus– MS mat was physically strong enough to maintain its form, 71） . Fungus–MS even if the vessel was overturned （Fig. 5D） mats were also formed on wider liquid surfaces in stainless steel trays（30×30 cm）. Matsumoto Yushi-Seiyaku, Co., Ltd., Osaka produces different types of MS, including MFL-80GCA（CaCO3-coated; mean diameter, 21 μm; density, 0.20）, MFL-80GTA（talccoated; mean diameter, 21 μm; density, 0.20）, and MMF-DE-1（non-coated; mean diameter, 30 μm; density, 0.06）. MS are made of polyacrylonitrile; they are synthesized through polymerization of emulsions or suspensions, followed by thermal expansion 70）. A recently developed porous microparticle, Advancel HB-2051（polymethylmethacrylate; diameter, 20 μm; Sekisui Chemical Co., Ltd., Tokyo）is currently being used in our laboratory. The fungus–MS mat has been used for the production of two useful enzymes, xylanase72）and β-glucosidase73）, by a transformant of Aspergillus oryzae RIB40 （Fig. 6A）. Both the enzymes were produced efficiently, compared to the conventional cultivation system, submerged cultivation （SmC）. Similar to solid-state cultivation, the digestion of the enzymes by inherent proteases was alleviated in the LSI system74）. 3.2 Principle and characteristics of L/L-IBR As mentioned in the previous section, the LSI system is useful for producing enzymes and water-soluble metabolites71−73）. A unique organic–aqueous interfacial cultivation system can be constructed by adding a harmless hydrophobic organic solvent onto a fungus–MS mat. In this system, the aqueous phase under the fungus–MS mat can be easily controlled or exchanged, unlike in the S/L-IBR. However, the organic phase above the fungus–MS mat acts as a reaction solvent, similar to the S/L-IBR. This interfacial microbial transformation system was called the liquid–liquid interface bioreactor（L/L-IBR; Fig. 6B）71）, and has been used in many types of fungal bioconversions. The L/L-IBR has shown excellent product accumulation and regio- and stereoselectivity in many cases; the protocol for L/L-IBR, similar to that for S/L-IBR, is easy, as aeration,

820

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

Fig. 5　F ormation of a fungal mat together with ballooned microspheres. Numerous number of polyacrylonitrile microspheres（MS; diameter, 20–100 μm; density, 0.03–0.20）suspended in an aqueous phase rapidly come to the liquid-surface together with fungal cells（A） . The fungal cells immobilized into the MS layer grow and differentiate to vegetative and aerial hyphae, sporangia, and spores during appropriate cultivation period to form a physically strong fungal cells–MS mat on the liquid-surface（B, C） . The fungus–MS mat did not collapse even by rolling of the vessel （D）.

Fig. 6　A genealogy of three types of interfacial cultivation/application systems using ballooned microsphere（MS） . In the LSI system （A）, fungal cells produce hydrophilic metabolites and/or enzymes into an aqueous phase. The L/L-IBR system （B）affords hydrophobic products into an organic phase via interfacial biotransformation of lipophilic substrate dissolved in the organic solvent. In the Ext-LSI system（C）, hydrophobic metabolites are produced via interfacial fermentation to accumulate into an organic phase. 821

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

agitation, and solvent extraction are not required（Fig. 7）. The concentration of the product and the residual substrate in the organic phase can be directly assessed using HPLC and GLC, without extraction and condensation. 3.3 Application of L/L-IBR in hydrolytic reactions L/L-IBR was first used for the hydrolysis of 2-ethylhexyl acetate into 2-ethyl-1-hexanol by Absidia coerulea NBRC 442371）. The type culture fungus was screened from approximately 400 strains using the S/L-IBR, to yield 51.1 g/L of 2-ethyl-1-hexanol from a 50％（w/v）solution of 2-ethylhexyl acetate in n-decane in 5 days. The hydrolytic reaction was performed using submerged cultivation（SmC）, organic–aqueous two-liquid phase system（TLP）, S/L-IBR, . As shown in and L/L-IBR （MS, CaCO3-coated MFL-80GCA） Fig. 8, the L/L-IBR showed the most efficient performance among the four studied systems; it yielded 107 g/L of 2-ethyl-1-hexanol after 8 days of cultivation71）. Later, the performance of L/L-IBR was attributed to the activating lipase of A. coerulea, which used Ca2＋ from the CaCO3coated MFL-80GCA MS75）, similar to the other lipases76）. 3.4 Application of L/L-IBR in reductive reactions Next, L/L-IBR was used in the asymmetric reduction of benzil to （S） -benzoin by Penicillium claviforme IAM 7294 （Fig. 9）77）. Although（S）-benzoin, a useful chiral auxiliary, has been produced through asymmetric reduction by fungi and bacteria, the low solubility of benzil and the toxicity of benzil and benzoin have limited the practical applications78）. Therefore, we tried to achieve a high concentration and ee of（S）-benzoin using L/L-IBR. Among the 25 fungal strains tested with S/L-IBR, Penicillium claviforme IAM

Fig. 8　C omparison of 2-ethylhexyl acetate-hydrolytic activities among submerged, organic-aqueous twoliquid phase, S/L-IBR and L/L-IBR systems. 7294 was the most effective; it efficiently reduced 3％ benzil to yield 12.9 g/L （S） -benzoin （95.2％ ee） in di-n-hexyl ether in 3 days77）. Reduction was also compared among four cultivation systems, SmC, TLP, S/L-IBR, and L/L-IBR（MS, MFL80GCA）systems. As shown in Fig. 9, the L/L-IBR showed the highest accumulation and ee of（S） -benzoin, and many needle-like crystals of excess（S）-benzoin were found on the fungus-MS mat. Thus, the efficacy of the L/L-IBR for asymmetric microbial reduction was also confirmed.

