Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

Effect of Deletion of Chitin SynthaseGenes on Mycelial Morphology andCulture Viscosity in Aspergillus oryzae

Christian Muller,1 Kim Hansen,2 Peter Szabo,3 Jens Nielsen1

1Center for Process Biotechnology, BioCentrum-DTU, Technical Universityof Denmark, 2800 Kgs. Lyngby, Denmark; telephone: +45-45252696;fax: +45-45884148; e-mail: [email protected] A/S, 2880 Bagsværd, Denmark3Danish Polymer Center, Department of Chemical Engineering, TechnicalUniversity of Denmark, 2800 Kgs. Lyngby, Denmark

Received 21 May 2002; accepted 15 July 2002

DOI: 10.1002/bit.10491

Abstract: The objective of this study was to quantify theeffect of disrupting two chitin synthases, chsB and csmA,on the morphology and rheology during batch cultiva-tion of Aspergillus oryzae. The rheological propertieswere characterized in batch cultivations at different bio-mass concentrations (from 3.4–22.5 g kg−1 biomass) andthe power-law model adequately described the rheolog-ical properties. In the cultivations there were pellets,clumps, and freely dispersed hyphal elements. The dif-ferent morphological fractions were quantified using im-age analysis. The apparent viscosity of the fermentationbroth was significantly affected by the biomass concen-tration, the morphology, and also by pH. The chsB dis-ruption strain had lower consistency index K values forall biomass concentrations investigated, which is a de-sirable trait for industrial Aspergillus fermentations.© 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 525–534,2003.Keywords: Aspergillus; morphology; rheology; chitin;image analysis

INTRODUCTION

In nature, filamentous fungi are typically saprophytic or-ganisms that secrete large quantities of enzymes in order todegrade organic matter. Multinucleate filamentous fungi,e.g., Aspergilli, consist of hyphal, cylindrical cells that in-crease in length by extending at their apices. The hyphaeform branches, making them able to invade and colonizeorganic matter. Aspergilli are extensively used in industryfor enzyme production because high expression levels arepossible and because these fungi have a very effective se-cretion pathway. The enzymes are produced by cultivationof the fungi in large bioreactors where the fungus growsfilamentously; i.e., as freely dispersed structures called hy-phal elements (Nielsen, 1996). During the course of a cul-tivation the hyphal elements consist of a population of dif-

ferentiated cells ranging from growing, producing cells toinactive, vacuolated, nonproducing cells. The morphologyof the hyphal elements greatly affects the outcome of thecultivation process in two ways. First, on the microscopiclevel there are indications of a correlation between mor-phology and secretion of proteins (Carlsen et al., 1994;Spohr et al., 1997; Bocking et al., 1999); however, the ex-istence of such correlation is debated (reviewed in Gibbs etal., 2000). Second, the formation of long branched hyphalelements affects the fermentation broth rheology becausethey tend to interact or entwine, forming clumps or pellets(Thomas and Paul, 1996). In the case where large clumpsare formed the viscosity of the fermentation broth is in-creased, resulting in reduced oxygen mass transfer, an in-creased fraction of oxygen-limited cells, and resulting in adecreased productivity and/or production of undesirablemetabolites. In addition to oxygen limitation, highly viscousfermentation broths can lead to poor mixing, resulting inproduct toxicity and nutritional concentration gradients(Bryant, 1977), which have the potential to alter the myce-lial morphology. Although filamentous fungi have beenused for enzyme production for many years, the high cultureviscosity is still a major unresolved problem (Metz et al.,1979; Olsvik and Kristiansen, 1994) and it makes a fila-mentous fungal cultivation more difficult to aerate thansingle-cell cultivation with the same biomass concentration.

As the fungi grow, the increased biomass concentrationresults in a viscous fermentation broth that behaves in anon-Newtonian manner (i.e., the ratio between the shearstress and the shear rate is not constant), as observed byPedersen et al. (1993) and Goudar et al. (1999). Non-Newtonian behavior leads to relatively low viscosities inregions of high shear rate (near the impeller) and higherviscosities in regions with low shear rates (near the wall ofthe bioreactor) (Metz et al., 1979), complicating the mattereven further. Increasing the agitation rate, and thereby dis-persing the hyphal elements, may solve the high viscosity

Correspondence to: Jens NielsenContract grant sponsor: Novozymes A/S, Bagsværd, Denmark

© 2003 Wiley Periodicals, Inc.

problem, but as noted by Smith et al. (1990) and Lejeuneand Baron (1995) care must be taken not to negatively affectspecific growth rates and productivity by fragmenting thehyphae. Therefore, it would be beneficial if one could de-vise a way of optimizing the hyphal morphology for large-scale enzyme production.

