Interface Bioreactor Research Articles

Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Research Highlights

Research Highlights

Microbial transformation of water-insoluble substrates by two types of novel interface bioprocesses, tacky liquid-liquid interface bioreactor and non-aqueous sporular bioconversion system.

Although microbial transformation has been expected as a substitution technology for organic synthesis, microbial toxicity and water-insolubility of synthetic substrates prevent the practical application of the technology. For these problems, the authors have developed two types of interfacial bioprocesses, solid-liquid and liquid-liquid interface bioreactors and applied the systems to many microbial transformations. In the bioreactors, addition of substrates and accumulation of products were remarkably enhanced based on the toxicity alleviation effect on the interfaces and solubilization of substrates and/or products in an organic phase of the bioreactors. Recently, a novel tacky liquid-liquid interface bioreactor has been developed and applied to actinomycetes and yeasts. Furthermore, a novel bioconversion system with fungal spores in an organic phase has been constructed based on the combination of two facts as follows: (i) the fungal spores are never resting cells and are active ones like the vegetable cells, (ii) the fungal spores have the excellent solvent-tolerance. In this review, the tacky liquid-liquid interface bioreactor (L-L IBRtac) and the non-aqueous sporular bioconversion system with immobilized fungal spores (NASB) are mainly given outlines.

Modified liquid–liquid interface cultivation system with floating microspheres and binder micro-pieces for slow-growing or unicellular microorganisms: Application to interfacial bioconversions with an actinomycete and yeasts

Abstract Liquid–liquid interface bioreactor (L–L IBR) is a unique non-aqueous bioconversion system which comprises a hydrophobic organic solvent (upper phase), a fungal cells–floating microspheres (MS) layer (middle phase), and a liquid medium (lower phase). In this study, a modified L–L IBR with actinomycetes and yeasts was developed by using binder micro-pieces (BM) and estimated its availability through some bioconversions. This modified interface cultivation system was named a tacky liquid–liquid interface bioreactor (L–L IBRtac). After the detailed estimation of its characteristics, the system was applied to oxidation of citronellol to citronellal, 2-methylcyclohexanol to 2-methylcyclohexanone, and 2-octanol to 2-octanone with Rhodococcus hoagii NBRC 3730, oxidation of citronellal to citronellic acid with Candida viswanathii NBRC 10321, and transacetylation of citronellol by acetyl coenzyme A (acetyl-CoA) produced from glucose by Pichia kluyveri NBRC 1165. The accumulation of citronellal, 2-methylcyclohexanone, and 2-octanone reached 3.1 (16 days), 2.3 (12 days), and 32.9 g/l (12 days) in spite of strong biotoxicities of the substrates/products without collapse of a cells–MS–BM layer. On the other hand, 6.1 g/l of citronellic acid and 2.8 g/l of citronellyl acetate were produced from 5% citronellal and 10% citronellol for 12 days, respectively.

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.

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.

Relationship between Interfacial Hydrophobicity and Hydroxylation Activity of Fungal Cells Located on an Organic–Aqueous Interface

A Liquid–liquid interface bioreactor (L–L IBR), which consists of a hydrophobic organic solvent (an upper phase), a layer of fungal cellballooned polyacrylonitrile microsphere (diameter, 20-40 μm; density, 0.03-0.20; a middle phase), and a liquid medium (a lower phase), is a unique and effective cultivation system for the microbial transformation with fungi [1]. The system has some interesting and practically important characteristics, i.e., alleviation of toxicity of poisonous substrate and/or product solubilized in the organic phase, excellent productivity of valuable hydrophobic chemicals, efficient supply of oxygen from the organic phase to fungal cells, control and management of pH and nutrients in the liquid medium, depression of catabolize repression caused by easily metabolizable carbon sources [2], and easy recovery of product without troublesome solvent extraction. The system has been applied to various microbial reactions, such as hydrolysis of an acetate ester [1,3], asymmetric reduction of an aromatic dike tone [4], and regio- and stereo selective epoxidation of - caryophyllene to (–)-β-caryophyllene oxide so far [5]. In all cases, it has been observed that substrate concentration, product accumulation, and region- and stereo selectivity’s of reaction reached very high level compared with two traditional cultivation systems, submerged and organic–aqueous two-liquid-phase systems.

ChemInform Abstract: Morphological, Physiological, and Biochemical Properties of Fungal Cells Located on a Liquid‐Surface and an Organic‐Aqueous Interface

AbstractReview: 45 refs.

Morphological, physiological, and biochemical properties of fungal cells located on a liquid-surface and an organic–aqueous interface

In general, morphological, physiological, and biochemical properties of fungal cells are drastically different among solid-state (SSC), liquid-surface (LSC), and submerged cultivation (SmC) systems. In addition to these traditional cultivation systems, a unique liquid-surface cultivation [liquid-surface immobilization (LSI)] system and interface cultivation systems [extractive liquid-surface immobilization (Ext-LSI) system; liquid–liquid interface bioreactor (L–L IBR)] bring some interesting fungal morphological, physiological, and biochemical characteristics. The unique properties of fungal cells located on a liquid-surface and an organic–aqueous interface are very important to application of fungi to the production of enzymes, metabolites, and bioconversion products. In this review, the unique and important characteristics of fungal cells immobilized in the LSI, L–L IBR, and Ext-LSI systems are explained in detail. Moreover, some application of the cultivation systems to fermentation, enzyme production, and microbial transformation are introduced.