Fig. 7　General procedure of the experiment with L/L-IBR and Ext-LSI systems. 822

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

Fig. 9　Comparison of benzil-reduction activities among submerged, organic–aqueous two-liquid phase, S/ L-IBR and L/L-IBR systems. 3.5 Application of L/L-IBR in hydroxylation reactions Microbial hydroxylation of the methylene carbon atom by the cytochrome P450 system is known to preferentially occur at activated positions, generally benzyl and aryl sites79）. Hydroxylation of non-activated carbons by chemical or enzymatic methods is known to be difficult. However, n-alkanes with non-activated methyl and methylene groups are known to be decomposed by terminal and/or subterminal hydroxylation by many microorganisms80）. In addition, many bacteria such as Pseudomonas putida81）, Brevibacterium erythrogenes82）, and Corynebacterium sp.83）are known to catalyze the terminal hydroxylation of n-alkanes. In contrast, Arthrobacter sp. catalyzes the subterminal hydroxylation of n-hexadecane to yield 2-, 3-, and 4-hexadecanols84）. Furthermore, BM-3, an engineered cytochrome P450, has been reported to catalyze both terminal and subterminal hydroxylation of n-alkanes85）. Regarding the assimilation of n-alkanes by yeasts, different strains have been reported to catalyze monoterminal, diterminal, and/or subterminal hydroxylation of n-alkanes. For example, while Candida parasilosis86）, C. intermedia87）, and Lodderomyces elongisporus88）hydroxylate a terminal methyl group of n-alkanes, Candida lipolytica hydroxylates the terminal methyl and C-2 methylene groups89）. Regarding fungal hydroxylation of n-alkanes, Cladosporium resinae hydroxylates a terminal methyl group90）, while Cunninghamella blakesleeana hydroxylates both terminal methyl and subterminal methylene groups of n-alkanes 91）. There have been no reports of regio- and stereoselective subterminal hydroxylation of nalkanes. The authors have isolated a fungal strain, Monilliera sp. NAP 00702, which catalyzes the regio- and stereoselective subterminal hydroxylation of n-decane92）. This strain could

accumulate（–）-4-decanol into an n-decane layer with 99％ regioselectivity and almost 100％ stereoselectivity（Fig. 10）. The n-decane layer acted as both substrate and reac-4tion solvent, and prevented the decomposition of the（–） decanol produced（Fig. 10）. It was assumed that excess oxidation of（–）-4-decanol into 4-decanone and the subsequent Baeyer–Villiger oxidation 93）were effectively repressed in the L/L-IBR. Further investigations were carried out for the regioand stereoselective subterminal hydroxylation of n-decane by Monilliera sp. NAP 00702, in the L/L-IBR. It was found the hydroxylation activity of the strain was significantly affected by hydrophobicity and charge on the surface of the synthetic polymer microparticles mixed into the polyacrylonitrile MS layer94, 95）. Many reports have been published on the relationship between surface or interface hydrophobicity and microbial cells. For example, the adsorption of Escherichia coli cells onto droplets of n-alkanes such as n-decane and n-hexadecane was reported to cause an increase in respiration and a decrease in glucose uptake96）. The same phenomenon was also confirmed in cells that adhered onto the surface of hydrophobic synthetic polymers such as styrene–divinylbenzene copolymer and polytetrafluoroethylene97）. It was reported that the growth rate of Staphylococcus epidermidis and Pseudomonas aeruginosa 98）, the cell surface hydrophobicity of Fusarium solani 99）, and the morphology of Cylindrocarpon and Acremonium cells100） were significantly affected by the adhesion onto hydrophobic solid surfaces. In addition to the phenomena described previously, researchers have found that the subterminal hydroxylation activity of Monilliera sp. NAP 00702 is significantly affected by contact with hydrophobic polymer microparticles mixed into an MS layer in the L/L-IBR system94）. Higher the hydrophobicity of the polymer particles mixed, higher the （–）-4-decanol concentration observed. The coefficient of determination（R2）between the polymer hydrophobicity and the（–）-4-decanol accumulation was 0.747. Thus, the contact between fungal cells and the hydrophobic solid surface is moderately effective in improving the microbial hydroxylation activity94）. It has also been reported that the charge（cationic or anionic）on the solid-surface significantly affected the microbial cells. For instance, the adhesion of microbial cells onto an anionic resin significantly affected their growth rate101）, the optimum pH for the growth of Escherichia coli102）, the spore formation of Bacillus subtilis103）, and the oxidative activity of Nocardia coralline104）. Apart from these observations, researchers have observed a unique phenomenon: the adhesion of anionic resin microparticles onto an MS layer significantly enhances the hydroxylation activity of Monilliera sp. NAP 00702 in the L/L-IBR system95）. While the addition of cation-exchange 823

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

Fig. 10　Regio- and stereoselective subterminal hydroxylation of n-decane with L/L-IBR system.（A）Comparison of subterminal hydroxylation activities among SmC, TLP and S/L-IBR systems.（B）Production of high concentrations of （–）-4-decanol by a mutant prepared by UV-irradiation under the optimum conditions. and the chelating resin microparticles onto an MS layer caused an inhibition of fungal growth and hydroxylation activity, the addition of anion-exchange resin microparticles with moderate total capacity （≤ 1.00 meq/g） promoted the production of（–） -4-decanol in the L/L-IBR system. The positive effect of the anion-exchange resin microparticles was also observed during the fermentative production of a fungicidal secondary metabolite, 6-pentyl-α-pyrone（6PP）, by Trichoderma atroviride, using a novel interfacial fermentation system called the extractive liquid-surface imsystem, described in detail later（Fig. mobilization（Ext-LSI） 6C）105, 106）. In this case, spore formation by T. atroviride was repressed by the addition of anion-exchange resins.

