Journal of Biotechnology 163 (2013) 112123

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Characterization and control of fungal morphology for improved production

performance in biotechnology
Rainer Krull a, , Thomas Wucherpfennig a , Manely Eslahpazir Esfandabadi a , Robert Walisko a ,
Guido Melzer a , Dietmar C. Hempel a , Ingo Kampen b , Arno Kwade b , Christoph Wittmann a
a
Institute of Biochemical Engineering, Technische Universitt Braunschweig, Germany
b
Institute for Particle Technology, Technische Universitt Braunschweig, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Filamentous fungi have been widely applied in industrial biotechnology for many decades. In submerged
Received 5 March 2012 culture processes, they typically exhibit a complex morphological life cycle that is related to production
Received in revised form 2 May 2012 performance a link that is of high interest for process optimization. The fungal forms can vary from dense
Accepted 25 June 2012
spherical pellets to viscous mycelia. The resulting morphology has been shown to be inuenced strongly
Available online 4 July 2012
by process parameters, including power input through stirring and aeration, mass transfer characteristics,
pH value, osmolality and the presence of solid micro-particles. The surface properties of fungal spores
Keywords:
and hyphae also play a role. Due to their high industrial relevance, the past years have seen a substantial
Filamentous microorganisms
Morphology
development of tools and techniques to characterize the growth of fungi and obtain quantitative estimates
Mycel and pellet growth on their morphological properties. Based on the novel insights available from such studies, more recent
Image analysis studies have been aimed at the precise control of morphology, i.e., morphology engineering, to produce
Rheology superior bio-processes with lamentous fungi.
Environome 2012 Elsevier B.V. All rights reserved.

1. Introduction
Due to their metabolic diversity, high production capac-
ity, secretion efciency, and capability of carrying out post-
translational modications, lamentous fungi are widely exploited
as efcient cell-factories in the production of metabolites, bioac-
tive substances and native or heterologous proteins, respectively.
The commercial use of fungal microorganisms has been reported
for multiple industrial sectors, such as those involved in the pro-
duction of simple organic compounds (e.g., detergents), food and
beverages, or pharmaceuticals (Archer, 2000; Barry and Williams,
2011; Grimm et al., 2005b; Lubertozzi and Keasling, 2009; Meyer,
2008; Papagianni, 2004; Wucherpfennig et al., 2010). One of the
most sensitive process parameters is the complex fungal mor-
phology. The morphological characteristics of lamentous fungi
vary from freely dispersed mycelia to distinct pellets of aggre-
gated biomass. Depending on the desired product, the optimal
morphology for a given bioprocess varies and cannot be general-
ized (Gibbs et al., 2000). Because the optimal productivity correlates
Corresponding author at: Institute of Biochemical Engineering, Technische Uni-
versitt Braunschweig, Gaustrae 17, 38106 Braunschweig, Germany.
E-mail address: r.krull@tu-braunschweig.de (R. Krull).
with a specic morphological form (Kaup et al., 2008; Zhang et al.,
2007), the advantages and disadvantages of mycelial or pellet cul-
tivation should be carefully balanced for each biological system.
For example, mycelial growth creates undesired high viscosity
of the cultivation broth, potentially limiting the nutrient supply
due to insufcient mixing. In comparison, cultivation broths with
distinct pellets show Newtonian ow behavior. However, limited
nutrient availability might occur within the inner part of the biopel-
lets. Quantitative characterization, monitoring, and even dened
control of the morphology of lamentous growing fungi are dif-
cult to obtain, which is inherently linked to the highly complex
morphological types and the bi-directional interaction between
morphology and the process environment, e.g., via broth rheology
or mixing and mass transfer performance. The morphological type
and related physiology of fungal systems strongly depend on the
environmental conditions in the bioreactor, i.e., the environome
(Krull et al., 2010). Among other factors, the variable environmen-
tal cultivation conditions comprise the inoculum concentration,
spore viability, pH value, temperature, dissolved oxygen concen-
tration, and mechanical stress (Deckwer et al., 2006). This review
illuminates recently developed sophisticated techniques to char-
acterize fungal growth on the micro- and macromorphological
and process-scale levels. Furthermore, more recent strategies to
control particle shape and bioprocess performance by targeted

0168-1656/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.06.024
 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
morphology engineering will be discussed. These issues are high-
lighted by selected relevant examples from the use of lamentous
fungi in biotechnology.
2. Morphological life cycle in suspension culture
The morphology of lamentous organisms in submerged cul-
tivation is of considerable interest due to its impact on process
productivity (McIntyre et al., 2001). Fig. 1 shows different morpho-
logical forms of Aspergillus. Mycelial growth can be differentiated
into micro- and macroscopic morphology (Krull et al., 2010).
In submerged cultivations, the observed macroscopic morphol-
ogy of lamentous fungi varies from freely dispersed mycelium
over loose mycelial clumps to dense pellets (Papagianni, 2004).
The nal macromorphology is determined based on the strain
properties and chosen cultivation conditions (e.g., inoculum prop-
erties, pH, mechanical stress, cultivation temperature, and medium
composition), the process mode, microparticle addition and osmo-
lality (Papagianni, 2004; Wucherpfennig et al., 2011; Znidarsic and
Pavko, 2001). Different mechanisms of pellet formation have been
reported (Nielsen, 1996). The coagulating type is characterized by
the aggregation of spores at the very beginning of the cultivation.
Pellets are then formed by germination of just a fraction of the
coagulated spores. Coagulating pellet formation has been reported
for several Aspergilli (Carlsen et al., 1996; Grimm et al., 2004). The
aggregation step has been previously shown to be inuenced by
pH value (Carlsen et al., 1996) and mechanical stress by agitation
(Metz and Kossen, 1977). In the non-coagulating type, a pellet is
generated from each individual spore. This type of pellet formation
has been reported for several Streptomyces species (Vecht-Lifshitz
et al., 1990).