Relationship between interfacial hydrophobicity and hydroxylation activity of fungal cells located on an organic–aqueous interface

In a liquid-liquid interface bioreactor, fungal cells locate in a hydrophilic polyacrylonitrile microsphere layer on an aqueous-organic interface. In this article, effects of hydrophobicity of the interface on n-decane hydroxylation activity of Monilliera sp. NAP 00702 was examined. (-)-4-Decanol production was significantly enhanced to 132% by addition of polytetrafluoroethylene.

Enhancement of n-decane hydroxylation activity of Monilliera sp. NAP 00702 in a liquid–liquid interface bioreactor by mixing of anion-exchange resin microparticles

Fungal cells immobilized with ballooned polyacrylonitrile (PAN) microsphere (MS) on an interface between a liquid medium and a hydrophobic organic solvent efficiently catalyze various microbial transformations such as hydrolysis, reduction, hydroxylation, and epoxidation of lipophilic substrates dissolved in the organic phase (liquid–liquid interface bioreactor; L–L IBR). In this study, the electric charge of MS layer in the L–L IBR was modified by mixing 4 kinds of anion-exchange, 2 kinds of cation-exchange or a chelating resin microparticles, and applied to regio- and stereoselective subterminal hydroxylation of n-decane to (−)-4-decanol by Monilliera sp. NAP 00702. In general, the addition of cation-exchange and chelating resin microparticles into a PAN-MS layer led to the strong inhibition of fungal growth and hydroxylation activity. On the other hand, the addition of anion-exchange resin microparticles having moderate total capacity (≤1.00meq/g) significantly increased hyphal growth and hydroxylation activity. Especially, the addition of IRA910CT microparticles (total capacity≥1.00meq/g) into a PAN-MS layer led to 1.6-fold increase of 4-decanol production. Thus, it was concluded that the moderate addition of anion-exchange resin microparticles into an MS layer in the L–L IBR system was effective to enhancement of fungal growth and hydroxylation activity.

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)

Nemania aenea SF 10099-1, a basidiomycete isolated from a forest soil sample, regio- and stereoselectively epoxidized β-caryophyllene (Car) to (-)-β-caryophyllene oxide (Car-Ox) in a liquid-liquid interface bioreactor (L-L IBR) consisted of a liquid medium (a bottom phase), a fungus-ballooned microsphere (MS) mat (a middle phase), and an organic phase containing Car (a top phase). The cultivation conditions, such as carbon and nitrogen sources, kind of MS, initial medium pH and Car concentration, were optimized in the L-L IBR system. The best carbon and nitrogen sources were xylose and tryptone, respectively. The most suitable polyacrylonitrile MS was MMF-DE-1 (former MFL-80SDE; non-coated type). Although the strain could not grow below pH 5.5, the endocyclic epoxidation of Car efficiently proceeded at a wide range of initial medium pH (6.0 to 9.0). The bioconversion system exhibited an excellent alleviation effect toward substrate and product inhibitions. While Car could be added into an organic phase (KF-96L-1CS, dimethyl silicone oil) at 50% (w/v), the accumulation of Car-Ox reached over 30g/l in spite of these strong microbial toxicities. Moreover, the epoxidation reaction smoothly proceeded in a novel L-L IBR system, a multistory L-L IBR systems, consisted of 5 stacked reactor units. The optical rotation of Car-Ox produced was (-) and the enantiomeric excesses of (-)-β-Car-Ox purified by 1st and 2nd recrystallization from methanol reached 97.51 and 99.33%, respectively.

Efficient hydrolytic reaction of an acetate ester with fungal lipase in a liquid–liquid interface bioreactor (L–L IBR) using CaCO3-coated ballooned microsphere

In a liquid-liquid interface bioreactor using a CaCO₃-coated ballooned microsphere, 2-ethylhexyl acetate was efficiently hydrolyzed to 2-ethyl-1-hexanol with Absidia coerulea NBRC 4423 compared with using talc-coated or non-coated ballooned microsphere. It was assumed that CaCO₃ brought about stabilization of lipase by Ca²⁺ and maintenance of medium pH.

