J. Biol. Macromol., 13(1), 1-11 (2013)

ReviewMorphological, physiological, and biochemical properties of fungal cells located on aliquid-surface and an organic–aqueous interface
Shinobu Oda
Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho,
Hakusan, Ishikawa 924-0838

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 anorganic–aqueous interface are very important to application of fungi to the production of
enzymes, metabolites, and bioconversion products. In this review, the unique and importantcharacteristics 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.

Received April 1, 2013; Accepted June 5, 2013
Key words: filamentous fungi, interface bioreactor, liquid-surface cultivation, secondary metabolite,
microbial transformation
fungal cells are cultivated in submerged
cultivation (SmC) system to form pelleted,

Introduction
Filamentous fungi have been applied tofilamentous,orclumpedforms

asvarious industries, such as traditional food andmorphological pattern. In general, the pelleted

breweryofform measuring 1–2 mm in diameter is the ideal

metabolites, enzymes, cosmetic materials, andmorphology for fungi in the SmC because thispharmaceutical intermediates. In many cases,

form can maintain a low viscous broth andindustries,

theproduction

enables efficient oxygen supply(1). However, a_________________________________
E-mail: odas@neptune.kanazawa-it.ac.jp

large number of factors, such as type and sizeof inoculum, composition and pH of medium,

2

Characteristics of fungal cells located on an interface

agitation rate, incubation temperature, and anumber of additives (surfactant, polymer, andchelator) must be optimized for the formationof fungal pellets(2). Thus, although the controlof the fungal morphology is practically very
important, this process is troublesome to
implement in general.
On the other hand, in a traditional cultivation
system named solid-state cultivation (SSC),
fungal cells growing on solid substrate or inert
support particles such as polyurethane foam
naturally differentiate to form much aerial andvegetative hyphae, sporangia and spores(3). The
SSC has some practical advantages, such as
higher productivity, lower risk of contaminationand lower energy consumption, and lower costof equipment compared with the SmC(4).
Moreover, derepression of catabolite repression
and restraint of digestion of targeted proteins byinherent

proteasearefavorable

for

the

production of metabolites and enzymes in theSSC(5,6). However, although the SSC hasvarious advantages abovementioned, someserious disadvantages, such as poor heatdissipation, low water activity, and slow
diffusion rates of nutrients, products andoxygen in a packed bed have been observed(4).
Therefore, it is fervently desired to develop ahigher performance of cultivation system forfungi.
Recently, we have developed three types ofu n i q u e f u n ga l c u l t i va t i o n s ys t e ms , aliquid-surface immobilization (LSI) (7,8), an
extractive LSI (Ext-LSI) (9,10), and a liquid–
liquid interface bioreactor (L–L IBR) systems(Fig. 1)(11,12). While the LSI system consists of afungus–polyacrylonitrile (PAN) ballooned
microsphere (MS) mat (a top phase) and liquidmedium (a bottom phase), the Ext-LSI and L–L
IBR systems consist of hydrophobic organic

Fig. 1. Principles of three types ofcultivation/application devices for fungal
cells. LSI, liquid-surface immobilization; L–L
IBR, liquid–liquid interface bioreactor;
Ext-LSI,
extractive
liquid-surface
immobilization. In all systems, fungal cells aretrapped on a ballooned polyacrylonitrile
microsphere (MS; diameter, 20–100 µm;
density, 0.03–0.20) layer formed on aliquid-surface of medium. The fungal cells
grow and differentiate to vegetative and aerial
hyphae, sporangia, and spores during
appropriate precultivation period to form aphysically strong fungus-MS mat on theliquid-surface. The fungus-MS mat is used asa biocatalyst to produce hydrophilic
metabolites and enzymes (LSI system). In thecase of overlaying the fungus-MS mat with aharmless hydrophobic organic solvent, such as
middle-chain alkanes, esters, ethers, and low
viscous dimethylsilicone oils, two types of
unique cultivation/applica-tion devices can beconstructed. The L–L IBR is an interfacial
microbialtransformation
deviceofhydrophobic substrates dissolved in theorganic phase. The Ext-LSI system is aninterfacial fermentation device of hydrophobic
metabolites by using nutrients dissolved in theliquid medium. In all cases, targeted products
are remarkably accumulated into a liquidmedium (LSI system) or an organic phase(Ext-LSI and L–L IBR systems) because of
the alleviation of some unfavorable
phenomena, such as catabolite repression,feed-backinhibition,
substrate/productinhibition, and digestion of products byinherent proteases. In both the L–L IBR andthe Ext-LSI system, the organic phase plays asa reservoir, an extractant, and a solvent ofwater-insoluble substrates and products.