2.1. Micromorphology
Fungal growth starts with a single or several spores, which
remain dormant until the nutrients necessary for activation are
supplied. The process of germination then begins by a swelling pro-
cess during which much water is absorbed, and the spores start
to extend in an isotropic manner, after which a singular germ-
tube with a tubular shape emerges (Osherov and May, 2001). The
growth of the hyphae is polarized, with a linear extension rate
at the hyphal apex. Along the length of the hypha, precursors
for the cell wall and proteins involved in cell wall synthesis are
transported in vesicles from the endoplasmic reticulum (ER) to
the hyphal tip. As a result of its gradient distribution, the ER net-
work supports the directed transportation of the vesicles to the tip
(Maruyama and Kitamoto, 2007), where they accumulate as the
so-called Spitzenkrper. The Spitzenkrper determines the tubu-
lar shape and the growth direction and rate of the hyphal cell
(Reynaga-Pena and Bartnicki-Garcia, 1997; McIntyre et al., 2001).
The elongation of a hyphal element leads to the formation of septae
and hyphal compartments. Due to an accumulation and excess of
transport vesicles, which cannot pass a septum, branching occurs
in subapical compartments (Aynsley et al., 1990). When a hyphal
element has grown to a certain length, a branch is formed lat-
erally to the parent (Barry and Williams, 2011). The length of a
hyphal element between two branching points is referred to as the
hyphal growth unit (HGU) (Caldwell and Trinci, 1973), which is
approximately equal to the total hyphal length divided by the total
number of tips. Low HGU values indicate an increased number of
growing tips in the mycelium (Aynsley et al., 1990), which might
be preferred in industrial production processes as secretion is pri-
marily related to the apical zone of the hypha at the sites of cell
wall synthesis (Barry et al., 2009; McIntyre et al., 2001), which is
assumed to contain larger pores due to the plasticity of the cell wall
113
(White et al., 2002). However, other studies have found no corre-
lation between the number of tips and protein secretion (Bocking
et al., 1999). Micromorphological growth in general can be char-
acterized by the sum of all single hyphal length in the mycelium,
the hyphal diameter, the number of tips and the tip extension and
branching rate of individual hyphae (Grimm et al., 2005b). The over-
all mycelium exhibits exponential growth with a constant hyphal
growth rate as long as no substrate limitation occurs and hyphal
density and diameter stay the same (Grimm et al., 2005a). Micro-
morphological parameters can be determined by microscopy and
digital image analysis methods to obtain information about cul-
ture performance and the establishment of structured models (see
Section 3.2).
2.2. Macromorphology
The apparent macromorphology, which is discernible by the
size and structure of the growth form, ultimately results from
micromorphological events and from other physico-chemical envi-
ronmental treatments (see Section 4). In general, both mycelial
and pellet growth increase the viscosity of the cultivation medium
(Pazouki and Panda, 2000). Although a pelleted cultivation broth
may have the advantages of Newtonian low viscosity and an easier
separation of the biomass during downstream processing, prob-
lems might arise with the transportation of nutrients into pellet
cores, thus leading to dead-zones with greatly reduced productiv-
ity. The lamentous growth forms therefore predominate in most
industrial cultivations because of increased cell growth and higher
productivities (Riley et al., 2000; Wucherpfennig et al., 2011). For
some bulk chemicals, such as citric acid, however, pellet or clump
morphology is advantageous (Papagianni, 2004). The formation of
pellets or clumps can result from the aggregation of spores prior
to germination, the aggregation of spores and germ tubes or the
aggregation of mycelia, depending on the organism. Pellets can be
described as stable spherical agglomerates composed of a branched
network of hyphae. Their shape can vary from smooth and spher-
ical to elongated and hairy. The macroscopic growth of fungi to
pellets is generally dened by an increase in the pellet radius and
the so-called critical radius. The critical radius indicates the
point at which diffusion limitations occur in the outer shell of the
pellet (Nielsen, 1996). One of the rst introduced macromorpholog-
ical growth models was the cube-root law (Emerson, 1950), which
describes pellet growth as it occurs in the outer nutrient-supplied
shellcore of constant width. Biomass in the inner coreshell is sub-
strate limited and does not participate in the growth process. Pellets
grow exponentially with a constant specic growth rate until a
critical radius is reached and mass transport limitations lead to a
depletion of nutrients at the pellet core. Assuming a spherical pellet
with a homogenous density along the radial coordinate, the pellet
biomass concentration is proportional to the cultivation time to
the power of 3. With continued exponential and unlimited growth
of the peripheral pellet layer, the inner zone, in which the oxygen
supply is limited, increases, and nally autolysis occurs, leaving
pellets with a hollow core (Barry et al., 2009). Different physio-
logical zones within a pellet have been quantied (Bizukojc and
Ledakowicz, 2010). Driouch and colleagues used a GFP-expressing
Aspergillus niger strain to visualize the active areas of these pellets
(Driouch et al., 2010a, 2012).
3. Characterization of fungal morphology on micro-,
macro- and process-scale level
Advances in particle and image analysis and in micromechanical
devices have provided quantitative morphological data. The broad
portfolio of techniques thus enables analysis on different particle
114 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
Table 1
Advanced particle characterization techniques on the micro-, macro-, and process-scale levels.