During the last decade there has been a focus on howstrains can be genetically engineered that are not just betterproducers, but also better adapted for growth in submergedcultivation. In filamentous fungi, random mutagenesis hasyielded many strains with interesting morphological pheno-types (reviewed by McIntyre et al., 2001). However, rarelyhas the gene been identified and only in a few cases has thegene been cloned, the phenotype methodically character-ized, and the gene product function found. Furthermore,only in a very few cases has the organism been studied insubmerged cultivation and the effect of the morphologicalalteration on productivity examined (reviewed by McIntyreet al., 2001). As far as we know, no studies have beenpublished where the rheological effect of disrupting specificmorphological genes has been examined during submergedcultivation.

The present study reports on the effect of deleting twochitin synthase genes from Aspergillus oryzae, designatedchsB and csmA, on branching and culture viscosity duringbatch cultivation. Chitin synthases (EC 2.4.1.16) are a fam-ily of enzymes which catalyze the polymerization of N-acetylglucosamine residues linked by �(1→4) glycosidicbonds. The product is chitin, which in fungi is aggregatedinto microfibrils (Rudall, 1969), forming a major structuralcomponent of the fungal wall. Aspects of the chitin metabo-lism such as synthesis, degradation, and cross-linking toother cell wall components are thought to be very importantfor maintaining the shape and physical strength of fungi(reviewed in Bulawa, 1993). In A. nidulans, chsB was foundto be essential for normal hyphal growth (Borgia et al.,1996), while lack of the csmA (chitin synthase with a myo-sin tail) gene product caused morphological abnormalitiessuch as ballooning and irregular septum formation (Horiu-chi et al., 1999). This suggests that controlling the expres-sion of chitin synthases might be used in controlling thefungal morphology.

MATERIALS AND METHODS

Strains

The A. oryzae A1560 (originally named IFO4177, Institutefor Fermentation, Osaka, Japan), HowB101 (a pyrG dele-tion strain originating from A1560), and CM100 (chsB::pyrG) strains were donated by Novozymes A/S (Bagsværd,Denmark). The strains disrupted in chsB (CM100) and incsmA (CM101) were constructed from HowB101. TheCM100 strain was constructed by transformation ofHowB101 with a plasmid containing 1.9 kb of the chsB witha 2.9 kb fragment containing pyrG inserted into

a HindIII site located approx. 0.7 kb within the chsB frag-ment. The CM101 strain was constructed by transformationwith a plasmid containing a 3.6 kb csmA fragment with a 2.1kb fragment containing pyrG inserted into a NotI site be-tween the two essential catalytic domains in the csmA. Thetransformants were selected as morphological mutants andthe gene disruption were checked by Southern and Northernanalysis. For further details on construction of the strainsCM100 and CM101 and characterization of the strains andgenes, see Muller et al. (2002b).

Bioreactors and Media

Batch cultivations were done in 5-L in-house bioreactorswith a working volume of 4.5 L. During cultivation the pH,temperature, agitation, and aeration were controlled and off-gas was monitored. The medium composition was KH2PO4

(1.5 g L−1), MgSO4, 7 H2O (1.0 g L−1), NaCl (1.0 g L−1),CaCl2, 2 H2O (0.10 g L−1), (NH4)2SO4 (7.3 g L−1), glucose,1 H2O (22.5 g L−1), trace metals (0.5 ml L−1). The tracemetal solution was ZnSO4, 7 H2O (14.3 g L−1), CuSO4, 5H2O (2.5 g L−1), NiCl, 6 H2O (0.5 g L−1), FeSO4, 7 H2O(13.8 g L−1). This medium was the standard defined mediumas described by Carlsen (1996a). For the high biomass cul-tivations (at “production pH conditions” and at “constantpH conditions”) the concentration of all components (exceptantifoam) was three times higher in order to obtain a highbiomass. pH in all cultivations was controlled by addition ofeither 2 M NaOH or 2 M HCl. Biomass measurements weremade by measuring dry matter per kg medium by dryingfiltered (Whatman GF/C filter paper, pore size 70 �m,Maidstone UK) biomass samples for 24–48 h in an oven at100°C, to constant weight.