S. Oda3

solvent (a top phase), fungus–MS mat (a middle

morphologies such as filamentous, clump, or

phase), and liquid medium (a bottom phase). In

pelleted forms in the SmC, the SSC generally

all systems, almost all fungal cells suspended inleads fungal cells to natural differentiation toa liquid medium are floated together withvegetative and aerial hyphae, sporangia, andnumerous PAN-MS onto the surface of thespores. Thus, the SSC brings about the superiorliquid medium to form a physically strong

productivities

fungus–MS mat onto the medium.

metabolite.

In this article, the principles, characteristics,

Recently,of
wetargeted
have

enzymes

observed

that

and
the

and applications of the LSI, L–L IBR, andproductivities of lipase by Absidia coerulea

Ext-LSI systems are reviewed in detail.

IFO

Furthermore, the relationship between the

β-glucosidase

physicochemical properties (electric charge and

LSI system are significantly higher than thosehydrophobicity)

organic–aqueous

in the SmC. It is easily assumed that theinterface and the morphological, physiologicalproduction of inherent proteases is repressed inand biochemical properties of fungal cells is

the LSI system likely to the SSC(6). Moreover,discussed.

fungal cells trapped in an MS layer differentiateofan4423(7),
(8)

and

of

xylanase(18)andby Aspergillus oryzae RIB40 into natural forms, i.e., vegetative and aerial
Liquid-surface immobilization (LSI) systemhyphae, sporangia, and spores likely to the SSC.

In many cases, the SmC causes strong carbon

The natural differentiation in the LSI system

catabolite repression in the presence of excess

must be favorable to display the inherent

easily metabolizable carbon sources, such as

abilities.

glucose,fructose,andsucrose

(13)

.

The

unfavorable phenomenon, which is brought

Extractive liquid-surface immobilization

about by some repressor proteins such as(Ext-LSI) system(14), induces a decrease in enzyme andFungal cells can grow and differentiate on an

metabolite productivities. Furthermore, targetedinterface between a liquid medium and a

enzymes produced by fungi are generally

harmless hydrophobic organic solvent such asdigested

producedn-alkanes, middle-chain fatty esters and ethers,. Both phenomena

and lowly viscous dimethyl silicone oil by theare serious problems for many microbial

aid of the MS. Firstly, the fungal cells locatedindustries.

on the organic–aqueous interface were applied

CreA

by

inherent

proteases(15)

simultaneously in the SmC

On the other hand, it has been reported thatto the production of a fungicidal secondary

the unfavorable phenomena above-mentioned

metabolite, 6-pentyl-α-pyrone (6PP)(9) which

can be avoided by adopting other traditional

was useful for coconut-aroma. 6PP was(16,17)cultivation system, the SSC. Indeed, theSSC generally enables superior productivities
of enzymes and metabolites compared with the
SmC(16,17). The morphology control of fungal

produced by some Trichoderma spp. in the
SmC(19),aqueousorganic–aqueous
(22)

LSC

two–liquid–phase

(20)

(21)

the

two–liquid–phase, and the SSC systems

,

,

(23)

. However, thecells in SSC is generally easier compared withaccumulation of 6PP was generally limited to

the SmC. While fungal cells form unnaturalvery low levels because of its strong fungicidal

4Characteristics of fungal cells located on an interface

Fig. 2. Derepression of carbon catabolite repression in Ext-LSI
system. The incubation was done at 25 °C without shaking. While strong
carbon catabolite repression by excess glucose appeared in the Ext-SmC
system, Ext-LSI system could alleviate the unfavorable phenomenone.