Characterization level Method portfolio Accessible parameter
Microscopic Fluorescence lifetime Fluorescence lifetimes of
imaging (FLIM) melanin, free and
protein-bound NAD(P)H
Atomic force microscopy Adhesion forces and local
(AFM) mechanical properties,
Youngs modulus
Zeta potential Electrostatic properties of
spores and aggregates
Contact angle Surface hydrophobicity,
measurement adhesion energy
Macroscopic Digital image analysis Apical growth, septation,
pellets branching of mycel and
pellet morphology,
projected area, solidity,
maximal diameter, aspect
ratio, morphology number
Focused beam reectance Chord lengths of individual
measurement (FBRM) particles and distributions
Laser diffraction technique Scattering intensity of
diffracted laser beam,
refraction index
Microelectrode technique Oxygen concentration,
diffusion coefcient,
oxygen penetration depth
Confocal laser scanning Pellet surface
microscopy (CLSM) characterization, hyphal
gradient, localization of
protein-producing regions
Flow cytometry Whole-cell-uorescence,
viability, particle size and
density, auto-uorescence
Rheological technique Apparent viscosity, yield
stress, consistency and
ow index
Pellet slicing and Internal pellet and surface
sedimentation structure, sink resistance
Investigated system
A. ochraceus
A. fumigatus, A. niger, A.
nidulans, S. cerevisiae,
Saccharopolyspora
erythraea, Staphylococcus
aureus, Streptomyces
coelicolor
A. niger
A. niger, A. nidulans,
Candida parapsilosis
A. awamori, A. oryzae, A.
terreus, Coprinopsis cinerea,
Geotrichum candidum,
Mortierella sp., Phellinus sp.,
Trichoderma reesei
A. niger, shear sensitive
inorganic clay-oc system
A. niger
A. niger, P. chrysogenum
A. niger
A. niger
A. niger, A. sojae, A. terreus,
Paecilomyces sinclairii,
Paecilomyces japonica,
Penicillium citrinum
A. niger
References
Herbrich et al. (2012)
Arfsten et al. (2010), Dague
et al. (2007, 2008), Dufrne
(2010), Fang et al. (2012),
Kailas et al. (2009), Smith
et al. (1998), Stocks and
Thomas (2001), Sullivan
et al. (2007), Touhami et al.
(2004), Vadillo-Rodriguez
et al. (2008), Wargenau
and Kwade (2010), Wright
and Armstrong (2006),
Wright et al. (2010), Zhao
et al. (2005)
Lyklema (2003), Priegnitz
et al. (2012), Wargenau
et al. (2011)
Dynesen and Nielsen
(2003), Gallardo-Moreno
et al. (2002), Leckband and
Israelachvili (2001), Ryoo
and Choi (1999)
Ahamed and Vermette
(2009), Barry et al. (2009),
Barry and Williams (2011),
Bizukojc and Ledakowicz
(2010), Hwang et al.
(2004), Kes and Rhl
(2009), Metz et al. (1981),
Mller et al. (2003), Packer
and Thomas (1990), Park
et al. (2006), Prosser and
Trinci (1979), Spohr et al.
(1998), Trinci (1974),
Wucherpfennig et al.
(2011), Znidarsic and
Pavko (2001)
Grimm et al. (2004), Grimm
et al. (2005a), Heffels et al.
(1998), Hopfner et al.
(2010), Kelly et al. (2006b),
Lin et al. (2008), Mahnke
et al. (2000), Pilz and
Hempel (2005), Shrig and
Wissler (2001), Stintzing
et al. (2008)
Ley (2000), Lin et al.
(2010), Wucherpfennig
et al. (2011)
Cronenberg et al. (1994),
Cui et al. (1998), Hille et al.
(2005), Hille et al. (2006),
Hille et al. (2009), Yang and
Lewandowski (1995)
Hille et al. (2005), Villena
et al. (2010)
Chen and Seguin-Swartz
(2002), de Bekker et al.
(2011), Laamme et al.
(2005), Tung et al. (2007)
Gupta et al. (2007), Olsvik
and Kristiansen (1992),
Olsvik and Kristiansen
(1994), Oncu et al. (2007),
Oolman et al. (1986),
Papagianni (2004),
Petersen et al. (2008),
Rodrguez Porcel et al.
(2005), Wucherpfennig
et al. (2010)
Lin et al. (2010)
 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123 115

Table 1 (Continued)

Characterization level Method portfolio Accessible parameter Investigated system References

Process Power input Reactor geometry, stirrer
shape and size (agitation),
gassing rate (aeration)
Computational uid Mechanical stress, ow
dynamics (CFD) patterns
Population balance Coupling of inner system
modeling property (e.g., pellet
diameter) with
independent parameter
(e.g., time, coordinates),
distributive character of
particle system
A. niger, Penicillium Cui et al. (1998), Fujita
chrysogenum et al. (1994), Grimm et al.
(2005b), Kelly et al. (2004),
Kelly et al. (2006a), Wittler
et al. (1986)
A. awamori, E. coli Eslahpazir et al. (2011),
Johansen et al. (1998),
Lapin et al. (2006),
Wucherpfennig et al.
(2010), Xia et al. (2009)
A. niger Lin et al. (2008),
Ramkrishna (2000)

Fig. 1. Morphological forms of Aspergillus sp.: (a) prole view of conidiophores (diameter 200 m) on solid agar medium, (b) single spore, (c) spore package (spore diameter
5 m), (d) germinated tube (length approx. 250 m), (e) coagulated type of mycel, in which single ungerminated spores adhere to germinated hyphal tubes (length approx.
100 m), (f) dispersed mycel, (g) exposed hyphae of a pellet (pellet hair) (length approx. 100 m), (h) pellet slice (diameter approx. 1000 m), (i) hairy biopellet (pellet
diameter approx. 1000 m), and (j) submerged biopellets.
116 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
size levels the microscopic, macroscopic, and process levels as
discussed below in more detail (Table 1 ).
3.1. Particle characterization at the microscopic level
The characterization of fungal microscopic morphology should
begin with the spore inoculum, a typical starting point for the
industrial seed culture of a production process. Inferior seed cul-
tures will result in suboptimal processes. Inoculation with spores
of unknown properties will turn the result of any cultivation into a
process of trial and error while also strongly hindering the correct
interpretation of the same. Fluorescence liftetime imaging (FLIM),
atomic force microscopy (AFM) and the investigation of spore sur-
faces by the measurement of Zeta potential or contact angle have
strongly contributed to our current knowledge on spore properties.
3.1.1. Fluorescence lifetime imaging microscopy (FLIM)
Herbrich et al. (2012) have used FLIM and two-photon exci-
tation microscopy to investigate the autouorescence of fungal
spores. The quantication of the uorescence lifetimes of melanin,
free NAD(P)H and protein-bound NAD(P)H within individual spores
provided new insights into their metabolic properties. Most strik-
ingly, this technique revealed tremendous differences between
spores that have been subjected to different treatments, phe-
nomena that could not be visualized by conventional optical
microscopy. Fig. 2 shows the relative abundance of bound and
free NAD(P)H determined via FLIM, in which a decreasing average
lifetime stands for decreasing amounts of protein-bound NAD(P)H
and hence impaired metabolic activity. This technique allows sig-
nicant metabolic differences between conidial populations to be
determined.