β-Caryophyllene oxides belong to a group of sesquiterpene oxides that have valuable biological activities, including antifungal112）, antibacterial113）, anti-inflammatory114）, insecticidal115）, and cytotoxic activities116）. From approximately 400 strains tested, a basidiomycete, Nemania aenea SF 10099-1, was selected as the best strain for producing（–）-β-caryophyllene oxide. After optimizing the culture conditions, 35.4 g/L of the epoxide was obtained in an organic phase（dimethyl silicon oil; KF-96L1CS）, despite the biotoxicity of the epoxide（MIC, 66–200 mg/L）112, 113）. Despite the high biotoxicity of the sesquiterpene, the optimal initial substrate concentration was 30％ （Fig. 11） . （w/v）

3.6 Application of L/L-IBR in epoxidation In general, enzymatic epoxidation selectively yields optically active regioisomers107）. Epoxy groups in many terpenes are known to enhance various biological activities, including antibacterial108）, antifungal109）, and cytotoxic activities110）. L/L-IBR has been used for producing optically active β-caryophyllene oxides through regio- and stereoselective epoxidation of β-caryophyllene（ Fig. 11）111）.

3.7 Construction of multistory L/L-IBR system A unique interface bioreactor, a multistory L/L-IBR, was constructed and used for producing（–）-β-caryophyllene oxide. In this system, five L/L-IBR units were stacked and connected by overflow and circulation lines. A 10％ solution of β-caryophyllene in low-density dimethylsilicone oil （KF-96L-1CS）naturally overflowed from the upper unit into the lower ones; it was then directed from a reservoir

824

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

Fig. 11　Regio- and stereoselective epoxidation of high concentrations of β-caryophyllene with L/L-IBR system. at the bottom to a head unit using a circulation pump, at a circulation rate of 5 ml/min（Fig. 12）. The multistory L/ L-IBR system was functioned stably for 14 days, and the yield of（–）-β-caryophyllene oxide in the organic phase reached 14 g/L（Fig. 12）111）. The multistory interface bioreactor might be practically useful for large-scale interfacial bioconversions.

4 ‌Extractive liquid-surface immobilization（Ext-LSI） system A unique interfacial fermentation system, called the Ext-LSI system, is stably constructed by adding a hydrophobic organic solvent onto a fungal cell-MS layer in the LSI system（Fig. 6C）. In this system, lipophilic secondary metabolites are spontaneously extracted from fungal cells into an organic phase, thus inhibiting feedback and/or endproduct inhibition by the metabolites. The Ext-LSI has been used for producing a fungicidal coconut-like aroma 105, 106） . compound, 6-pentyl-α-pyrone（6PP） Fungicidal secondary metabolites have been produced by some Trichoderma spp. in various fermentation systems, including submerged 117）, aqueous two-liquid phase118）, organic–aqueous two-liquid phase119）, liquid-surface120）, and solid-state cultivation systems121）. However, only low levels of 6PP were accumulated, because of its strong anti-fungal activity120, 122）. Indeed, the maximum reported levels of accumulation of 6PP in submerged, liquidsurface, and solid-state fermentations were 474 mg/L122）, 455 mg/L120）, and 3 g/kg123）, respectively. In contrast, the accumulation of 6PP in the Ext-LSI system reached 7.1 g/L in a dimethylsilicon oil layer, despite its strong fungicidal 124） . activity（MICs against some fungi, 0.1–0.5 g/L） （Fig. 13）

5 Future prospects Bioprocess such as enzymatic and microbial transformations are expected to replace organic synthesis because of their advantages such as resource conservation, energy efficiency, and environmentally harmonic properties. However, the substrate concentration, product accumulation, reaction rate, biocatalyst stability, and productivity in bioprocesses are generally inferior to those in organic syn-

Fig. 12　S ynthesis of（–）-β-caryophyllene oxide in a multistory L/L-IBR system.（A）Reactor unit,（B）overflow line, circulation pump,（E） circulation line. （C）reservoir,（D） 825

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

Fig. 13　Production of 6-pentyl-α-pyrone in an Ext-LSI system. theses. The insolubility of synthetic substrates in the aqueous phase also limits the practical use of bioprocesses. Despite these shortcomings of traditional bioprocesses, the S/L-IBR and the L/L-IBR have some practically important advantages, compared to SmC and TLP. For example, high concentrations of substrates and products are found in the organic phase, aeration and agitation are not required in the reactor, oxygen supply is efficient, product recovery is easy, and the microbial reactions have high regio- and stereoselectivity as well as wide versatility. Recently, it was found that it might be possible to use fungal spores as“tough”biocatalysts in moderately polar organic solvents125）. This possibility is considered very important to the pharmaceutical industry, because the intermediates of many drugs are insoluble in both water and hydrophobic organic solvents. The unique non-aqueous microbial transformation systems, S/L-IBR, L/L-IBR, ExtLSI, and fungal spore biotransformation systems in organic solvents, might be practically useful for the production of industrially useful chemicals.

REFERENCES 1）Fernandez, P.; Cruz, A.; Angelova, B.; Pinheiro, H.M.; Cabral, J.M.S. Microbial conversion of steroid compounds: recent developments. Enzyme Microb. （2003） . Technol. 32, 688-705 2）Kuriata-Adamusiak, R.; Sturb, D.; Lochnski, S. Application of microorganisms towards synthesis of chiral terpenoid derivatives. Appl. Microbiol. Biotechnol. 95, 1427-1436 （2012） . 3）Nakahara, T.; Kawashima, H.; Sugisawa, T.; Takamori, Y.; Tabushi, T. Induction and characterization of mutants enhanced in assimilability of n-alkanes in shake cultures from a strain of Candida sp. J. Ferment.