Fig. 3. Effect of mixing of ion-exchange resin microparticles into a MS layer on the productionof 6-pentyl-α-pyrone (6PP) and the formation of spores of Trichoderma atroviride
AG2755-5NM398. Control: PAN only; anion-exchange resins: IRA904 (total capacity, ≥ 0.65 meq/g),
IRA958 (≥ 0.80), IRA910CT (≥ 1.00), IRA98 (≥ 1.35); cation-exchange resins: 252 (≥ 1.80), IRC76 (≥3.90); chelating resin: IRC748 (≥ 1.35). All ion-exchange resins (50 µm pass) were mixed in an MS
layer at the ratio of 5-fold weight on PAN-MS. Thus, thickness of the fungus–MS-resin layer wasapproximately 3 mm in all cases. The incubation was performed at 25 °C without shaking.

S. Oda5

activity (MICs = 0.2–0.5 g/L)(19,24). Indeed, themetabolites.

maximum accumulations of 6PP produced in

Ballooned

the SmC and the SSC are only 474 mg/L and 3(25,26)g/kg, respectively(MMF-DE-1,

Matsumoto Yushi Seiyaku, Co., Ltd., Osaka) is
electrically.

PAN-MS
neutral(27)

and

has

relatively

To the contrary, the accumulation of 6PP inhydrophilic surface (contact angle, 50 °). The

an organic phase (dimethyl silicone oil) in theelectric charge of an MS layer can be modified

Ext-LSI system reached to 7.1 g/L because theby mixing ion-exchange resin microparticles.

organic phase played as a reservoir and an

All ion-exchange resins (50 µm pass) tested(9)extractant of toxic 6PP . Recently, it waswere mixed in the MS layer at the ratio ofobserved that fungal cells located on the

5-fold weight on the PAN-MS. Thus, thickness

organic–aqueous interface in the Ext-LSIofsystem are liberated from carbon catabolite

approximately 3 mm in all cases. In the case ofrepression to produce much 6PP compared withmixing cation-exchange (252 and IRC76;

(10)

the

fungus–MS-resin

layer

was

. It

Organo Co., Ltd., Tokyo) and chelating resin

is concluded that the Ext-LSI system may be(IRC748) microparticles into the PAN-MS layer,

liberate fungal cells from product toxicity,

fungal morphology and fermentative activity

feed-back inhibition, and catabolite repression

drastically changed as shown in Fig. 3(28).

traditional extractive fermentation (Fig. 2)

to afford large quantities of lipophilic secondary

Fig. 4. Application of L–L IBR to various fungal bioconversions and aphotograph and a schematic diagram of a multi-story L–L IBR system. The
system consisted of stacked reactor units, diaphragm-pump, and solvent-circulationlines to connect an organic phase.

6Characteristics of fungal cells located on an interface

Fig. 5. Effect of mixing of ion-exchange resin microparticles into a MS layer
on production of (–)-4-decanol and the formation of aerial mycelia of
Monilliera sp. NAP 00702. All ion-exchange resins (50 µm pass) were mixed in
an MS layer at the ratio of 5-fold weight on PAN-MS to form approximately 3mm-thickness of fungus–MS-resin layer. The correlation coefficient (R2) was
calculated among PAN, IRA904, IRA958, and IRA910CT. The vertical sections ofMS-layers were photographed with a stereomicroscope. The incubation was
carried out at 25 °C without shaking for 14 days.

For the addition of anion-exchange resin

morphological, physiological, and biochemical

microparticles (IRA904, 958, 910CT, and 98)

properties of microorganism, such as oxidation

into the PAN-MS layer, the higher the total

activity(29), optimum pH for the bacterial

capacity of anion-exchange resin, the higher thegrowth(30), spore formation(31), and growth

6PP production and the lower the spore

rate(30,32), drastically change in the presence offormation were observed. The correlation

anion-exchange resin. Although the exact

coefficient (R2) between the total capacity andmechanisms of the phenomena caused by

the 6PP production reached 0.724. On the otheranion-exchange resin are not clear at the present

hand, the addition of cation-exchange (252 andtime, it is assumed that electric signal generated

IRC76)

resinby adhesion onto the charged resin surface

microparticles decreased the 6PP production,

causes abovementioned interesting phenomena.

andchelating(IRC748)and IRC748 in particular exhibited the strongest
inhibitory activity on fungal growth. Thus, it isLiquid–liquid interface bioreactor (L–L IBR)

concluded that the quaternary ammonium ion of

The L–L IBR is defined as a bioreactor usingtheanion-exchange

resin

microparticles(28)

enhanced the production of 6PP.

living fungal cells located in an MS layer (amiddle phase) formed on an interface between