3.1.2. Atomic force microscopy (AFM)
Atomic force microscopy enables the imaging of single microor-
ganisms with a sub-nanometer resolution to directly measure
adhesion forces and local mechanical properties (Arfsten et al.,
2010; Wright and Armstrong, 2006; Wright et al., 2010). For this
investigation, the spores have to be xed to the tip of the cantilever,
which is used as an indentation probe, to withstand the lateral
forces encountered during the preceding AFM imaging. Mechan-
ical entrapping in lter pores (Arfsten et al., 2010; Touhami et al.,
2004), microstructures (Kailas et al., 2009), and polymers such as
gelatin (Sullivan et al., 2007) or glue (Fang et al., 2012) have been
used for this purpose. In addition, chemical immobilization onto
at substrates is used frequently (Vadillo-Rodriguez et al., 2008).
The recorded force/displacement curves of small indentations
into the cell wall are used to calculate Youngs modulus based on
Hertzian theory (Zhao et al., 2005), a value that is a function of the
whole cell and allows different cell types or environmental param-
eters (such as pH and ionic strength) to be compared. Even cell wall
parameters can be extracted from the obtained data via nite ele-
ment methods, as exemplied for cells (Smith et al., 1998), hyphae
(Zhao et al., 2005) or spores of different Aspergillus strains (Fang
et al., 2012; Zhao et al., 2005). Compared with the mycelia of the la-
mentous bacterium Streptomyces coelicolor (Wright and Armstrong,
2006), the mycelia of Aspergillus hyphae are much stiffer. To trans-
fer micromechanical behavior to macroscopic behavior such as
the breakage of hyphae due to hydrodynamic stress, tensile tests
are necessary. Until now, this process was only realized by Stocks
and Thomas (2001) with the hyphae of the lamentous bacterium
Saccharopolyspora erythraea. The direct measurement of adhesion
forces between spores was performed by Wargenau and Kwade
(2010) at different values of pH and ionic strength. A ne reso-
lution of adhesion forces across the surface, called adhesion force
mapping, revealed hydrophilic and hydrophobic domains on the
surface (Dague et al., 2007, 2008). Many of the investigated fungi
showed clearly textured surfaces. Patterns of thin (10 nm) and long
cylinders could be observed. These so-called rodlets are composed
of hydrophobins (moderately hydrophobic proteins) and deter-
mine the interaction forces in the dormant state. Experiments with
Aspergillus fumigatus revealed a dramatic change in surface com-
position during germination (Dague et al., 2008; Dufrne, 2010).
Adhesion measurements on mutants of this fungus helped reveal
that the formation of rodlets could be retraced to a certain gene
(Dague et al., 2008).
3.1.3. Zeta potential
The Zeta potential provides information about the electrostatic
properties of spores. It is calculated from electrophoretic mobility
using the Smoluchowski equation (Lyklema, 2003). The Zeta poten-
tial can be used to predict the agglomeration behavior of particles in
a suspension, whereby the pH and ionic strength of the surround-
ing medium are critical parameters. Measurements from Wargenau
et al. (2011) showed a negative Zeta potential of A. niger spores even
in acidic media, which could be retraced to a negatively charged
layer of melanin pigments that was adsorbed at the surface of the
spores (Priegnitz et al., 2012).
3.1.4. Contact angle measurement
Contact angle measurements are used to determine the
hydrophobicity of surfaces. Small drops of different liquids are
placed on a surface. From the angle of these drops toward the
surface, the adhesion energy can be calculated (Leckband and
Israelachvili, 2001). Ryoo and Choi (1999) produced small disks
out of lyophilized cells or spores from A. niger and used them for
macroscopic contact angle measurements. The contact angle mea-
surements of conidiospores from Aspergillus nidulans (Dynesen and
Nielsen, 2003) and Candida parapsilosis (Gallardo-Moreno et al.,
2002) were realized directly on sporulating colonies. Viewing the
microscopic behavior of single microorganisms gives insight into
the mechanisms of the macroscopic performance. Utilizing this
knowledge is an important issue to gain control over the morpho-
logical behavior of lamentous fungi.
3.2. Particle characterization at the macroscopic level
3.2.1. Digital image analysis systems
Image analysis has clear advantages in terms of speed, pre-
cision, labor intensity and quantitative assessment (Barry and
Williams, 2011). From early on, morphological investigations have
been undertaken to characterize the surface cultures of lamen-
tous fungi as well as hyphal growth and branching and to quantify
fungal morphology (e.g., Prosser and Trinci, 1979; Trinci, 1974).
Today, digital image analysis is currently the state of art method
to characterize apical growth, septation and branching of mycel
and pellet morphology (Znidarsic and Pavko, 2001). Powerful
approaches have become available today based on pioneering
studies on semiautomatic methods (Metz et al., 1981), software
development for automated image analysis (Packer and Thomas,
1990) and on-line image analysis systems (Spohr et al., 1998).
Many studies have quantied pellets in terms of their size and
the extent of the lamentous region (e.g., Mller et al., 2003;
Park et al., 2006), whereas others have focused on the high-
throughput, low-resolution quantication of suspended pellets
and aggregates (Bizukojc and Ledakowicz, 2010; Kes and Rhl,
2009). Barry et al. (2009) integrated novel algorithms to ana-
lyze membrane-immobilized cultures of Aspergillus oryzae. Today,
these measurements are strongly facilitated by convenient soft-
ware platforms (Ahamed and Vermette, 2009; Hwang et al.,
2004); in particular, the freely available, open source program
Image J appears useful (http://rsbweb.nih.gov/ij/, Wucherpfennig
et al., 2011). New developments even allow the extraction of
 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
Fig. 2. Fluorescent lifetime images (FLIM) of Aspergillus ochraceus spores. Distribution of free NAD(P)H (left), and distribution of enzyme-bound NAD(P)H (right). The color
code represents the average uorescence lifetime per pixel in ns (Herbrich et al., 2012).
Fig. 3. Correlation of specic productivity and fungal morphology using the mor-
phology number (MN) of the fructofuranosidase-producing strain Aspergillus niger
SKAn 1015.