Technol. 61, 19-23 （1983）. 4）Faramarzi, M.A.; Yazdi, M.T.; Jahandar, H.; Amini, M.; Monsef-Esfahani, H.R. Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum. J. Ind. Microbiol. Biotechnol. 33, 725-733（2006）. 5）Cruz, A.; Fernandes, P.; Cabral, J.M.S.; Pinheiro, H.M. Whole-cell bioconversion of β-sitosterol in aqueous– organic two-phase systems. J. Mol. Catal. B: Enzym. 11, 579-585（2001）. 6）Li, G.; Li, F.; Deng, L.; Fang, X.; Zou, H.; Xu, K.; Li, T.; Tan, G. Increased yield of biotransformation of exemestane with β-cyclodextrin complexation technique. Steroids 78, 1148-1151（2013）. 7）Converti, A.; Borghi, A.D.; Gandolfi, R.; Molinari, F.; Palazzi, E.; Zilli, M. Simplified kinetics and thermodynamics of geraniol acetylation by lyophilized cells of Aspergillus oryzae. Enzyme Microb. Technol. 30, 216-223（2002）. 8）Griffin, D.R.; Gainer, J.L.; Carta, G. Asymmetric ketone reduction with immobilized yeast in hexane: biocatalyst deactivation and regeneration. Biotechnol. Prog. 17, 304-310（2001）. 9）Inoue, A.; Yamamoto, N.; Horikoshi, K. Pseudomonas putida which can grow in the presence of toluene. Appl. Environ. Microbiol. 57, 1560-1562（1991）. 10）Rachadech, W.; Navacharoen, A.; Ruangsit, W.; Pongtharangkul, T.; Vangnai, A.S. An organic solvent-, detergent- and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5. Microbiology 79, 620-629 （2010）. 11）Watanabe, H.; Tanji, Y.; Unno, H.; Hori, K. Rapid conversion of toluene by an Acinetobacter sp. Tol 5 mutant showing monolayer adsorption to water–oil interface. J. Biosci. Bioeng. 106, 226-230（2008）.

826

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

12）Nielsen, L.E.; Kadavy, D.R.; Rajagopal, S.; Drijber, R.; Nickerson, K.W. Survey of extreme solvent tolerance in Gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene. Appl. Environ. Microbiol. 71, 5171-5176 （2005） . 13）Matsui, K.: Teranishi, S.; Kamon, S.; Kuroda, K.; Ueda, M. Discovery of a modified transcription factor endowing yeasts with organic-solvent tolerance and reconstruction of an organic-solvent-tolerant Saccharomyces cerevisiae strain. Appl. Environ. Mi. crobiol. 74, 4222-4225 （2008） 14）Pinheiro, H.M.; Cabral, J.M.S. Effects of solvent molecular toxicity and microenvironment composition on the Δ1 dehydrogenation activity of Arthrobacter simplex cells. Biotechnol. Bioeng. 37, 97-102 （1991） . 15）Aono, R.; Kobayashi, H.; Joblin, K. N.; Horikoshi, K. Effect of organic solvents on growth of Escherichia coli K-12. Biosci. Biotechnol. Biochem. 58, 20092014 （1994） . 16）Teh, J.S.; Lee, K.H. Effects of n-alkanes on Cladosporium resinae. Can. J. Microbiol. 20, 971-976 . （1974） 17）Bar, R. Phase toxicity in a water-solvent two-liquid phase microbial system. in Biocatalysis in Organic Media （Laane, C.; Tramper, J.; Lilly, M. D. ed.） , Else. vier, Amsterdam, pp. 147-153 （1986） 18）Silver, S.; Wendt, L. Mechanism of action of phenethyl alcohol: breakdown of the cellular permeability （1967） . barrier. J. Bacteriol. 93, 560-566 19）Galbraith, H.; Miller, T.B. Effect of long chain fatty acids on bacterial respiration and amino acid uptake. J. Appl. Bacteriol. 36, 659-675 （1973） . 20）Yamashiro, K.; Nishihara, H.; Tani, Y.; Fukui, S. Studies on the metabolism of Saccharomyces sake.（IV） Influence of various amino acids upon the inhibitory action of α-phenethyl alcohol; especially protecting effect of L-alanine. J. Ferment. Technol. 48, 98-102 （1971） .（in Japanese） 21）Jackson, R.W.; DeMoss, J.A. Effect of toluene on Escherichia coli. J. Bacteriol. 90, 1420-1425 （1965） . 22）Galbraith, J.; Miller, T.B. Physicochemical effects of long chain fatty acids on bacterial cells and their protoplasts. J. Appl. Bacteriol. 36, 647-658 （1973）. 23）Borst-Pauwels, G.M.F.H.; Dobbelmann, J. The mechanism of inhibition of anaerobic phosphate uptake by fatty acids in yeast. Biochim. Biophys. Acta 290, 348-354 （1972） . 24）Zheng, J. -Y.; Wu, J. -Y.; Zhang, Y. -J.; Wang, Z. Resolution of（R,S）-ethyl-2-（4-hydroxyphenoxy）propionate using lyophilized mycelium of Aspergillus oryzae WZ007. J. Mol. Catal. B: Enzym. 97, 62-66 . （2013） 25）Fatima, Y.; Kansal, H.; Soni, P.; Banerjee, U.C. Enanti-