Regio- and Stereoselective Subterminal Hydroxylations of n-Decane by Fungi in a Liquid–Liquid Interface Bioreactor (L–L IBR)

Abstract This article may be the first report to describe the excellent regio- and stereoselective subterminal hydroxylations of n-alkane with microorganisms. Approximately 2000 fungal strains were screened for the regioselective hydroxylation of n-decane with a solid–liquid interface bioreactor, which is a microbial transformation device on an interface between an agar plate and an organic phase (n-decane). Although Beauveria bassiana ATCC 7159, a typical fungus having a strong and versatile hydroxylating ability, mainly produced a mixture of 2-, 3-, 4-, and 5-decanone regioisomers into a n-decane layer, Monilliela sp. NAP 00702 and Rhizopus oryzae R-38-8 typically accumulated 4- and 5-decanols into the n-decane layer, respectively. Although a small amount of 4- and 5-decanones were also produced, the regioselectivity at C-4 and C-5 positions of n-decane reached 99 and 90%, respectively. Furthermore, the hydroxylation of n-decane with Monilliela sp. NAP 00702 stereoselectively proceeded to afford almost 100% ee of (−)-4-decanol.

Asymmetric Reduction of Benzil to (S)-Benzoin withPenicillium claviformeIAM 7294 in a Liquid-Liquid Interface Bioreactor (L-L IBR)

The asymmetric reduction of benzyl to (S)-benzoin with Penicillium claviforme IAM 7294 was applied to a liquid-liquid interface bioreactor (L-L IBR) using a unique polymeric material, ballooned microsphere (MS). The L-L IBR showed superior performance, as compared with suspension, organic-aqueous two-liquid-phase, and solid-liquid interface bioreactor (S-L IBR) systems, affording 14.4 g/l-organic phase of (S)-benzoin (99.0% ee).

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

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.

Biodesulfurization of Dibenzothiophene with Rhodococcus erythropolis ATCC 53968 and Its Mutant in an Interface Bioreactor.

An interface bioreactor was used in the biodesulfurization (BDS) of dibenzothiophene (DBT) with Rhodococcus erythropolis ATCC 53968. Although this strain could efficiently degrade DBT to 2-hydroxybiphenyl (2-HBP), the microbial film peeled off from the surface of the carrier (agar plate) to an organic phase (dodecane or tetradecane). To overcome this problem, a UV mutant, which strongly adhered to the carrier surface, was obtained by a new screening method. The best UV mutant (UM-021) could efficiently catalyze the BDS and the microbial film did not peel off from the surface of the carrier to tetradecane layer.

Biodesulfurization of dibenzothiophene with Rhodococcus erythropolis ATCC 53968 and its mutant in an interface bioreactor

An interface bioreactor was used in the biodesulfurization (BDS) of dibenzothiophene (DBT) with Rhodococcus erythropolis ATCC 53968. Although this strain could efficiently degrade DBT to 2-hydroxybiphenyl (2-HBP), the microbial film peeled off from the surface of the carrier (agar plate) to an organic phase (dodecane or tetradecane). To overcome this problem, a UV mutant, which strongly adhered to the carrier surface, was obtained by a new screening method. The best UV mutant (UM-021) could efficiently catalyze the BDS and the microbial film did not peel off from the surface of the carrier to tetradecane layer.

Preparation of Methyl Ursodeoxycholate via Microbial Reduction of Methyl 7-Ketolithocholate in an Anaerobic Interface Bioreactor.

An interface bioreactor, which is a device for the microbial transformation of water-insoluble substrates, was applied to an anaerobic bioconversion for the first time. Methyl 7-ketolithocholate [Me-7KLCA] was reduced with the human intestinal bacterium Eubacterium aerofaciens JCM 7790 in a convenient anaerobic interface bioreactor using a nutrient agar plate placed in a GasPak pouch. The resulting methyl ursodeoxycholate [Me-UDCA] is a precursor of ursodeoxycholic acid, which is used as a cholesterol gallstone-dissolving agent. The reaction conditions were optimized, and ABCM medium and dihexyl ether were selected as the best carrier and reaction solvent, respectively. The toxicity of the bile acid esters toward the human intestinal bacterium was effectively alleviated in the interface bioreactor, in which the maximal concentrations of Me-7KLCA and Me-UDCA in the dihexyl ether layer respectively reached to 12.0 and 6.1 g/l.

Synthesis of (S)-Ibuprofen via Enantioselective Degradation of Racemic Ibuprofen with an Isolated Yeast, Trichosporon cutaneum KPY 30802, in an Interface Bioreactor.

An interface bioreactor was used for the enantioselective degradation of (RS)-ibuprofen (IBU). An isolated yeast, Trichosporon cutaneum KPY 30802, preferentially degraded (R)-IBU to accumulate (S)-isomer. The addition of hydroquinone (10 mM) into a hydrophilic carrier was effective for the elevation of enantiomeric excess and the repression of excess degradation of (S)-IBU (E value, 9.3).

Synthesis of ( S)-ibuprofen via enantioselective degradation of racemic ibuprofen with an isolated yeast, Trichosporon cutaneum KPY 30802, in an interface bioreactor

An interface bioreactor was used for the enantioselective degradation of ( RS)-ibuprofen (IBU). An isolated yeast, Trichosporon cutaneum KPY 30802, preferentially degraded ( R)-IBU to accumulate ( S)-isomer. The addition of hydroquinone (10 mM) into a hydrophilic carrier was effective for the elevation of enantiomeric excess and the repression of excess degradation of ( S)-IBU ( E value, 9.3).