Redrawn from Wucherpfennig et al. (2011).
dimensionless morphology numbers to represent distinct mor-
phological parameters, such as projected area, solidity, maximal
diameter, and aspect ratio, which enabled, for the rst time, a com-
prehensive characterization of fungal morphology that correlated
closely with productivity (Fig. 3).
3.2.2. Focused beam reectance measurement (FBRM)
Focused beam reectance measurement is a size determining
optical method previously used in the chemical and pharmaceutical
industries (Heffels et al., 1998; Shrig and Wissler, 2001). It is an opti-
cal technique based on the detection of backscattering laser light
and provides chord lengths of individual particles and their distri-
bution (Hopfner et al., 2010). The rst studies using this method
involved inorganic model systems. Mahnke et al. (2000) described
the time-dependent disintegration kinetics of a shear sensitive
inorganic clay-oc system with FBRM to determine the mechan-
ical stresses in a two-phase bubble column reactor. The effects of
power input, uid phase viscosity and solid loading on the mechan-
ical stress on the suspended particles was examined with FBRM by
Pilz and Hempel (2005). Stintzing et al. (2008) expanded FBRM for
the determination of mechanical stress from disintegration kinet-
ics in stirred tank reactors and concluded that aeration-induced
117
stress has a greater impact on the particle destruction than stirring-
induced stress. More recently, fungal systems were also studied
by FBRM (Grimm et al., 2004, 2005a; Kelly et al., 2006b). Grimm
et al. (2004, 2005a) characterized the conidial inocula and seeding
cultures of A. niger to assess the impact of the aggregation pro-
cess. Based on the time-dependent development of the microscopic
measurements of particle concentration and their correlation with
the FBRM results, a kinetic model has been proposed for the coni-
dial aggregation of A. niger using population balance modeling (Lin
et al., 2008).
3.2.3. Laser diffraction technique
The laser diffraction technique, also known as Low Angle Laser
Light Scattering (LALLS), works on the basis of particles of a given
refraction index interacting with a laser beam passing through an
inverse Fourier-type lens. The angle of the diffracted laser beam
is inversely proportional to the particle size, and the scattering
intensity of the diffracted laser beam is directly proportional to the
particle size (Ley, 2000). Lin et al. (2010) and Wucherpfennig et al.
(2011) introduced the laser diffraction technique to characterize
the spore aggregation and pellet size of A. niger. The advantage of
this method is the quickness of the measurement and most impor-
tantly, the improved statistical evaluation of the measured data.
This technique appears to be a practical, rapid, robust, and repro-
ducible method for quality control or production purposes.
3.2.4. Microelectrode technique
Pellet morphology has an important inuence on the effective
diffusion coefcient and penetration depth of oxygen into the pellet
(Cui et al., 1998; Hille et al., 2006, 2009). These important proper-
ties with regard to process performance can be studied by oxygen
microelectrode measurements (Cronenberg et al., 1994; Hille et al.,
2005, 2009). This method allows spatially resolved measurements
of concentration gradients not only in the aggregate itself but also
in the bulk phase and boundary layer around the pellet. The major-
ity of models of mass transport and turnover in fungal pellets depict
mass transport as being purely diffusive on the basis that advection
and turbulence are negligible in submerged cultivations (Cui et al.,
1998). Following the assumption that diffusion is the dominating
mass transport mechanism, the effective diffusion coefcient can
be used as a parameter in equations for the description and calcu-
lation of mass transport and turnover (Hille et al., 2009). Within
pellets, the effective diffusion depends on the space available for
transport and is often expressed as a function of pellet porosity
or biomass density. Moreover, the deformation of pellet structure
118 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
was found to have a signicant impact on the penetration depth
of substrates. The diffusion limitation of whole pellets was found
to be mainly a function of size, with a pronounced inuence of
advection on the outer zone of pellets that is supplied with oxygen.
The effective diffusion coefcient was concluded to potentially be
insufcient for the description of mass transport within the pellet
periphery for a broad range of realistic uid dynamic conditions
(Hille et al., 2009). In addition, a convective mass transfer has to be
taken into consideration (Yang and Lewandowski, 1995).
3.2.5. Confocal laser scanning microscopy (CLSM)
A precise structural analysis of fungal morphology is achieved
by the application of confocal laser scanning microscopy (CLSM),
which provides superior surface characterization and thus a more
thorough image analysis (Villena et al., 2010). CLSM has been used
to dene the hyphal gradient within the outer pellet periphery,
which is a gradient describing the hyphal fraction at the surface of
the pellet (Hille et al., 2005). Because the determination of hyphal
gradients using CLSM is very accurate but time-consuming for rou-
tine applications, a standard method was established based on a
previous normalization with CLSM data, in which the pellet fraction
gradient, and hence the pellet density in the border area, was corre-
lated with the determination of the settling velocities of the pellets
with sufcient accuracy (see Section 3.2.8). Structure-border inves-
tigations were performed to investigate the interactions between
mass transport and the growth and development of the biolm
structure. The data generated with CLSM were of great importance
for understanding the underlying growth and reaction processes
(Hille et al., 2005). To localize the protein-producing regions in
biopellets, an A. niger strain that produces green uorescent pro-
tein (GFP) was used to examine the specic relationships between
morphology and productivity. GFP-producing regions in equato-
rial pellet slices were analyzed with CLSM. Only the peripheral
regions of the pellets contributed to protein production, which
seems plausible given that the peripheral regions feature a good
nutrient supply and therefore develop good growth and productiv-
ity. Based on that result, the generation of uffy pellets with a high
gradient of the hyphal fraction within the outer pellet periphery
seems desirable.
3.2.6. Flow cytometry
Flow cytometry is a high-throughput characterization method.
Spores, aggregates or pellets are investigated as they pass through
a ow chamber. After the particles are exposed to a laser beam,
whole-cell-uorescence is generated and detected, and the cell
then exits the ow chamber, making room for the next particle.
The development and application of ow cytometry, however, were
long focused primarily on medical purposes (Tung et al., 2007).