As for the effects of anion-exchange resin on

an aqueous phase (a bottom phase; liquid

microbial cells, some reports have been

medium) and an organic phase (a top phase;published so far. It has been reported that somehydrophobic organic solvent) likely to theS. Oda7

Ext-LSI system. In the L–L IBR, lipophilicorganic–aqueous two–liquid–phase system (Fig.

substrates were dissolved in the organic phase4).

products

Among these applications of the L–L IBR to(accumulated in the organic phase) or tofungal bioconversion, regio- and stereoselectivehydrophilic products (accumulated in thehydroxylation of n-decane to (–)-4-decanol byaqueous phase). The reactor exhibited various

Monilliera sp. NAP 00702 is very interesting.

merit for the fungal bioconversion, such as the

The regio- and stereoselectivities of the reaction

alleviation of toxicity of substrates and products

reachedandconvertedtolipophilic

99%

and

almost

100%

ee,

(34)

and much accumulation of poisonous products

respectively

in the organic phase, high dissolution of

the established theory insisting on the shortage

lipophilic substrates and products into theof regio- and stereoselectivities of microbialorganic phase, high oxygen supply in stationary

subterminal hydroxylation(37–39).

cultivation,

excellent

regio-. The discovery firstly overturnedandAs for the hydroxylation of n-decane bystereoselectivities, and very wide applications.

Monilliera sp. NAP 00702, recently, it was

The bioreactor has been applied to various

reported that interfacial charge in the L–L IBR

fungal bioconversions, such as hydrolysis(11)(34-36),

reductionhydroxylation

(12)

,

(7,33),

anddrasticallyaffectedthegrowth

and

the
(35)

hydroxylation activity of the fungal cells.

epoxidationand has exhibited the excellent

Although the addition of cation-exchange

productivityofvarioususefulchemicals(IRC76; Organo Co., Ltd., Tokyo) and chelatingcompared

with

the

SmC

and

resins (IRC748) strongly inhibited the fungal

anFig. 6. Relationship between hydrophobicity of resin added and(–)-4-decanol production. All hydrophobic resins (50 µm pass) weremixed in an MS layer at the ratio of 5-fold weight on PAN-MS toform approximately 3 mm-thickness of fungus–MS-resin layer. The
incubation was done at 25 °C without shaking for 14 days

8

Characteristics of fungal cells located on an interface

growth, the addition of anion-exchange resinof fungal cells, such as increase of growth(IRA98, 910CT, 958, and 904) led therate(40) and respiration activity(41), decrease ofsignificant increase of hydroxylation activity

the rate of glucose uptake(41), initiation of thewith the enhancement of hyphal growth (Fig. 5).

formation

However,theeffectofthestrongest

spores(42)

of

and

extracellular(43)

matrix. It is also known that hydrophobic

anion-exchange resin, IRA98 (total capacity ≥organic solvents, such as n-dodecane and1.35 meq/g), declined the hydroxylation activity

n-tetradecane, lead the increase of respiration

2

to some extent. The correlation coefficient (R )activity and the decrease of the rate of glucose

between the 4-decanol production and the total

uptake(44).

anion-exchange

capacity

of

PAN

only,

Although the exact mechanisms of the

PAN+IRA904 (1:5), PAN+IRA958 (1:5), andphenomena caused by hydrophobic resin are

PAN+IRA910CT (1:5) was 0.994(35). It isnot clear at this time, it is assumed that theconcluded that the moderate anion-exchange

physicochemical properties of cell surface

capacity (total capacity = 1.0 meq/g) in an MSchangelayer enhanced the growth and hydroxylationhydrophobic substrata. Indeed, it was reported

activity

tothat the surface hydrophobicity of fungal cells

fermentative activity, while the cation-exchange

was remarkably increased by the adaptation toand chelating activities in the MS layer arehydrophobic circumstance(45). It is expected thatunfavorable to fungal growth, hydroxylation

mechanisms of the change of morphological,

and fermentative activities.

physicochemical,offungalcells

inadditionbyadhering

onto

surfaceandofbiochemical

Recently, it was reported that the interfacialcharacteristics of microbial cells containinghydrophobicity in an MS layer of the L–L IBR

fungal cells are clarified and reflected to thesystemindustrial application of microorganisms.

