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A Unique Pattern of Mycelial Elongation ofBlakeslea trisporaand ItsEffect on Morphological Characteristics andb-Carotene Synthesis

Jae-Cheol Jeong, Jeewon Lee, Young-Hoon Park

Microbial and BioProcess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115,Yusong, Taejon 305-600, South Korea

Received: 6 July 2000 / Accepted: 21 August 2000

Abstract. The mycelial morphology ofBlakeslea trisporawas of crucial importance in the productionof b-carotene in submerged cultures ofB. trispora. After the spores were inoculated, the time-coursevariation of mycelial morphology was closely examined under the microscope. With the addition of thenon-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern ofmycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance ofmycelia with very short length, which allowed a well-dispersed growth ofB. trisporawithout significantpellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungalculture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But theshort mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition,because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation.The 20-fold increase in specific yield ofb-carotene (mgb-carotene produced per g mycelium) wasachieved with this drastic change in the pattern of mycelial elongation. The reason for this result mightbe more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in thewell-dispersed mycelial cultures ofB. trispora.

Filamentous fungi have been extensively used in thecommercial production of a wide range of secondarymetabolites, and their morphological type (i.e., pelletaggregates, rough pellets, smooth pellets, mycelialflocks, and so on) is closely related to metabolite pro-duction [1]. Although the various methodologies forpellet formation have been studied with many molds, itremains very difficult to elucidate a mechanism ofmorphological type change during the mycelial growthbecause many intrinsic and external parameters are in-terrelated with high complexity. Therefore, most exper-imental efforts have been made to optimize various ex-ternal factors at the macroscopic scale. As a result, thefactors such as bioflocculant, medium composition andviscosity, various additives (e.g., polymers, surfactants,and chelators), shear forces, temperature, pressure, etc.have been reported to be effective in changing mycelial

morphology type [1, 2, 10, 11]. However, several impor-tant results of microscopic study on mycelial morphol-ogy have been recently reported. Spohr et al. [7, 8]reported an increase ofa-amylase production from amore densely branched mutant ofAspergillus oryzaeanddesigned a flow-through cell to measure the growth ki-netics of hyphae at the microscopic level. Digital imageanalysis has also been developed and used for morpho-logical characterization and biomass estimation of fila-mentous microorganisms [9].

In the present work, the mycelial morphology ofB.trispora, a potentb-carotene producer, was examined indetail, focusing on the elongation patterns of the myce-lium. b-Carotene is a secondary metabolite, widely usedin the preparation of feed-additives, food products, andcosmetics. It has an antioxidant activity and serves as aprecursor for biosynthesis of vitamin A as well. Re-cently, it has been reported that the nonionic surfactantSpan 20 plays a significant role in enhancingb-caroteneCorrespondence to:J. Lee;email: jwlee@mail.kribb.re.kr

CURRENT MICROBIOLOGY Vol. 42 (2001), pp. 225–228DOI: 10.1007/s002840010208 Current

MicrobiologyAn International Journal© Springer-Verlag New York Inc. 2001

production, which was merely presumed owing to itseffect on either cell wall permeability or metabolic in-termediate (trisporic acid) synthesis [6]. Through thevarious photomicrographic analyses of mycelial growthof B. trispora, this study found that the action of Span 20is directly related both to the elongation pattern of my-celium, leading to a significant morphological change,and to the production of the secondary metabolite,b-car-otene.

Materials and Methods

Fungal strains and culture media.(1) and (2) mating-type strains ofBlakeslea trispora(ATCC 14271 and 14272, respectively) were usedfor the various flask-culture experiments. From the spores of eachB.trispora strain on potato-dextrose agar plates incubated for 2–3 days,5 3 106 and 13 107 spores of (1) and (2) type strain respectivelywere collected and inoculated together into 50-ml GAY medium in250-ml Erlenmeyer flasks. The flask cultures were incubated at 180rpm at 27°C. The GAY medium contained, per liter: 70 g glucose(Sigma), 2 gL-asparagine (Sigma), 1 g yeast extract (Difco), 1.5 gKH2PO4, 0.5 g MgSO4 z 7H2O, and 5 mg thiaminz HCl (Sigma). Theinitial culture pH was adjusted at 5.5, and Span 20 (Sigma) was addedat 1% (vol/vol) where applicable.

Photomicrographic analysis of mycelial morphology.The morpho-

logical characteristics of mycelial cultures grown in various flasks wereexamined at the microscopic level under the microscope (ModelECLIPSE E600, Nikon Co., Tokyo, Japan) equipped with a digitalcamera (Model C-2000 ZOOM, Olympus America Inc., Melville, NY,USA). From the photo files generated by the digital camera, themycelial morphology was visualized by Microsoft Powerpoint97.

Estimation of dry mycelial mass andb-carotene concentrations.Ex-periments for the estimation of dry mycelial mass and synthesizedb-car-otene concentrations were made in triplicate, and the estimations weredone by following the procedure described by Jeong et al. [4]. Themycelial cultures were filtered through muslin in a Buchner funnel, washedthoroughly with distilled water, and the filtered mycelium was lyophilizedat 270°C for estimating the concentration of the dry mycelial mass. Thesynthesizedb-carotene was solvent-extracted from the freeze-dried myce-lium by using a solvent mixture of hexane (Merck, Darmstadt, Germany)and methanol (J.T. Baker, Phillipsburg, NJ, USA) (1:1, vol/vol). Theb-carotene concentration was measured by using a reversed-phase HPLC(Z-module C18 column, Waters Model 486) at 450 nm.