With the application of viability tests for the spores of various
fungal strains by ow cytometry (Chen and Seguin-Swartz, 2002)
and the introduction of autouorescence (Laamme et al., 2005),
spore populations with higher autouorescence were shown to
have signicantly increased viability as determined by the num-
ber of colony forming units. Flow cytometry has recently become
feasible to use with fairly large particles, such as A. niger pel-
lets up to 2000 m. Current instruments are able to analyze and
even sort particles on the basis of the physical characteristics of
size, density, and uorescence signals. Flow cytometry has shown
heterogeneities in A. niger pellets with respect to size and gene
expression (de Bekker et al., 2011).
3.2.7. Rheological technique
Correlations between broth rheological and morphological
parameters and properties have been studied extensively due to
the large variability of mycelia growth in submerged cultures,
which makes the characterization challenging (Papagianni, 2004).
The increased culture broth viscosity leads to heterogeneous,
stagnant, non-mixed zone formations, which make the cultiva-
tion challenging and more expensive to operate (Oncu et al.,
2007). Variations in the broth characteristics affect the bioreactor
hydrodynamic properties, which include mixing and mass trans-
fer performances (Kilonzo and Margaritis, 2004; Petersen et al.,
2008). The efciency and productivity of the entire cultivation
process depends on these hydrodynamic properties (Gupta et al.,
2007). Mycelial cultivation broths generally show non-Newtonian
rheological characteristics and shear thinning or pseudoplas-
tic behavior (Olsvik and Kristiansen, 1994). Pseudoplastic uids
exhibit a decrease in viscosity with an increase in the shear rate.
Non-Newtonian viscosity is usually characterized by apparent vis-
cosity (Petersen et al., 2008). The rheology of mold suspensions
are commonly described using the Ostwaldde Waele power law
approach (Oncu et al., 2007; Petersen et al., 2008; Rodrguez Porcel
et al., 2005), HerschelBulkley correlation (Petersen et al., 2008),
Bigham model (Olsvik and Kristiansen, 1994) and Casson model
(Oolman et al., 1986). Typically, the Ostwaldde Waele power
law and the HerschelBulkley model most adequately describe
the broth rheological prole over the whole range of shear rates.
In some studies, the consistency index K has been used as the
sole indicator for the viscosity of lamentous cultivations (Olsvik
and Kristiansen, 1992). Because the ow index n is an exponent,
changes will have a larger effect on shear stress than a similar
change in K. K-values as single indicator of uid viscosity are lim-
ited to data sets where n is constant (Olsvik and Kristiansen, 1994).
Most of the measured morphological parameters were population
averages based on selected samples and assumptions, with the dis-
advantage that much of the information from the image analysis
data is lost by taking a population average or by excluding part
of the population. A multivariate approach to the size distribution
data for the prediction of the rheological characteristics of lamen-
tous cultivation broths was taken by Petersen et al. (2008), and a
detailed overview of the correlation between the morphology and
rheology of lamentous growing biological systems was given by
Wucherpfennig et al. (2010).
3.2.8. Pellet slicing and sedimentation
To investigate the internal pellet structure, pellet slicing was
applied by Lin et al. (2010). The images of the A. niger pellets slices
(thickness of 70 m) from the equatorial region, cultivated at differ-
ent mechanical stresses of stirring and aeration, are shown in Fig. 4.
Biopellets of the combination at the highest aeration are relatively
unstructured and irregular at the periphery and have a much larger
structure overall, i.e., a more equal biomass distribution over the
entire radial coordinate of the pellet slice. In contrast, those pellets
that were treated with increased agitation ratios are rather circu-
lar and have a more compact pellet surface structure. To determine
the inuence of the pellet surface structure on the particle behavior
in liquid media, the sedimentation velocities of pellets were mea-
sured in a water-lled sedimentation column (Lin et al., 2010). The
periphery of the pellets that were treated with the highest share of
aeration was the least compact, whereas the pellets treated with
a lower share of the aeration showed much more compact pellet
surface structures. This investigation is in good agreement with
the results of the qualitative analysis of the overall pellet structure
regarding the compactness of the pellet peripheral regions.
3.3. Characterization at the process level
At the process level, the identication and quantication of
mechanical stresses, which are related to a distinct morphology
of the fungal elements within the process, are the main appli-
cation elds of numerical simulation and modeling. The specic
power input, in turn, inuences the Kolmogorov length scale, which
 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
Fig. 4. Microscopic images of pellet slices over the cultivation time of Aspergillus niger AB1.13 in a stirred bioreactor at different combinations of volumetric power input
P/V (aeration [aer] and agitation [agi]) of the total volumetric power input of 143 W m3 (bottom row: 52 [aer] + 91 [agi] W m3 , middle row: 78 [aer] + 65 [agi] W m3 , upper
row: 103 [aer] + 40 [agi] W m3 ), scale bar: 500 m.
Taken from Lin et al. (2010).
can correspond with the eddies (Kelly et al., 2006a). The reactor
geometry, stirrer shape and size, gassing rate and subsequent dis-
sipated energy are among the uid dynamic-related criteria that
can affect the morphology and productivity. The stirrer geome-
try plays a key role in stirred tank bioreactors and can inuence
cultivation in two ways: lower stirring speeds lead to inadequate
nutrient distribution and gas dispersion, whereas vigorous stirring
can cause cell wall damage (Grimm et al., 2005b; Wittler et al.,
1986). Particle stress in different bioreactors (stirred tank reactor,
bubble column, loop reactor) under different volumetric power
inputs and impellers of various types and geometries have been
investigated in detail by Henzler (2000). For stirred tank reactors,
the stress produced by different stirrers can be predicted with the
aid of a geometrical correlation derived from experimental particle
disintegration kinetics: impellers with a large blade area relative to
the tank dimensions produce less shear stress because of their uni-
form power input compared with small and especially axial-ow
inclined-blade impellers.
The effect of agitation on fungal morphology has been the sub-
ject of several studies (Cui et al., 1998; Fujita et al., 1994; Kelly et al.,
2004). The authors have concluded that the total hyphal length of
a fungal mycel decreases with increasing specic power input.
To obtain the detailed data on the parameters contributing to
mechanical stress, more sophisticated methods, such as computa-
tional uid dynamics (CFD) (Eslahpazir et al., 2011), are required.