also

affectedthesubterminalhydroxylation of n-decane to (–)-4-decanol by
Monilliera sp. NAP00702. As shown in Fig. 6,

References

the mixing of hydrophobic resin particles such

1. Li, Z. J., Shukla, V., Fordyee, A. P., Pedersen,aspolypropylene

(PP)

and

A. G., Wenger, K. S., Marten, M. R.:

polytetrafluoroethylene (PTFE) in the PAN-MS

Fungalenhanced the hydroxylation activity. The

behavior in a fed-batch Aspergillus oryzae

positive correlation between the contact angle

fermentationof

Biotechnol. Bioeng., 70, 300-312(2000).

hydrophobic

resin

and

(–)-4-decanol

2

production was observed (R = 0.747). However,morphology
at

and

the

fragmentation

production

scale.

2. Liao, W., Liu, Y., Chen, S.: Studying pellet

the mixing of hydrophobic resin microparticles

formation

did not accelerate hyphal growth.

Rhizopus oryzae to enhance organic acid

Concerning the effect of hydrophobic resinon microbial cells, some interesting phenomena
have

been

hydrophobic

reported.
resin

The

into

addition

microbial

of
cell

of

a

filamentous

fungusproduction. Appl. Biochem. Biotechnol.,
137-140, 689-701(2007).
3. Sakurai, Y., Misawa, S., Shiota, H.: Growthandrespiratory

activity ofAspergillus

suspension leads some unique morphological,

oryzae grown on solid state medium. Agric.

physiological, and biochemical characteristics

Biol. Chem., 49, 745-750(1985).

S. Oda9

4. Adinarayana, K., Prabhakar, T., Srinivasulu,

11. Oda, S., Isshiki, K.: Asymmetric reduction

V., Rao, M. A., Lakshmi, J., Ellaiah, P.:of benzil to (S)-benzoin with Penici- llium

Optimization of process parameters forclaviforme IAM 7294 in a liquid–liquid

cepharosporin C production under solidinterface bioreactor (L–L IBR). Biosci.

state

Biotechnol.

fermentation

chrysogenum.

fromProcess

Acremonium

Biochem.,

12. Oda, S., Fujinuma, K., Inoue, A., Ohashi,(2003).
5. de Souza, D. F., de Souza, C. G. M., Peralta,

S.:

Synthesis

oxide

via

of

(–)-β-caryophyllene

regio-

andstereoselective

R. M.: Effect of easily metabolizable

endocyclic epoxidation of β-caryophyllene

sugars in the production of xylanase bywith Nemania aenea SF 10099-1 in a

Aspergillus tamarii in solid-state fermenta-liquid–liquid interface bioreactor (L–L

tion. Process Biochem., 36, 835-838(2001).

IBR).

Aguilar,561-565(2011).

C.

N.,

J.

Biosci.

Bioeng.,112,

Favela-Torres,

E.,

G.:

and

13. Pitson, S. M., Seviour, R. J., McDougall, B.

repression patterns of fungal tannase in

M.: Induction and carbon source control ofsolid-state and submerged cultures. Process

extracellular β-glucosidase production in

Biochem., 36, 565-570(2001).

Acremonium persicinum. Mycol. Res., 103,

Oda,

161-167(1999).

Viniegra-González,

7.

72,

1364-1367(2008).

39,

171-177

6.

Biochem.,S.,

Isshiki,Induction

K.:

Liquid–surface

immobilization system and liquid–liquid

14. Piñaga, F., Fernández-Espinar, M. T., Vallés,inter- face bioreactor: application to fungal

S., Ramón, D.: Xylanase production inhydrolysis.

Aspergillus nidulans: induction and carbon

Process

Biochem.,42,

catabolite repression. FEMS Microbiol.

1553-1560 (2007).

Lett., 115, 319-323(1994).

8. Ozeki, K., Kondo, K., Nakagawa, H., Ozeki,
K., Oda, S., Ohashi, S.: Production of
β-glucosidase

byAspergillus

oryzae15. Xu, J., Wang, L., Ridgway, D., Gu, T.,transformantof

Moo-Young, M.: Increased heterologous

RIB40

a

protein production in Aspergillus niger

liquid-surface immobilization (LSI) system.

fermentation through extracellular proteases

J. Biol. Macromol., 11, 23-30(2011)

inhibition by pelleted growth. Biotechnol.

ainProg., 16, 222-227(2000).