oselective reduction of aryl ketones using immobilized cells of Candida viswanathii. Process Biochem. 42, 1412-1418（2007）. 26）Dias, A.C.P.; Cabral, J.M.S.; Pinheiro, H.M. Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immiscible organic solvents. Enzyme Microb. Technol. 16, 708-714（1994）. 27）Ceen, E.G.; Herrmann, J.P.R.; Dunnill, P. Solvent damage during immobilized cell catalysis and its avoidance: studies of 11α-hydroxylation of progesterone by Aspergillus ochraceus. Appl. Microbiol. Biotechnol. 25, 491-494（1987）. 28）Fernandes, P.; Cabral, J.M.S.; Pinheiro, H.M. Bioconversion of a hydrocortisone derivative in an organic– aqueous two-liquid-phase system. Enzyme Microb. Technol. 17, 163-167（1995）. 29）Fragnelli, M.C.; Hoyos, P.; Romano, D.; Gandolfi, R.: Alcántara, A.R.; Molinari, F. Enantioselective reduction and deracemisation using the non-conventional yeast Pichia glucozyme in water/organic solvent biphasic systems: preparation of（S）-1,2-diaryl-2-hydroxy-ethanones（benzoins）. Tetrahedron 68, 523528（2012）. 30）Déziel, E.; Comeau, Y.; Villemur, R. Two-liquid-phase bioreactors for enhanced degradation of hydrophobic/toxic compounds. Biodegradation 10, 219-233 （1999）. 31）Angelova, B.; Fernandes, P.; Cruz, A.; Pinheiro, H.M.; Hutafov, S.; Cabral, J.M.S. Hydroxylation of androstenedione by resting Rhodococcus sp. cells in organic media. Enzyme Microb. Technol. 37, 718-722 （2005）. 32）Guieysse, B.; Girne, M.D.T.G.; Mattiasson, B. Microbial degradation of phenanthrene and pyrene in a twoliquid phase-partitioning bioreactor. Appl. Microbiol. Biotechnol. 56, 796-803（2001）. 33）Oda, S.; Ohta, H. Microbial transformation on interface between hydrophilic carriers and hydrophobic organic solvents. Biosci. Biotechnol. Biochem. 56, 2041-2045（1992）. 34）Oda, S.; Sugai, T.; Ohta, H. Interface bioreactor: microbial transformation device on an interface between a hydrophilic carrier and a hydrophobic organic solvent. in Enzymes in Nonaqueous Solvents （Vulfson, E.N.; Halling, P.J.; Holland, H.L. eds.）Humana Press, Totowa, pp. 401-416（2001）. 35）Oda, S.; Ohta, H. Alleviation of toxicity of poisonous organic compounds on hydrophilic carrier/hydrophobic organic solvent interface. Biosci. Biotechnol. Biochem. 56, 1515-1517（1992）. 36）Sugai, T.; Yamazaki, T.; Yokoyama, M; Ohta, H. Biocatalysis in organic synthesis: the use of nitrile- and amide-hydrolyzing microorganisms. Biosci. Biotechnol. Biochem. 61, 1410-1427（1997）. 827

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

37）Wang, D.-L.; Nag, A.; Lee, G.-C.; Shaw, J.-F. Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent. J. Agric. Food Chem. 50, 262-265 （2002） . 38）Miyazawa, T.; Kurita, S.; Sakamoto, H.; Otomatsu, T.; Hirose, K.; Yamada, T. Resolution of 2,2-diphenyl-1,3dioxolane-4-methanol via Rhizopus sp. lipase-catalyzed enantioselective transesterification with vinyl （1999） . butanoate. Biotechnol. Lett. 21, 447-450 39）Oda, S.; Tanaka, J.; Ohta, H. Interface bioreactor packed with synthetic polymer pad: application to hydrolysis of neat 2-ethylhexyl acetate. J. Ferment. Bioeng. 86, 84-89 （1998） . 40）Katoh, O.; Sugai, T.; Ohta, H. Application of microbial enantiofacially selective hydrolysis in natural product synthesis. Tetrahedron: Asymm. 5, 1935-1944 （1994） . 41）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 in an interface bioreactor. Appl. Environ. Micribiol. 62, . 2216-2220 （1996） 42）Oda, S.; Ohta, H. Double coupling of acetyl coenzyme A production and microbial esterification with alcohol acetyltransferase in an interface bioreactor. J. （1997） . Ferment. Bioeng. 83, 423-428 43）Oda, S.; Ohta, H. A new double coupling system: synthesis of citronellyl acetate via transacetylation to citronellol from acetyl coenzyme A produced from glucose and free fatty acids. Biosci. Biotechnol. Biochem. 65, 1917-1919 （2001） . 44）Oda, S.; Sugai, T.; Ohta, H. Synthesis of citronellyl acetate via transacetylation to citronellol from acetyl coenzyme A produced from glucose and acetate in growing yeasts. Chem. Lett. 2001, 500-501 （2001）. 45）Yoshioka, K.; Hashimoto, N. Ester formation by alcohol acetyltransferase from brewer’ s yeast. Agric. Biol. Chem. 45, 2183-2190 （1981） . 46）Oda, S.; Sugai, T.; Ohta, H. Optical resolution of racemic citronellol via a double coupling system in an interface bioreactor. J. Biosci. Bioeng. 87, 473-480 . （1999） 47）Mori, K.; Wu, J. Synthesis of the four stereoisomers of 6,10,13-trimethyl-1-tetradecanol, the aggregation pheromone of predatory stink bug, Sriretrus anchorago. Liebigs Ann. Chem. 1991, 783-788 （1991）. 48）Parmar, V.S.; Prasad, A.K.; Singh, P.K.; Gupta, S. Lipase-catalyzed transesterification using 2,2,2-trifluoroethylbutyrate: effect of temperature on rate of reaction and enantioselectivity. Tetrahedron: Asymm. （1992） . 3, 1395-1398 49）Yamaguchi, Y.; Oritani, T.; Tajima, A.; Komatsu, A.; Moroe, T. Screening of dl-menthyl ester hydrolyzing