Cultivations

Freeze-dried spores were used to inoculate rice cultures us-ing the method of Carlsen et al. (1996a) and 6–8 days afterinoculation the rice grains were covered with green or whitespores, depending on the strain. The spores were harvestedby washing with 0.1% Tween solution and were used as aninoculum for submerged cultivation in a final concentrationof 2–6*109 spores L−1. The aeration rate was 1 vol. air (vol.culture)−1 min−1. Temperature was controlled at 30°Cthroughout. The pH for inoculation was 3.5, which, during“production pH conditions,” was increased slowly to 6.0when the biomass concentration was approx. 1 g kg−1. ThepH was increased to 6.0, because �-amylase is stable at pH6.0. At “constant pH conditions” the pH was started at 3.5and then kept at 4.5 throughout the cultivation. Stirrer speedin all processes was initially 100 rpm during germination.When the biomass concentration was approx. 1 g kg−1 theaeration was set to 500 rpm. The agitation was then in-creased as the fermentations progressed to 800–900 rpm.

�-Amylase Activity

Filtered fermentation broth was diluted 10 times with 0.1 gL−1 BSA and 0.1 g L−1 CaCl2 and frozen after sampling. The

526 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 81, NO. 5, MARCH 5, 2003

mixture was then thawed and the extracellular �-amy-lase activity was determined by a modified BoehringerMannheim method using a Cobas Mira (Roche Diagnostics,Nutley, NJ) as previously described (Carlsen and Nielsen,2001). This method was based on cleavage of the artificialsubstrate 4,6-ethylidene (G7)-p-nitrophenyl (G1)-�-D-maltoheptaoside by �-amylase and glucoamylase (added inthe kit), which results in release of p-nitrophenol. The unitof �-amylase activity is FAU ml−1 (one FAU is the amountof �-amylase which at 37°C hydrolyses 5.26 g of starchper h).

Morphological Sample Preparation

Eight ml fermentation broth was taken from the batch cul-tivation and two drops of Lactophenol cotton blue added(Fluka Chemie, 61335, Buchs, Switzerland) and the samplewas stored at 4°C. For measurements, the sample was di-luted with water to a biomass concentration of approx. 0.4g kg−1 and mixed with one drop of commercial detergent inorder to disperse the clumps and hyphal elements and lowerthe water surface tension. Samples were dispensed as dropsdirectly on a microscope slide (no cover glass) and mea-sured using a ×4 objective and a magnification on-screen of220 times. Samples were dispensed with pipette tips withcut-off ends in order to eliminate preferential mycelial sizeselection during dilution and slide preparation.

Image Analysis

Morphological characterization of the cultures was carriedout using a Quantimet 5501W Image Analyser (Leica, Cam-bridge, UK) connected to a Nikon (Nikon Optiphot 2, NikonCorp., Japan) fluorescence microscope via a Kappa CF 8/1FMC (Kappa Messtechnik GmbH, Germany) monochromevideo camera. Images were obtained, enhanced, and mea-sured using a Quantimet 5501W Image Analysis softwareroutine. The program measured the (projected) area, convexarea, fullness ratio, and number of branches (manually) ofthe dispersed hyphal elements and clumps in each sample.For each sample all objects above 200 �m2 were measureduntil more than 100 hyphal elements or clumps were mea-sured. The fraction of objects below 200 �m2 was insig-nificant at all time points. All measurements were made inpixels and then converted to �m by a calibration using aNikon Objective Micrometer. The projected area of a hy-phal element is the number of pixels multiplied by thesquare of the calibration constant for that magnification.The hyphal growth unit area (AHGU) (�m2 tip−1) was mea-sured for the freely dispersed hyphal elements. It was foundthat measuring the projected area instead of the hyphallength was more accurate since it eliminated the errorsource that lies in skeletonizing the images when, e.g., thehyphae were swollen. Swollen hyphal cell walls and swol-len conidia occurred frequently in the CM101 strain in thestart of the cultivation and this caused the average diameterto change during the cultivation. This made measurement of

AHGU more attractive than the length of the hyphal growthunit (lHGU) (Cadwell and Trinci, 1973; Trinci, 1974) sincethese hyphal defects could be included in the measurement.