The main advantage of CFD is fewer resource demands, with the
exibility and generality of turbulence models representing var-
ious geometries and cultivation conditions. To simulate the ow
patterns in bioreactors, some simplifying assumptions are needed.
Thus, the similarities to stirred tank reactors are mainly focused so
that the gross ow patterns will be obtained (Lapin et al., 2006; Xia
et al., 2009). The morphological range of A. niger and how it is inu-
enced by different environmental parameters has been frequently
discussed in numerous investigations (e.g., Johansen et al., 1998;
Wucherpfennig et al., 2010).
Another suitable method for describing the morphological
changes of lamentous fungi is population balancing, which is
mainly focused on how to couple an inner property (diameter,
in the case of pellets) with an independent parameter (e.g., time,
coordinates) so that a distributive property of the particles can
be captured (Ramkrishna, 2000). Based on intracellular reactions
up to physico-chemical and uid dynamic phenomena at the
119
macroscopic level, the morphogenesis of mycelial growth and pel-
let formation via distinct aggregation steps ought to be completely
covered by population balancing and veried by different particle
size analysis techniques. Lin et al. (2008) simulated the aggregation
mechanisms of A. niger AB1.13 and validated the model with the
kinetic data of Grimm et al. (2004). Improvements are necessary,
particularly regarding the description of the aggregate breakage.
4. Control of fungal morphology
From the engineering point of view, distinct fungal morphol-
ogy has to be controlled by dened environmental conditions.
Table 2 shows the most relevant approaches to control fungal
morphology at the process level. These investigations focused
on variations in the environome, changing the most important
operating parameters, such as the spore concentration, pH value,
mechanical stress due to power input by agitation and/or aeration,
cultivation temperature, medium composition or process mode.
New sophisticated techniques to tailor the fungal morphology for
enhanced productivity investigated the addition of microparticles
or variation of osmolality of the cultivation broth.
Many research groups correlated morphology and productivity
to the concentration of spores in the inoculum (e.g., Bizukojc and
Ledakowicz, 2010; Grimm et al., 2004; Liu et al., 2008; Papagianni
and Mattey, 2004; Tucker et al., 1992). Grimm et al. (2004) inves-
tigated the inuence of spore concentration on the aggregation
velocity. This parameter is strain dependent and increases with an
increasing concentration of inoculum spores until a maximum is
reached.
The pH of the medium can signicantly affect the morphol-
ogy and productivity (e.g., Bizukojc and Ledakowicz, 2009; Carlsen
et al., 1996; Mainwaring et al., 1999; Papagianni, 2004; Vecht-
Lifshitz et al., 1990). It has an enormous effect on the aggregation
of spores at the beginning of the cultivation, which dictates the
nal morphology (Carlsen et al., 1996; Galbraith and Smith, 1969;
Grimm et al., 2005a). However, problems related to product insta-
bility at extreme pH values limit its applicability.
Determining the inuence of agitation (e.g., Amanullah et al.,
2002; Casas Lpez et al., 2005; Cui et al., 1998; Kelly et al., 2004,
2006a; Lejeune and Baron, 1995; Mitard and Riba, 1986; Nielsen
et al., 1995; Papagianni et al., 1998; Wang et al., 2003) and aer-
ation (e.g., Casas Lpez et al., 2005; Cui et al., 1998; Li et al.,
120 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123

Table 2
Most relevant approaches to control fungal morphology on the process level.

Investigated system Comment References

Classical approaches
Spore concentration (inoculum)
pH and pH shifting
Mechanical stress (agitation and
aeration induced power input)
A. terreus, A. niger, Rhizopus
oryzae
A. terreus, A. niger, A.
oryzae, Streptomyces tendae
A. oryzae, A. terreus, A.
niger, Trichoderm reesei,
Penicillium chrysogenum
Growth and morphology development
strain dependent, e.g., increased
aggregation velocity with rising spore
concentration, max. aggregation velocity at
a certain inoculum concentration
Growth and morphology development
strain dependent
Dependent on agitation- and aeration
induced volumetric power input,
increasing kL a and biomass concentration
with increasing agitation rate, with
increasing aeration pellet size and surface
structure decrease, pellet concentration
increase
Bizukojc and Ledakowicz (2010),
Grimm et al. (2004), Liu et al.
(2008), Nielsen et al. (1995),
Papagianni and Mattey (2004),
Tucker et al. (1992)
Bizukojc and Ledakowicz (2009),
Carlsen et al. (1996), Galbraith and
Smith (1969), Grimm et al. (2005a),
Mainwaring et al. (1999),
Papagianni (2004), Vecht-Lifshitz
et al. (1990)
Amanullah et al. (2002), Casas
Lpez et al. (2005), Cui et al.
(1998), Kelly et al. (2004), Kelly
et al. (2006a), Lejeune and Baron
(1995), Li et al. (2008), Lin et al.
(2010), Mitard and Riba (1986),
Nielsen and Krabben (1995),
Nielsen et al. (1995), Papagianni
et al. (1998), Wang et al. (2003)

New approaches
Microparticle-enhanced A. niger, Caldariomyces Precise formation of fungal morphology Driouch et al. (2010a,b, 2011,
cultivation (MPEC) fumago through ne-tuned variation of particle 2012), Kaup et al. (2008)
size and concentration
Osmolality A. niger Formation of fungal morphology and Bobowicz-Lassociska and Grajek
increased productivity inuenced by (1995), Fiedurek (1998),
osmotic pressure in the cultivation broth, Wucherpfennig et al. (2011)
change in fungal physiology

2008), and therefore of varying mechanical power input (Lin et al.,
2010; Nielsen and Krabben, 1995), on morphology and productiv-
ity has been an objective in several studies. However, increased
stirring, which is eventually required, increases the energy costs of
a process, which is an undesired feature linked to such a process
modication. At the large scale, a homogenous distribution of the
cultivation broth is further limited by the relatively low level of
energy input that is practically feasible.