microorganisms and species specificity of microbial esterases. Nippon Nogeikagaku Kaishi 50, 475-480 （1976） .（in Japanese） 50）Schindler, J. Terpenoids by microbial fermentation. Ind. Eng. Chem. Prod. Res. Dev. 21, 537-539 （1982） . 51）Oda, S.; Kikuchi, Y.; Nanishi, Y. Synthesis of optically active mandelic acid via microbial oxidation of racemic 1-phenyl-1,2-ethanediol. Biosci. Biotechnol. Biochem. 56, 1216-1220（1992）. 52）Oda, S.; Kato, A.; Matsudomi, N.; Ohta, H. Production of aliphatic carboxylic acid via microbial oxidation of 1-alkanols with interface bioreactor. J. Ferment. Bioeng. 78, 149-154（1994）. 53）Freese, E.; Sheu, C.W.; Galliers, E. Function of lipophilic acids as antimicrobial food additives. Nature 241, 321-325（1973）. 54）Kato, N.; Shibasaki, I. Comparison of antimicrobial activities of fatty acids and their esters. J. Ferment. Technol. 53, 793-801（1975）.（in Japanese） 55）Oda, S.; Inada, Y.; Kato, A.; Matsudomi, N.; Ohta, H. Production of（S）-citronellic acid and（R）-citronellol with an interface bioreactor. J. Ferment. Bioeng. 80, 559-564（1995）. 56）Oda, S.; Kato, A.; Matsudomi, N.; Ohta, H. Enantioselective oxidation of racemic citronellol with an interface bioreactor. Biosci. Biotechnol. Biochem. 60, 83-87（1996）. 57）Oda, S.; Sugai, T.; Ohta, H. Syntheses of optically active citronellol, citronellal, and citronellic acid by microbial oxidation and double coupling system in an interface bioreactor. Bull. Chem. Soc. Jpn. 73, 28192823（2000）. 58）Goldberg, K.; Schroer, K.; Lütz, S.; Liese, A. Biocatalytic ketone reduction̶a powerful tool for the production of chiral alcohols̶part II: whole-cell reductions. Appl. Microbiol. Biotechnol. 76, 249-255 （2007）. 59）Sugai, T.; Katoh, O.; Ohta, H. Chemo-enzymatic synthesis of（R,R） （–） -pyrenophorin. Tetrahedron 51, 11987-11998（1995）. 60）Ondetti, M.A.; Cushman, D.W. Inhibition of the reninangiotensin system. A new approach to the therapy of hypertension. J. Med. Chem. 24, 355-361（1981）. 61）Oda, S.; Inada. Y.;Kobayashi, A.; Ohta, H. Production of ethyl（R） -2-hydroxy-4-phenylbutanoate via reduction of ethyl 2-oxo-4-phenylbutanoate in an interface bioreactor. Biosci. Biotechnol. Biochem. 62, 17621767（1998）. 62）Nakayama, F.; Facs, M.M. Oral cholelitholysis̶chemo versus urso: Japanese experience. Dig. Dis. Sci. 25, 129-134（1980）. 63）Oda, S.; Sugai, T.; Ohta, H. Synthesis of methyl ursodeoxycholate via microbial reduction of methyl 7-ke-

828

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

tolithocholate with Eubacterium aerofacience JCM 7790 grown on two kinds of carbon and hydride sources, glucose and mannitol. J. Biosci. Bioeng. 91, 178-183 （2001） . 64）Oda, S.; Sugai, T.; Ohta, H. Preparation of methyl ursodeoxycholate via microbial reduction of methyl 7-ketolithocholate in an anaerobic interface bioreac. tor. J. Biosci. Bioeng. 91, 202-207 （2001） 65）Oda, S.; Sugai, T.; Ohta, H. Preparation of methyl ursodeoxycholate in an interface bioreactor: use of a mixed solvent system to increase the solubilities of substrate and product. J. Biosci. Bioeng. 92, 86-88 （2001） . 66）Evans, A.M. Comparative pharmacology of S（＋）ibuprofen and（ RS）-ibuprofen. Clin. Rheumatol. 2001, S9-S14 （2001） . 67）Tanaka, J.; Oda, S.; Ohta, H. Synthesis of （S）-ibuprofen via enantioselective degradation of racemic ibuprofen with an isolated yeast, Trichosporon cutaneum KPY 30802, in an interface bioreactor. J. Biosci. （2001） . Bioeng. 91, 314-315 68）Oda, S.; Ohta, H. Biodesulfurization of dibenzothiophene with Rhodococcus erythropolis ATCC 53968 and its mutant in an interface bioreactor. J. Biosci. Bioeng. 94, 474-477 （2002） . 69）Oshiro, T.; Hine, Y.; Izumi, Y. Enzymatic desulfurization of dibenzothiophene by cell-free system of Rhodococcus erythropolis D-1. FEMS Microbiol. Lett. （1994） . 118, 341-344 70）Jonsson, M.; Nordin, O.; Kron, A.L.; Malmström, E. Thermally expandable microspheres with excellent expansion characteristics at high temperature. J. Appl. Polym. Sci. 117, 384-392 （2010） . 71）Oda, S.; Isshiki, K. Liquid-surface immobilization system and liquid–liquid interface bioreactor: application to fungal hydrolysis. Process Biochem. 42, 1553. 1560 （2007） 72）Ozeki, K.; Takagi, Y.; Oda, S; Ohashi, S. Production of xylanase with a transformant of Aspergillus oryzae RIB40 in a liquid-surface immobilization （LSI）system. J. Biosci. Bioeng. 109, 224-226 （2010） . 73）Ozeki, K.; Kondo, K.; Nakagawa, H.; Ozeki, K; Oda, S.; Ohashi, S. Production of β-glucosidase by a transformant of Aspergillus oryzae RIB40 in a liquid-surface immobilization（LSI）system. J. Biol. Macromol. 11, 23-30 （2011） . 74）Asther, M.; Haon, M.; Roussos, S.; Record, E.; Delattre, M.; Lesage-Meessen, L.; Labat, M.; Asther, M. Feruloyl esterase from Aspergillus niger: a comparison of the production in solid state and submerged （2002）. fermentation. Process Biochem. 38, 685-691 75）Oda, S.; Wakui, H.; Ohashi, S. Efficient hydrolytic reaction of an acetate ester with fungal lipase in a liquid–liquid interface bioreactor. J. Biosci. Bioeng.