Results and Discussion

From the standpoint of mass transfer efficiency, themycelial morphology leading to dispersed fungal growthis greatly advantageous for the production of target me-tabolites with high yield. The various factors such as

Fig. 1. Photomicrographs showing mycelial elongation ofBlakeslea trisporagrown for 12 h in GAY medium either supplemented with Span 20(1% vol/vol) (A) or without any non-ionic surfactant (B). (Both 340 and3100 resolutions were used and compared. Magnification bars shown at340 and3100 resolution were 1 mm and 400mm, respectively.)

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electrostatic forces, steric interaction forces, excretion ofmaterial between cells, and hydrodynamic forces (shearstress) have been shown to favor the dispersion by pre-venting mycelial aggregation and pellet formation [1].For example, Kosakai et al. [5] used mineral support toincrease an electrostatic repulsion between the myceliaof Rhizopus oryzaeand achieved dispersed growth aswell as enhanced production of L(1)-lactic acid. In thisstudy, it was found that dispersed growth could beachieved by changing an intrinsic property of mycelialelongation.

Photomicrographic illustrations of the mycelial mor-phology of B. trispora. After the spores were inoculatedinto the growth medium (50 ml), the morphologicalchange was closely monitored under the microscopeduring the course of spore germination and mycelialgrowth. As is evident from Fig. 1, the grown myceliawere already heavily tangled 10 h after the inoculum wasadded to the surfactant-free medium. However, myce-lium elongation was significantly restricted in the pres-ence of the non-ionic surfactant (Span 20), and the my-celium looks much shorter compared with that observedin the surfactant-free cultures (Fig. 1). After 2-day cul-tivation, the surfactant-free cultures produced a bigclump of tangled mycelium irrespective of nutrient glu-cose concentrations (Fig. 2B and 2C), whereas well-dispersed growth occurred in the flask cultures contain-ing Span 20 (Fig. 2A).

The addition of the non-ionic surfactant (Span 20)drastically changed the pattern of mycelial elongation:the mycelial length was markedly reduced with muchless branching. This intrinsic change of mycelial elonga-tion was favorable to dispersed growth. Since the signif-icant tangle between long mycelia also occurred in theglucose-deficient medium with severe growth inhibition(Fig. 3A and 2C), the reduced mycelial length by Span20 seems to have nothing to do with the reduced growthrate at the early stages of growth (Fig. 3A). The mech-anism of Span 20’s action on mycelial elongation iscurrently not clear, even though its influence on cell wallpermeability has already been reported [6]. As reportedby Jacques et al. [3], sometimes the fatty acid moiety ofthe detergent was incorporated into the lipids of thecellular membranes when various Tween detergentswere added to batch cultures ofStreptococcus salivarius.But in this case, it is too early to give a plausible reasonfor the morphological changes of microorganisms bySpan 20. A common effect of a surfactant (as a paramor-phogen) on the cellular membrane is to increase itsleakiness, and this would result in a perceived stress tothe fungus. Therefore, the change in cell wall morphol-

ogy might be a response to this stress following theincreased leakiness by Span 20.

Effect of morphological characteristics on fungalgrowth and b-carotene production. In the surfactant-containing cultures, the estimation of dry myceliumweight also showed a significant lag period (8 h) in thebeginning of fungal growth, but the much higher biomass(dry mycelium) concentration was finally achieved after3-day cultivation (Fig. 3A). With the dispersed myce-lium growth, the production ofb-carotene was highlyenhanced, and the amount ofb-carotene produced pergram mycelium increased 20-fold (Fig. 3B). Without

Fig. 2. Macroscopic views of growth morphology in flask cultures ofBlakeslea trisporagrown for 48 h in three different media: (A) GAYmedium supplemented with Span 20 (1% vol/vol); (B) GAY medium;and (C) glucose-deficient GAY medium.

J.-C. Jeong et al.: Mycelial Elongation ofBlakeslea trispora 227

glucose in the culture medium, both the mycelial growthand b-carotene production were significantly inhibited(Fig. 3).

Since it is generally accepted that dispersed growthleads to increases in overall mass transfer efficiency andhence reduced nutrient gradients, it is not surprising thatthe highest biomass concentration ofB. trispora wasobtained in dispersed growth. The secondary metabolismfor b-carotene (a strong anti-oxidant) synthesis would bemore stimulated by environmental oxidative stress, as

previously reported [4]. Therefore, the uniform and ef-ficient transfer of dissolved oxygen and hence the moreintensive oxidative stress to the mycelium might havedrastically increased the synthesis level ofb-carotene perunit mycelial mass. The membrane stress caused by asurfactant, mentioned above, can also directly interferewith electron transfer and hence oxygen radical defenseas well as with growth, and thus Span 20 might affect theintracellular generation of oxygen radicals or defensesagainst them. Consequently, the oxidative stress could beof both external and internal origin.

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8. Spohr A, Dam-Mikkelsen C, Carsen M, Nielsen J, Villadsen J(1998) On-line study of fungal morphology during submergedgrowth in a small flow-through cell. Biotechnol Bioeng 58:541–553

9. Treskatis S-K, Orgeldinger V, Wolf H, Gelles ED (1997) Morpho-logical characterization of filamentous microorganisms in sub-merged cultures by on-line digital image analysis and patternrecognition. Biotechnol Bioeng 53:191–201

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Fig. 3. Time-course variations of (A) dry mycelial mass (g/1-L culture)of Blakeslea trisporaand (B) specificb-carotene yield (mg per g drymycelial mass in the flask cultures.B. trisporawere cultivated in threedifferent media: GAY medium supplemented with Span 20 (1% vol/vol) (F); GAY medium (E); and glucose-deficient GAY medium (�).

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