9. Oda, S., Isshiki, K., Ohashi, S.: Production of
6-pentyl-α-pyrone

withTrichoderma

16.

Aguilar,C.

N.,

Favela-Torres,atroviride and its mutant in a novel

Viniegra-González,extractive liquid–surface immobilization

repression patterns of fungal tannase in(Ext–LSI) system. Process Biochem., 44,

solid-state and submerged cultures. Process

625–630(2009).

Biochem., 36, 565-570(2001).

10. Oda, S., Araki, H., Ohashi, S.: Derepression

17.

Hölker,U.,G.:

Höfer,

Induction

E.,

M.,

Lenz,

advantages

and

J.:

of carbon catabolite repression in an

Biotechnologicalextractive liquid–surface immobilizationlaboratory-scale(Ext–LSI) system. J. Biosci. Bioeng., 113,

with fungi. Appl. Microbiol. Biotechnol., 64,

742-745(2012).

175-186(2004).

solid-stateoffermentation

10

Characteristics of fungal cells located on an interface

18. Ozeki, K., Takagi, Y., Oda, S., Ohashi, S.:
Production of xylanase with a trans-

6-n-pentyl-2H-pyran-2-one. J. Nat. Prod.,60, 1242–1244(1997).

formant of Aspergillus oryzae RIB40 in a

25. Serrano-Carreón, L., Flores, C., Rodríguez,liquid-surface immobilization (LSI) system.

B., Galindo, E.: Rhizoctonia solani, an

J. Biosci. Bioeng., 109, 224-226(2010).

elicitor of 6-pentyl-α-pyrone production by

19. Cutler, H. G., Cox, R. H., Crumley, F. G.,

Trichoderma harzianum in a two liquid

Cole,

P.

D.:

6-Pentyl-α-pyrone

fromphases, extractive fermentation system.

Trichoderma harzianum: its plant growth

Biotechnol. Lett., 26, 1403-1406(2004).

inhibitory and antimicrobial properties.

26. De Aráujo, Á. A., Pastore, G. M., Berger, R.

Agric. Biol. Chem., 50, 2943-2945(1986).

G.: Production of coconut aroma by fungi

20. Rito-Palomarces, M., Negrete, A., Miranda,

cultivation in solid–state fermentation. Appl.

L., Flores, C., Galindo, E., Serrano-

Biochem. Biotechnol., 99. 747-752(2002).

Carreon, L.: The potential application of

27. Hsu, H. -L., Yang, W. -C., Lee, Y. -L., Yew,

aqueous two-phase systems for in situ

T. -R.: Polyacrylonitrile as a gate dielectric

recoveryof

6-pentyl-infinity

material.

producedbyTrichoderma

EnzymeMicrob.

pyroneharzianum.

Technol.,

28,

Appl.

Phys.

Lett.,

91,

23501-23503(2007).
28. Oda, S., Michihata, S., Sakamoto, N.,
Horibe, H., Kono, A., Ohashi, S.: Enhance625-631(2001)
21. Rocha-Valadez, J. A., Estrada, M., Galindo,

ment of 6-pentyl-α-pyrone fermentation

shake

activity in an extractive liquid-surface

flasks to stirred fermentors: scale-up of animmobilization (Ext-LSI) system by mixing

extractiveforanion-exchange resin microparticles. J.

byBiosci. Bioeng., 114, 596–599(2012).

E.,

Serrano-Carreón,L.:

fermentation

6-pentyl-α-pyrone

From
process

production

Trichoderma harzianum using volumetric
power

input.

Process

Biochem.,

41,

29. Raymond, R. L., Jamison, V. W., Hudson, J.
O.: Microbial hydrocarbon co-oxidation. II.
Use

1347-1352(2006).
22. Kalyani, A., Prapulla, S. G., Karanth, N. G.:ofion-exchange

resins.

Appl.

Microbiol., 17, 512-515(1969).

Study on the production of 6-pentyl-α-

30. Hattori, R., Hattori, T., Furusaka, C.:pyrone using two methods of fermentation.

Growth of bacteria on the surface of anion-Appl.

exchange resin. I. Experiment with batch

Microbiol.

Biotechnol.,53,

culture. J. Gen. Appl. Microbiol., 18,

610-612(2000).
23. Cooney, J. M., Lauren, D. R., Jensen, D. J.,

271-283(1972).