112, 151-153（2011）. 76）Khattabi, M.E.; van Gelder, P.; Bitter, W.; Tommassen, J. Role of the calcium ion and the disulfide bond in the Burkholderia glumae lipase. J. Mol. Catal. B: Enzym. 22, 329-338 （2003）. 77）Oda, S.; Isshiki, K. Asymmetric reduction of benzil to （S） -benzoin with Penicillium claviforme IAM 7294 in a liquid–liquid interface bioreactor（ L–L IBR）. Biosci. Biotechnol. Biochem. 72, 1364-1367（2008）. 78）Demir, A.S.; Hamamci, H.; Ayhan, H.; Duygu, A.N.; Igdir, A.C.; Capanoglu, D. Fungi mediated conversion of benzil to benzoin and hydrobenzoin. Tetrahedron: Asymm. 15, 2579-2582 （2004）. 79）Herath, W.; Ferreira, D.; Mikell, J.R.; Khan, I.A. Microbial metabolism. Part 5. Dihydrokawain. Chem. Pharm. Bull. 52, 1372-1374（2004）. 80）van Beilen, I.B.; Li, Z.; Duetz, W.A., Smits, T.H.M.; Witholt, B. Diversity of alkane hydroxylase systems in the environment. Oil & Gas Sci. Technol. 58, 427440（2003）. 81）Grund, A.; Shapiro, J.; Fennewald, M.; Leahy, J.; Markbreiter, K.; Nieder, M.; Toepfer, M. Regulation of alkane oxidation in Pseudomonas putida. J. Bacteriol. 123, 546-556（1975）. 82）Pirnik, M.P.; Atlas, R.M.; Bartha, R. Hydrocarbon metabolism by Brevibacterium erythrogenes: normal and branched alkanes. J. Bacteriol. 119, 868-878 （1974）. 83）Bacchin, P.; Robertiello, A.; Viglia, A. Identification of n-decane oxidation products in Corynebacterium cultures by combined gas chromatography-mass spectrometry. Appl. Microbiol. 28, 737-741（1974）. 84）Klein, D.A.; Henning, F.A. Role of alcoholic intermediates in formation of isomeric ketones from n-hexadecane by a soil Arthrobacter. Appl. Microbiol. 17, 676-681（1969）. 85）Peters, M.W.; Meinhold, P.; Glieder, A.; Arnold, F.H. Regio- and enantioselective alkane hydroxylation with engineered cytochrome P450 BM-3. J. Am. Chem. Soc. 125, 13442-13450（2003）. 86）El-Assar, S.A.; Omar, S.H.; Rehm, H.-J. Oxidation of n-tetradecane by Candida parapsilosis KSh 21 adsorbed on different glass rings. Appl. Microbiol. Biotechnol. 29, 442-446（1988）. 87）Liu, C.-M.; Johnson, M.J. Alkane oxidation by a particulate preparation from Candida. J. Bacteriol. 106, 830-834（1971）. 88）Mauersberger, S.; Schunck, W.-H. Müller, H.-G. The induction of cytochrome P-450 in the alkane-utilizing yeast Lodderomyces elongisporus: alterations in the microsomal membrane fraction. Appl. Microbiol. Biotechnol. 19, 29-35（1984）. 89）Klug, M.J.; Markovetz, A.J. Degradation of hydrocarbons by members of the genus Candida. II. Oxida829

J. Oleo Sci. 66, (8) 815-831 (2017)

S. Oda

tion of n-alkanes and 1-alkenes by Candida lipolyti. ca. J. Bacteriol. 93, 1847-1852 （1967） 90）Walker, J.D.; Cooney, J.J. Pathway of n-alkane oxidation in Cladosporium resinae. J. Bacteriol. 115, . 635-639 （1973） 91）Allen, J.E.; Markovetz, A.J. Oxidation of n-tetradecane and 1-tetradecene by fungi. J. Bacteriol. 103, 426-434 （1970） . 92）Oda, S.; Isshiki, K.; Ohashi, S. Regio- and stereoselective subterminal hydroxylation of n-decane by fungi in a liquid–liquid interface bioreactor （L-L IBR） . Bull. Chem. Soc. Jpn. 82, 105-109 （2009） . 93）Mihovilovic, M.D.; Snajdrova, R.; Grötzl, B. Microbial Baeyer-Villiger oxidation of 4,4-disubstituted cyclohexan and cyclohexenones by recombinant wholecells expressing monooxygenases of bacterial origin. J. Mol. Catal. B: Enzym. 39, 135-140 （2006） . 94）Oda, S.; Sakamoto, N.; Horibe, H.; Kono, A.; Ohashi, S. Relationship between interfacial hydrophobicity and hydroxylation activity of fungal cells located on an organic–aqueous interface. J. Biosci. Bioeng. 115, 544-546 （2013） . 95）Oda, S.; Sakamoto, N.; Horibe, H.; Kono, A.; Ohashi, S. Enhancement of n-decane hydroxylation activity of Monilliera sp. NAP 00702 in a liquid–liquid interface bioreactor by mixing of anion-exchange resin microparticles, Process Biochem. 47, 2494-2499 （2012）. 96）Morisaki, H. Effect of a liquid–liquid interface on the metabolic activity of Escherichia coli. J. Gen. Appl. Microbiol. 30, 35-42 （1984） . 97）Morisaki, H. Effect of solid–liquid interface on metabolic activity of Escherichia coli. J. Gen. Appl. Mi. crobiol. 29, 195-204 （1983） 98）Gottenbos, B.; van der Mei, H.C.; Busscher, H.J. Initial adhesion and surface growth of Staphylococcus epidermidis and Pseudomonas aeruginosa on biomedical polymers. J. Biomed. Mater. Res. 50, 208214 （2000） . 99）Vergara-Fernández, A.; van Haaren, B.; Revah, S. Phase partition of gaseous hexane and surface hydrophobicity of Fusarium solani when grown in liquid and solid media with hexanol and hexane. Biotech. nol. Lett. 28, 2011-2017 （2006） 100）Gigelis, R.; He, H.; Yang, H.Y.; Chang, L.-P.; Greenstein, M. Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation. J. Ind. Microbiol. Biotechnol. 33, 815-826 （2006） . 101）Hattori, R. Growth of Escherichia coli on the surface of an anion-exchange resin in continuous flow system. J. Gen. Appl. Microbiol. 19, 319-330 （1972）. 102）Hattori, R.; Hattori, T.; Furusaka, C. Growth of bacteria on the surface of anion-exchange resin. I. Experiment with batch culture. J. Gen. Appl. Microbiol.