The clumps/pellet compactness was measured on allclumps/pellets and it was estimated as the ratio of the pro-jected area of the clump and the projected convex area ofthat clump. The convex area of an object is its area afterfilling in any internal voids and any concavities in its ex-ternal perimeter.

Rheological Measurements

Viscosity measurements were carried out in a Bohlin CSRheometer (Bohlin Rheologi, AB, Sweden) connected to aPC and equipped with a C25 concentric cylinder geometry(with a bob diameter of 25 mm and a cup diameter of 27.5mm). Twenty-five ml fermentation broth samples were pe-riodically withdrawn from the batch cultivations and usedfor rheological analysis. All measurements were made di-rectly after a sample had been taken since the properties andstructure of the fermentation broth might deteriorate duringstorage. During measurement the sample temperature waskept at 22°C. The measurements were carried out using the“yield stress” option of the rheometer in which the shearstress was first increased with time and then decreasedwhile the shear rate and strain was measured and the vis-cosity was calculated. The total sweep time (up/down) was120 sec in all experiments. For one sample three measure-ments were made (each with a new sample) in order tominimize scatter.

RESULTS

Table I compiles the measured specific growth rate, the�-amylase yield on biomass and the measured AHGU (pro-jected hyphal area per tip) for the three strains investigated.Compared to the wild-type strain, A1560, the two disruptionstrains CM100 and CM101 had a lower specific growth rate,but the �-amylase titer and the yield of �-amylase on bio-mass under production conditions were approximately thesame. For the two disruption strains the specific growthrates were higher when cultivated at pH 4.5 than at 6.0.AHGU was similar for each strain at the different pHconditions but when comparing the three strains theCM100 strain was hyperbranched (it had more branches perprojected hyphal area than the CM101 and the A1560strains).

Production pH Conditions

The strains were examined at “production pH conditions”(at 3× the standard medium concentration used by Carlsenet al. [1996a]) in order to investigate the rheology and themorphology at high biomass concentrations. Figure 1 showsthe dispersed hyphal elements from these cultivations andillustrates the tendency of the A1560 strain to form largeclumps and the CM100 strain to hyperbranch. The CM101

MULLER ET AL.: CHITIN SYNTHESIS, MORPHOLOGY, AND VISCOSITY 527

strain formed small compact elements and branched simi-larly to the A1560 strain. Figure 2 shows the (projectedarea) size distribution results of all hyphal elements dividedafter their size. Each distribution is an average value of 2–3samples in the time interval indicated. The x-axis legendsindicate the time that the first and the last sample weretaken. Plotted on the y-axis is the fraction of the total bio-mass (measured as projected area). For each single samplemore than 100 elements were measured. The 0–37,001 �m2

size intervals consist of all small hyphal elements where thenumber of branches could be counted (from these the AHGU

was calculated) and all small clumps where the number ofbranches could not be counted. Then follows clumps withsizes from 37,001–74,000 �m2 and over 74,001 �m2.

As Figure 2a for the A1560 strain indicates, the biomass

was initially present mainly as dispersed hyphal elements ofsmaller sizes. However, after 39.9–42.0 h of cultivation ofthe A1560 strain (biomass concentration of 6.1–8.3 g kg−1)and after 32.1–38.6 h of cultivation of the CM101 strain(biomass concentration of 5.7–8.6 g kg−1) (Fig. 2c), thefungal biomass was mainly present as large, inseparableclumps/pellets (>37,000 �m2). In the case of the A1560strain more than 50% of the biomass consisted of large,inseparable clumps/pellets. Later in these two cultivationsthe fraction of clumps decreased and the fraction containingsmaller clumps increased. In contrast to this, the cultivationwith the CM100 strain primarily contained smaller clumpsthroughout the cultivation.

The structure of the biomass was further investigated bymeasuring the AHGU and the clump/pellet compactness as a

Table I. Summary of overall strain characteristics during the six batch cultivations at high biomassconcentration.

