Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry—the fast alternative to microscopic image analysis

Appl Microbiol Biotechnol DOI 10.1007/s00253-017-8475-2 METHODS AND PROTOCOLS Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry—the fast alternative to microscopic image analysis Daniela Ehgartner 1,2 & Christoph Herwig 1,2 & Jens Fricke 1,2 Received: 3 April 2017 / Revised: 27 July 2017 / Accepted: 5 August 2017 # The Author(s) 2017. This article is an open access publication Abstract An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-017-8475-2) contains supplementary material, which is available to authorized users. * Jens Fricke 1 CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, TU Wien, Gumpendorferstrasse 1a/166, 1060 Vienna, Austria 2 Research Area Biochemical Engineering, Institute for Chemical, Environmental and Biological Engineering, TU Wien, Gumpendorferstrasse 1a/166, 1060 Vienna, Austria accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments. Keywords Filamentous fungi . Flow cytometry . Morphology . Pellets . Microscopy . Image analysis . Hyphae Introduction An important parameter in filamentous bioreactor cultivations is the morphology of the fungi. Morphology and productivity are highly interlinked and depend on process conditions. Filamentous fungi exhibit a large variety of morphological forms in submerged culture. These forms range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the aimed product, different characteristics of morphology are favorable (Papagianni 2004). Dispersed growth was described to achieve better production performance of glucoamylase (Gibbs et al. 2000), while pellets were related to citric acid production (Papagianni 2004). However, it is not just that the productivity is directly linked to the morphology, but also the process is affected. Several studies were conducted, investigating the connection of morphology and viscosity. The latter is linked to mass transfer and energy input (Chain et al. 1966; Petersen et al. 2008; Riley et al. 2000). As a general trend, it can be stated that filamentous growth with high amounts of hyphae causes increased viscosity (Quintanilla et al. 2015). Furthermore, high fraction of pellets results in better mass and heat transfer, and lower power input levels needed for mixing (Znidarsic and Pavko 2001). Various factors build a complex system of interactions. Operation conditions Appl Microbiol Biotechnol influence the growth, the product formation, and the morphology. In addition, filamentous growth directly influences the morphology, which, consequently, further changes the viscosity and which in turn has an impact on the operation conditions (Quintanilla et al. 2015). In the recent decades, the investigation of the fungal morphology in submerged bioreactor cultures is a central issue. Microscopy in combination with image analysis is the most common method (Cox et al. 1998; Paul and Thomas 1998; Posch et al. 2012; Vanhoutte et al. 1995). Automated image recording and automated analysis of images allow a highthroughput, statistically verified morphological analysis (Posch et al. 2012). Furthermore, online methods for image analysis exist like for example the quantification of morphology in flowthrough cells. These last mentioned online analyses focused only on hyphal morphology. The flow cell with a height of 40 μm limits these tools to dispersed growing cultures (Christiansen et al. 1999; Spohr et al. 1998). Most common morphological classifications distinguish between freely dispersed mycelia and aggregates. Freely dispersed mycelia include hyphae, which are long and can have branches (Cox et al. 1998). Simple clumps, also called small clumps or entanglements, are larger freely dispersed mycelia where the main hypha is not identifiable. These are often referred as Bartificially overlapping hyphae^ (Cox et al. 1998; Paul and Thomas 1998; Posch et al. 2012). A further dispersed morphological class is clumps, also called large clumps (Cox et al. 1998; Paul and Thomas 1998; Posch et al. 2012). These consist of aggregated or clumped hyphae (Cox et al. 1998). Large clumps are distinguished from socalled pellets by the missing of a dense core. Latter is a central dark region in the center of the aggregate, which is typical for pellets. The core is surrounded by a brighter outer mycelial region, the Bhairy^ annular region. Pellets have the size of several hundred micrometer to more than 1 ml (Cox et al. 1998; Paul and Thomas 1998). Cox et al. (1998) pointed out that pellets are three-dimensional, which possibly cannot be sufficiently covered by image analysis based on microscopy. As a more appropriate investigation, a chamber on the microscope stage to preserve the shape is proposed. Methods making pictures on microscope slides assume pellets to be nearly spherical (Cox et al. 1998). Various morphological parameters are evaluated concerning length/size/diameter of hyphae and hyphal aggregates. Morphological evaluation of pellets focuses apart from size evaluation, especially on the description of the annular area and the annular area compared to the core (Paul and Thomas 1998). Although flow cytometry has often been applied for the morphological description of microorganisms as bacteria (Ehgartner et al. 2015; Langemann et al. 2016), investigations of filamentous organisms apart from the spore stadium (Ehgartner et al. 2016a, b) are scarce. Therefore, the main reason is the (...truncated)