Perry-Meyer, L. J.: Effect of harvest time,

31. Hattori, R.: Growth and spore formation oftemperature, light, and spore inoculum

Bacillus subtilis adsorbed on an anion-

concentrationexchange resin. J. Gen. Appl. Microbiol., 22,on

6-n-pentyl-2H-pyran-

2-one production by Trichoderma spp. J.

215-226(1976).

Agric. Food Chem., 45, 2802-2806(1997).

32. Hattori, R.: Growth of Escherichia coli on

24. Cooney, J. M., Lauren, D. R., Poole, P. R.,

the surface of an anion-exchange resinin

Whitaker, G.: Microbial transformation ofcontinuous flow system. J. Gen. Appl.

theMicrobiol., 18, 319-330(1972).

Trichoderma

metabolite

S. Oda11

33. Oda, S., Wakui, H., and Ohashi, S.:

40. Gottenbos, B., van der Mei, H. C., Busscher,

Efficient hydrolytic reaction of an acetate

H. J.: Initial adhesion and surface growth ofester with fungal lipase in a liquid–liquid

Staphylococcus

interface bioreactor (L–L IBR) using

Pseudomonas aeruginosa on biomedical

CaCO3-coated ballooned microsphere. J.

polymers. J. Biomed. Mater. Res., 50,

Biosci. Bioeng., 112, 151-153(2011).

208-214(2000).

34. Oda, S., Isshiki, K., Ohashi, S.: Regio- andstereoselective
lationsofsubterminal

n-decanebyininterface

Bull.

Chem.

Soc.

Jpn.,

82,

on

metabolicactivityofEscherichia coli. J. Gen. Appl. Microbiol.,a29, 195-204(1983).

liquid–liquid interface bioreactor (L–L
IBR).

and

41. Morisaki, H.: Effect of solid–liquidhydroxy-

fungi

spidermidis

42.

Shaw,

B.

D.,

Hoch,

H.

C.:

The

pycnidiospore of Phyllosticta ampelicida:105-109(2009).
35. Oda, S., Sakamoto, N., Horibe, H., Kono,

surface properties involved in substratum

A., Ohashi, S.: Enhancement of n-decane

attachment and germination. Mycol. Res.,

hydroxylation activity of Monilliera sp. NAP
00702

in

a

liquid–liquidinterfacebioreactor by mixing anion-exchange resin
microparticles.

Process

Biochem.,

47,

103, 915-924(1999).
43. Clement, J. A., Martin, S. G., Porter, R.,
Butt, T. M., Beckett, A.: Germination andthe role of extracellular matrix in adhesion ofurediniospores of Uromyces viciae- fabae

2494-2499(2012).
36. Oda, S., Sakamoto, N., Horibe, H., Kono,
A., Ohashi, S.: Relationship between

to synthetic surfaces. Mycol. Res., 97,
585-593(1993).

and44. Morisaki, H.: Effect of a liquid–liquidhydroxylation activity of fungal cells

interface on the metabolic activity oflocated on an organic–aqueous interface. J.

Escherichia coli. J. Gen. Appl. Microbiol.,

Biosci. Bioeng., 115, 544-546(2013).

30, 35-42(1984).

interfacialhydrophobicity

37. Klein, D. A., Henning, F. A.: Role of

45. Vergara-Fernández, A., van Haaren, B.,

alcoholic intermediates in formation of

Revah, S.: Phase partition of gaseous

isomeric ketones from n-hexadecane by ahexane and surface hydrophobicity ofsoil Arthrobacter. Appl. Microbiol., 17,

Fusarium solani when grown in liquid and676-681(1969).

solid media with hexanol and hexane.

38. Farinas, E. T., Schwaneberg, U., Glieder, A.,

Biotechnol. Lett., 28, 2011-2017(2006).

Arnold, F. H.: Directed evolution of acytochrome

P450

monooxygenaseforalkane oxidation. Adv. Synth. Catal., 343,
601-606(2001).
39. Peters, M. W., Meinhold, P., Glieder, A.,
Arnold, F. H.: Regio- and enantio- selective
alkanehydroxylation

withengineered

cytochrome P450 BM-3. J. Am. Chem. Soc.,
125, 13442-13450(2003).

Communicated by Ueno Hiroshi