A. oryzae strain

A1560 CM100 CM101

Wild-type �chsB �csmA

Production �max h−1 0.23 0.16 0.16pH �-amylase FAU ml−1 1.1 1.2 1.3conditions Ypx FAU (g DW)−1 0.06 0.06 0.06

AHGU �m2 tip−1 360 ± 25 190 ± 12 378 ± 25

Constant �max h−1 0.23 0.18 0.22pH AHGU �m2 tip−1 323 ± 17 195 ± 7 365 ± 20

AHGU is given as projected mycelial area pr. tip (�m2 tip−1). Mean AHGU are measured ondispersed mycelia approx. 30 h after inoculation.

Figure 1. Images of dispersed fungal hyphae taken at a biomass concentration of 10 g kg-1 from cultivation at production pH conditions. Scale bar �

50 �m.


function of cultivation time. Figure 3a shows that the AHGU

decreased during cultivation of the strains A1560 andCM101, indicating that the number of branches per pro-jected area increased. The cultivation of the CM100 strain

had a constant AHGU. In Figure 3b it is observed that theclump compactness of all strains remain relatively constantduring the cultivations. However, the compactness ofCM100 was higher than that of the A1560 and the CM101strains, indicating that hyperbranched morphology leads tomore compact clumps.

Constant pH Conditions

The strains were examined at “constant pH conditions” (atpH 4.5) and at these conditions the (projected area) sizedistribution of the biomass were as in Figure 4. Throughoutthe cultivations of the strains A1560 and CM101 large parts(approximately 50%) of the biomass were present as large,inseparable pellets/clumps (>37,000 �m2). However, dur-ing the cultivations with these two strains there was nodecrease in this fraction of the biomass, signifying that frag-mentation was not as pronounced at “constant pH condi-tions” as at “production pH conditions.” The biomassthroughout the cultivation with the CM100 strain waspresent predominantly as smaller clumps, as was also thecase for this strain at “production pH conditions.” In Figure5 the mean AHGU and the mean clump/pellet compactness ofthe strains are shown as functions of the cultivation time.

Figure 3. “Production pH conditions” (a) mean AHGU and (b) meanclump compactness as a function of time in the three cultivations: �

A1560, � CM100, � CM101. Error bars show the 95% confidence inter-val.

Figure 2. “Production pH conditions” size (projected area) distributionof the biomass during batch cultivation as function of time interval. A1560(a), CM100 (b), CM101 (c). Size indicators are in projected area (�m2).The biomass concentration increases during the measurements from 1.5–19.0 g kg−1 in (a), 2.7–18.0 g kg−1 in (b), and 5.7–22.6 g kg−1 in (c).


The AHGU was quite constant for the A1560 and the CM101strain, in contrast to the result at “production pH condi-tions,” perhaps because there were no large changes in thebiomass size distributions at “constant pH conditions.” The

clump compactness (Fig. 5b) for the strains also remainedstable for the strains; however, it is not understood why theCM100 strain morphology was less compact and theCM101 strain morphology was less “loose” than at “pro-duction pH conditions.”

Rheological Results

The shear-rate dependent nature of the fermentation broth ofthe “constant pH conditions” cultivation with the strainA1560 is shown in Figure 6. The biomass concentrations of7.7, 16.2, and 23.1 g kg-1 correspond to cultivation times of27.6, 33.6, and 39.7 h, respectively. The viscosity of thefermentation broth was characterized over four decades ofshear rate, making it usable for rheological characterization.The typical shear rates encountered in stirred-tank reactorsare in the range of 20–180 s−1 (Petersen et al., 1993); how-ever, above a shear rate of approx. 12 s−1 a steady valuecould not be reached. Therefore, the measurement was car-ried out at lower shear rates by first increasing the shearstress while measuring shear rate and then decreasing it

Figure 4. “Constant pH conditions” size (projected area) distribution ofthe biomass during batch cultivation as a function of time interval. A1560(a), CM100 (b), CM101 (c). Size indicators have the unit �m2. The bio-mass concentration increases during the measurements from 4.4–23.1 gkg−1 in (a), 2.6–15.5 g kg−1 in (b), and 4.7–15.0 g kg−1 in (c).