A pioneering contribution toward the customization of lamen-
tous morphology was the intentional addition of insoluble inert
microparticles (Kaup et al., 2008). Microparticle-enhanced culti-
vation (MPEC) was applied as a novel method to control fungal
morphology development to improve productivity. As shown for
the model process of chloroperoxidase (CPO) formation by the
fungi Caldariomyces fumago, the presence of microparticles (alu-
minum oxide and hydrous magnesium silicate) caused a dispersion
of the cells up to a level of single hyphae and a 5-fold enhanced
CPO production. Additionally, Driouch et al. (2010a) expanded
the supplementation of silicate microparticles to control the mor-
phological development of A. niger. Inoculated from spores, the
morphology of A. niger could be precisely adjusted to a num-
ber of different distinct morphological forms by the addition of
this micromaterial (Fig. 5). In addition to previous ndings, the
authors showed that the use of microparticles enables not only
free mycelium but also a rather precise engineering of morphology
through a ne-tuned variation of particle size and concentration
(Driouch et al., 2010a). Using a mutant co-expressing glucoamy-
lase GFP under the control of the same promoter, the effects of
microparticles on the spatial distribution of protein production in
the cellular aggregates could be visualized, providing a detailed
picture of the strongly altered underlying metabolism in A. niger.
For pelleted growth, protein production was maximal only within
a thin layer at the pellet surface and markedly decreased in the
pellet interior, whereas the interaction with the microparticles
created a highly active biocatalyst with the dominant fraction of
cells contributing to production. This strategy could be successfully
transferred to fed-batch bioreactors (Driouch et al., 2010b, 2011),
resulting in a 10-fold higher production performance than that of
any other process reported so far.
More recently, highly active coreshell pellets of A. niger could
be designed by the addition of titanate microparticles to the growth
medium (Driouch et al., 2012). As tested for two recombinant
strains, the enzyme titer by the titanate-supplemented cultivation
broth was increased 3.7-fold (for fructofuranosidase) and 9.5-fold
(for glucoamylase) compared with the unsupplemented control. In
stirred tank bioreactors, the microparticles increased the nal glu-
coamylase activity to 1080 U mL1 , which was 7-fold higher than
the conventional processes. The major reason for the enhanced
production was concluded to be the close association between
the titanate particles and the fungal cells. Below a microparti-
cle concentration of 2.5 g L1 , the particles were found inside the
pellets, including single particles embedded as 50150 m parti-
cle aggregates in the center, resulting in coreshell pellets. With
increasing titanate levels, the pellet size decreased from 1700
(control) to 300 m. The uorescence-based resolution of GFP
expression revealed that the large pellets of the control were only
active in a 200 m surface layer. Compared with the control, the
fungal pellet supplemented with titanate microparticles was com-
pletely active. The reduced pellet size and the tailor-made change
of the inner structure to a more open pellet structure enabled a
higher mass transfer and total penetration of the pellet. There-
fore, this work opens a broad range of previously unimplemented
advantages for the improvement of biotechnological processes.
Bobowicz-Lassociska and Grajek (1995) were able to increase
the protein secretion of washed and ltered A. niger mycelia by the
addition of KCl. In addition, Fiedurek (1998) was able to increase
the activity of A. niger-expressed glucose oxidase 2-fold by adding
NaCl to centrifuged mycelia, thereby administering an osmotic
shock to the fungus. A further accurate and reproducible way to
tailor-make fungal morphology was found to be the manipulation
of osmolality within the culture medium as increased NaCl con-
centrations led to mycelial growth and enhanced productivity for
 R. Krull et al. / Journal of Biotechnology 163 (2013) 112123
Fig. 5. Morphology engineering of Aspergillus niger SKAn1015 by supplementation with hydrous magnesium silicate (6 m particle size) in submerged culture. The addition
of various microparticle concentrations (g L1 ): (A) control without talc, (B) 0.01, (C) 0.1, (D) 0.4, (E) 0.6, (F) 1.0, (G) 2.0, (H) 3.0, and (I) 5.0. The image analysis was performed
by light microscopy after 72 h of cultivation.
Taken from Driouch et al. (2010a).
two A. niger strains (Wucherpfennig et al., 2011). The morphol-
ogy and productivity of both strains was shown to be considerably
inuenced by osmotic pressure. In all cases, the increased osmo-
lality led to more mycelial growth. The specic productivity of
the fructofuranosidase-producing strain A. niger SKAn 1015 could
be increased approximately 18-fold from 0.5 to 9 U mg1 h1 . The
specic productivity of the glucoamylase-producing strain A. niger
AB1.13 could be elevated using the same procedure. However, the
observed changes in productivity might not be due to the change
in morphology alone. The external parameter of osmolality might
have affected the fungal physiology, which could in turn affect the
morphology independently. The increase in the observed produc-
tivity, however, was shown to correlate with the active surface area
of the fungus.
5. Conclusions and future prospects
The huge efforts in the characterization of fungal morphology
show an evident connection between the operating environment of
the bioprocess and the morphology and metabolic properties of the
individual cells. Closing the gap between the metabolic and process
levels remains the most interesting aspect for future work. Still this
eld is far from understanding the underlying metabolic and regu-
latory mechanisms. Experimental and computational technologies
in systems biology and systems biotechnology, however, provide
a powerful toolbox to step toward a better understanding of this
complex link between the biological and engineering aspects of
fungal cultures.
In addition, the methods introduced for morphology analysis
still leave room for improvement as they depend on partly agglom-
erated mycelia and underestimate various fractions of the biomass,
making a description of the whole population impossible. More
121
powerful and automated image analysis techniques will help to fur-
ther categorize fungal morphology and eventually lead to a more
certain prediction of the culture viscosity.
Future fungal bio-processes might involve the targeted engi-
neering of fungal morphology as a platform technology. Interesting
approaches are microparticle addition and osmolality variation.
These techniques have already proven useful and now await further
proof-of-concept for industrial application a promising starting
point for the next level of fungal biotechnology.
Acknowledgments
The authors gratefully acknowledge the nancial support pro-
vided by the German Research Foundation (DFG) through the
Collaborative Research Center SFB 578 From Gene to Product
at the Technische Universitt Braunschweig, Germany. In addi-
tion, Robert Walisko and Christoph Wittmann thank the Allianz
Industrie Forschung Micro-particle based cultivation of lamen-
tous fungi (No. 16926/N) for nancial support.
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