18, 271-283（1972）. 103）Hattori, R. Growth and spore formation of Bacillus subtilis adsorbed on an anion-exchange resin. J. Gen. Appl. Microbiol. 22, 215-225（1976）. 104）Raymond, R. L.; Jamison, V. W.; Hudson, J. O. Microbial hydrocarbon co-oxidation. II. Use of ion-exchange resins. Appl. Microbiol. 17, 512-515（1969）. 105）Oda, S.; Isshiki, K.; Ohashi, S. Production of 6-pentyl-α-pyrone with Trichoderma atroviride and its mutant in a novel extractive liquid-surface immobilization（Ext-LSI）system. Process Biochem. 44, 625630（2009）. 106）Oda, S.; Michihata, S.; Sakamoto, N.; Horibe, H.; Kono, A.; Ohashi, S. Enhancement of 6-pentyl-α-pyrone fermentation activity in an extractive liquid-surface immobilization（Ext-LSI）system by mixing anion-exchange resin microparticles. J. Biosci. Bioeng. 114, 596-599（2012）. 107）Demyttenaere, J.C.R.; Adams, A.; Vanoverschekde, J.; de Kimpe, N. Biotransformation of（S） （＋） -linalool by Aspergillus niger: an investigation of the culture conditions. J. Agric. Food Chem. 49, 5895-5901 （2001）. 108）Canttell, C.L.; Lu, T.; Fronczek, F.R.; Fischer, N.H. Antimycobacterial cycloartanes from Bottrichia frytescens. J. Nat. Prod. 59, 1131-1136（1996）. 109）Isaka, M.; Chinthanom, P.; Boonruangprapa, T.; Rungjindamai, N.; Pinruan, U. Eremphilane-type sesquiterpenes from the fungus Xylaria sp. BCC 21097. J. Nat. Prod. 73, 683-687（2010）. 110）Bruno, M.; Rosselli, S.; Maggio, A.; Racchuglia, R.A.; Bastow, K.F.; Lee, K.-H. Cytotoxic activity of some natural and synthetic guaianolides. J. Nat. Prod. 68, 1042-1046（2005）. 111）Oda, S.; Fujinuma, K.; Inoue, A.; Ohashi, S. Synthesis of（–）-β-caryophyllene oxide via regio- and stereoselective endocyclic epoxidation of β-caryophyllene with Nemania aenea SF 10099-1 in a liquid–liquid interface bioreactor（L–L IBR）. J. Biosci. Bioeng. 112, 561-565（2011）. 112）Arrhenius, S.P.; Langenheim, J.H. Inhibitory effects of Hymenaea and Copaifera leaf resins on the leaf fungus, Pestalotia subcuticularis. Biochem. Syst, Ecol. 11, 361-366（1983）. 113）Magiatis, P.; Skaltsounis, A.-L.; Chinou, L.; Haroutounian, S.A. Chemical composition and in-vitro antimicrobial activity of the essential oils of three Greek Achillea species. Z. Naturforsch. 57c, 287-290 （2002）. 114）Chavan, M.J.; Wakte, P.S.; Shinde, D.B. Analgesic and anti-inflammatory activity of caryophyllene oxide from Annona sauqmosa L. bark. Phytochemistry 17, 149-151（2010）. 115）Cheng, S.-S.; Wu, C.-L.; Chang, H.-T.; Kao, Y.-T.;

830

J. Oleo Sci. 66, (8) 815-831 (2017)

Production of Aroma Compounds in Interface Bioprocesses: Review

Chang, S.-T. Antitermitic and antifungal activities of essential oil of Calocedrus formosana leaf and its composition. J. Chem. Ecol. 30, 1957-1967 （2004） . 116）Zheng, G.-Q.; Kenney, P.M.; Lam, L.K.T. Sesquiteras potenpenes from clove （Eugenia caryophyllata） tial anticarcinogenic agents. J. Nat. Prod. 55, 9991003 （1992） . 117）Cutler, H.G.; Cox, R.H.; Crumley, F.G.; Cole, P.D. 6-Pentyl-α-pyrone from Trichoderma harzianum: its plant growth inhibitory and antimicrobial proper. ties. Agric. Biol. Chem. 50, 2943-2945 （1986） 118）Rito-Palomares, M.; Negrete, A.; Miranda, L.; Flores, C.; Galindo, E.; Serrano-Carreon, L. The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum. Enzyme Microb. Tech. nol. 28, 625-631 （2001） 119）Rocha-Valadez, J.A.; Estrada, M.; Galindo, E.; Serrano-Carreón, L. From shake flasks to stirred fermentors: scale-up of an extractive fermentation process for 6-pentyl-α-pyrone production by Trichoderma harzianum using volumetric power input. Process Biochem. 41, 1347-1352 （2006） . 120）Kalyani, A.; Prapulla, S.G.; Karanth, N.G. Study on

the production of 6-pentyl-α-pyrone using two methods of fermentation. Appl. Microbiol. Biotechnol. 53, 610-612（2000）. 121）Cooney, J.M.; Lauren, D.R.; Jensen, D.J.; Perry-Meyer, L.J. Effect of solid substrate, liquid supplement, and harvest time on 6-n-pentyl-2H-pyran-2-one （6PAP）production by Trichoderma spp. J. Agric. Food Chem. 45, 531-534 （1997）. 122）Serrano-Carreón, L.; Flores, C.; Rodríguez, B.; Galindo, E. Rhizoctonia solani, an elicitor of 6-pentyl-α-pyrone by Trichoderma harzianum in a two liquid phases, extractive fermentation system. Biotechnol. Lett. 26, 1403-1406（2004）. 123）De Aráujo, Á.A.; Pastore, G.M.; Berger, R.G. Production of coconut aroma by fungi cultivation in solidstate fermentation. Appl. Biochem. Biotechnol. 99, 747-752（2002）. 124）Parker, S.R.; Cutler, H.G.; Jacyno, J.M.; Hill, R.A. Biological activity of 6-pentyl-2H-pyran-2-one and its analogs. J. Agric. Food Chem. 45, 2774-2776（1997）. 125）Oda, S.; Sugitani, A.; Ohashi, S. Solvent-tolerance of fungi located on an interface between an agar plate and an organic solvent. Biosci. Biotechnol. Biochem. 78, 1971-1974（2014）.

831

J. Oleo Sci. 66, (8) 815-831 (2017)