Figure 5. “Constant pH conditions” (a) mean AHGU and (b) mean clumpcompactness as a function of time in the three cultivations: � A1560, �

CM100, � CM101. Error bars show the 95% confidence interval.


again. Figure 6 shows that an apparent yield stress thresholdwas observed in the start of the measurement series (whenthe slope is changing). At that shear rate a certain shearstress had to be applied before the fermentation broth wasmoved. As is seen in Figure 6, the yield stress thresholdgenerally increased as the biomass concentration increased,but with some deviation. Except for the yield stress, therheology of the samples could be described by the power-law model:

µ = K(�)n−1

where � (Pa s) is the apparent viscosity, � (s−1), the shearrate, K (Pasn) the consistency index, and n the dimension-less power-law index. The best fit (using linear regression)was made for each sample in order to estimate n and K. Thepower-law was used to describe the flow behavior becauseit gave a good fit to the curve and because most transportsystems for non-Newtonian liquids make use of this equa-tion. During the fit to the power-law the decreasing shearrate curve was used since this was most stable and becausethis curve was not affected by the initial yield stress. Asseen on the curves, the slope of the three plots is approxi-mately the same, signifying that the power-law index n isthe same for these biomass concentrations.

The power-law parameters for the cultivations at “pro-duction pH conditions” are shown on Figure 7. The power-law index n was between 0.05–0.38 for biomass concentra-tion from 3.9 g kg−1 to 22.5 g kg−1. There was no clearcorrelation between the power-law index n and the strain.

Figure 8 shows that at “constant pH conditions” therewere rheological differences between media with the threestrains. The power law index was between 0.2–0.03 for allmeasurements. There was no clear correlation between thepower-law index and the biomass concentration or betweenthe power-law index and the strain.

DISCUSSION

The development of the cultivations (Figs. 2, 4) show thatthe distribution between different morphological forms

(freely dispersed hyphal elements, clumps, and pellets) iscomplex and the distribution of clump sizes depends onboth growth and fragmentation. In several studies it hasbeen observed that the fraction of clumps changes duringthe cultivation (Smith et al., 1990; Riley et al., 2000; Li etal., 2000). Our data shows that the directed genetic changesin chitin synthesis in the strains CM100 and CM101 altersthe microscopic morphology of the strains compared to thewild-type (Table I and Muller et al., 2002a) and this altersthe clump formation processes so the clumps size and clumpcompactness is altered during cultivation.

Effect of pH

One of several factors that induce clumps/pellet formation(reviewed by Gibbs et al., 2000) is high pH (above 5.0). Thereason for this is not completely understood but high pHmay influence the interactions between hyphae and spores,

Figure 6. Apparent viscosity of the fermentation broth during cultivationof the A. oryzae A1560 strain at “constant pH conditions” at differentbiomass concentrations: � 7.7 g kg−1, � 16.2 g kg−1, � 23.1 g kg−1.

Figure 7. “Production pH conditions” power-law constants as a functionof biomass concentration. a: Power-law index n. b: Consistency index K.� A1560, � CM100, � CM101. The trendlines are power-law functionsfitted to the data.


causing these to be more prone to adhesion. That high pHincreases agglomeration has been observed for germlings(Gerin et al., 1993) and spores (Galbraith and Smith, 1969;Carlsen et al., 1996b). We investigated the culture morphol-ogy and viscosity at “production pH conditions” (increase inpH from 3.5 to 6.0) for the three strains. Because pH mightinfluence the culture morphology and rheology we also con-ducted experiments at “constant pH conditions” (pH 4.5) inorder to examine the direct influence of the chitin synthesisdisruption strains. In the present study at “production pHconditions,” pellet and clump formation or agglomerationappear to be induced in all three strains by the pH increase(or by high pH), as seen in the size distributions in Figure 2.A possible reason for this is that remaining spores adhere toexisting clumps and dispersed hyphal elements and formlarger clumps and pellets. When comparing with “constantpH conditions,” this effect was not as pronounced for theA1560 and the CM100 strains as seen in the size distribu-tions in Figure 4a,b, probably due to less adhesion of hy-phae and spores at constant pH. In contrast, in the CM101strain cultivation there were more clumps at “constant pHconditions” (Fig. 4c) than at “production pH conditions”(Fig. 2c). This could be due to that the CM101 strain frag-

mented more easily at “production pH conditions,” as alsoindicated by the low specific growth rate (Table I) becauseit may be sensitive to changes in pH or high pH (Table I).Further arguments for this are presented in the followingsection.

Fragmentation

The data shows that mycelial fragmentation is dependent onthe physiological state of the fungus, as also observed byPaul et al. (1994), Nielsen and Krabben (1995), and Mc-Intyre et al. (2000); at “production pH conditions” the AHGU

of the strains A1560 and CM101 decreased (Fig. 3a,c) dur-ing cultivation, indicating that the number of branches perprojected area increased. For these strains it was also foundthat the average projected area of the dispersed hyphal el-ements decreased during the cultivation by 52% and 50%,respectively (data now shown). Taken together with thedevelopment of the size distribution during the cultivations(Fig. 2), the decreasing AHGUs for strains A1560 andCM101 seems to be caused by breakup of clumps/pelletsinto fragmented hyphal elements and perhaps breakup ofhyphal elements. A fragmented hyphal element is smallerand has more “tips” than before it was fragmented, althoughsome of these “tips” may actually be fragmented hyphalends. This mechanism for fragmentation is comparable tothat described in fed-batch cultivation of A. oryzae by Cui etal. (1997) and Li et al. (2000); here, hyphal elements areformed when they are “shaved off” pellets/clumps and whenhyphal elements are fragmented. In contrast to the behaviorof the A1560 and the CM101 strains, the cultivation of theCM100 strain had a constant AHGU (Fig. 3a) and duringcultivation the average projected area of the dispersed hy-phal elements only decreased by 23% (data not shown).

At “constant pH conditions” for the A1560 and CM100strains there were no increase in the population of smallclumps and mycelia (<37,000 �m2) later in the cultivations(Fig. 4), signifying that fragmentation was not as pro-nounced at “constant pH conditions” as at “production pHconditions.” In support of this, the average size of the dis-persed hyphal elements decreased by 22% and 28% for theCM100 and A1560 strains, respectively (data not shown).For the CM101 strain “constant pH conditions” there was anincrease in the population of small clumps and mycelia. Inaddition, the average size of the dispersed hyphal elementsdecreased by 53% for the CM101 strain (data not shown).This indicates that in cultivations at “constant pH condi-tions” it was mainly in cultures with the CM101 strain thatthere was fragmentation of the hyphal elements, indicatingthat the CM101 strain is more vulnerable to agitation-induced fragmentation than the A1560 and CM100 strains.This effect could be due to a weak cell wall of the CM101strain, as previously found (Muller et al., 2002b); the strainfrequently had swollen conidial and hyphal cell walls, co-nidial scars, and it was hypersensitive to osmotic stress andthe chitin-binding reagent Calcofluor White.

Figure 8. “Constant pH conditions” power-law constants as a function ofbiomass concentration. a: Power-law index n. b: Consistency index K. �

A1560, � CM100, � CM101. The trendlines are power-law functionsfitted to the data.


Increased Branching

Bocking et al. (1999) reported in A. oryzae that as thebranching frequency increased the viscosity decreased, sug-gesting that highly branched fungal strains could reduceviscosity and hence increase the O2 supply. Metz (1976)found a relationship that indicates that cultures that containmoderately branched hyphal elements are more viscous thanthose that contain highly branched hyphae. Our findingsagree with Bocking et al. (1999) and Metz (1976) that in-creased branching leads to less hyphal interaction. Wefound that the consistency index was lowest in fermentationbroth containing the CM100 strain and this might be due tothe finding that this cultivation had smaller hyphal elementsand clumps than the cultivations with the two other strains.Due to increased branching, the CM100 strain did not reachlong hyphal lengths, resulting in fewer interactions withother hyphal elements.

However, the branching pattern of CM100 also affectedthe internal structure of the hyphal element. In all cultiva-tions the clump compactness was quite constant for thestrains, but at both pH conditions the clump compactness ofthe CM100 strain was consistently higher than that of thetwo other strains (Figs. 3b, 5b). However, this did not affectthe productivity or the specific growth rate.

Rheology: Yield Stress

In Figure 6 it is seen that an apparent yield stress thresholdwas observed at the start of the measurement series (whenthe slope is changing). A plausible explanation for the yieldstress threshold is that before measurement the fermentationbroth had a structure originating from hyphal interactions,which was degraded under increasing shear stress. A yieldstress threshold has also been observed by Roels et al.(1974) and Allen and Robinson (1990) for Penicilliumchrysogenum. In Figure 6 it is also seen that a yield stresswas not observed in the decreasing shear rate curve at theend of the experiment. We speculate that this is because the“hyphal interactions” structure was reformed with a slowerrate than the experiment time.

Rheology: The Power-Law Index n

As seen in Figure 7a, the power-law index n values at “pro-duction pH conditions” were between 0.05–0.38 for bio-mass concentrations from 3.9–22.5 g kg−1 and at “constantpH conditions” (Fig. 8a) they were between 0.03–0.18 forbiomass concentrations from 3.4–20.5 g kg−1. At both pHconditions there was no clear correlation between thepower-law index and the strain. The power-law index valuesfit reasonably well with results for dispersed hyphal ele-ments in P. chrysogenum: 0.20–0.25 by Roels et al. (1974)and 0.46–0.17 from 4–17.71 g l−1 biomass by Goudar et al.(1999). In contrast to these results and in agreement with theresults by Olsvik and Kristiansen (1992) and Pedersen et al.(1993), we did not find any clear correlation between bio-

mass concentration and the power-law index. The differentfindings for the behavior of the power-law index could bedue to the difference in strains and morphology and possiblyin the measuring method. The power-law index valuesfound at “constant pH conditions” (Fig. 8a) seems to beslightly lower than at “production pH conditions” (Fig. 7a)but the reason for this is unknown; all samples were run inthe same way and the fit to power-law using linear regres-sion was done at the same time for all samples. It could beexplained by variation in surface charge with pH (as ob-served by Gerin et al., 1993) and thereby with hyphal in-teractions.

Rheology: The Consistency Index K

The consistency index K increased with biomass concen-tration for all three strains at both pH conditions. The con-sistency index values at “production pH conditions” (Fig.7b) were between 0.3–55 Pa sn for biomass concentrationfrom 3.9 g kg−1 to 22.5 g kg−1 and at “constant pH condi-tions” (Fig. 8b) they were between 0.2 and 43 Pa sn forbiomass concentrations from 3.4–20.5 g kg−1. These valuesfit reasonably well with data for A. awamori (Wang andWebb, 1995), who found consistency index values from0–14 Pa sn for biomass concentrations from 3–22 g kg−1,and Goudar et al. (1999), who found consistency index val-ues from 0–6 Pa sn for biomass concentrations from 0–18 gkg−1 in P. chrysogenum.

The profiles of the consistency index vs. the biomassconcentrations for the different strains show that the con-sistency index values were generally lower for CM100 thanfor A1560 and CM101; however, with some scatter. Thissuggests that the fermentation broth containing the CM100strain had a lower viscosity. The reason might be that thehyphal elements of CM100 are much more branched (andhave shorter hyphae) and therefore do not form large hyphalclumps as easily as A1560 and CM101. When comparingthe CM100 strain cultivations at the two conditions it is seenthat at “constant pH conditions” where the clumps were thesmallest the consistency index was also smaller. This sug-gests that particularly low biomass concentration but alsosmall clumps result in a low consistency index value.

CONCLUSION

The aim of the study was to examine how disruption of twochitin synthase genes (chsB and csmA) affects the fungalmorphology and the rheological properties during batch cul-tivation of A. oryzae. The measured rheology is a result ofa variety of microscopic and macroscopic interactions of thebiomass in the fermentation broth and, therefore, care mustbe taken when correlating the rheological properties to par-ticular morphological variables. Fundamental problemswith developing better correlations between cultivation rhe-ology and biomass concentration are: 1) the influence of thediverse hyphal interactions on rheology remains empirical,and 2) fungal morphology develops during the cultivation in


processes that are difficult to control. Despite these prob-lems, this study demonstrates that single genes in the chitinsynthesis pathway can be used to alter the morphologicalpopulation and the rheological properties of the cultivation.In all three strains there was a relationship between theconsistency index K and the biomass concentration and aneffect of the morphological structure on the rheologicalproperties. This indicates that a more desirable morphologycan be obtained by making directed genetic changes in ei-ther the control or fabrication of the fungal cell wall.

ACKNOWLEDGMENT

The authors thank Mhairi McIntyre at the Center for ProcessBiotechnology for help with structuring the morphologicalmeasurements and data.

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Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae