- Technical Notes
- Open Access
Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites
© The Author(s) 2018
- Received: 12 June 2018
- Accepted: 9 August 2018
- Published: 21 August 2018
Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger.
We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors d-2-hydroxyvaleric acid and l-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300–800 μm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50–150 μm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5–2.3 g L−1 were achieved in the CELL-tainer® system, which is about 30–50% of the titres achieved under STR conditions.
This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.
- Single-use wave-mixed bioreactor
- Aspergillus niger
- Heterologous gene expression
- Talcum microparticle
- Tet-on system
Filamentous fungi are of great economic importance as cell factories in industrial biotechnology. Due to their metabolic diversity, high production capacity, secretion efficiency, and the capability of conducting post-translational modifications, filamentous fungi like Aspergillus niger are widely exploited as cell factories for the production of organic acids, proteins and enzymes [1, 2]. Of interest is also their natural ability to synthesize bioactive secondary metabolites such as non-ribosomal peptides (NRPs) in large amounts [3, 4]. Due to the dramatic increase in the amount of pathogenic bacteria, which are resistant to commonly used antibiotics, the search for new antibiotics and other pharmaceuticals from fungal resources became one recent focus of the fungal research community [5–7].
An interesting class of bioactive fungal NRPs are the cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide and PF1022, all of which consist of alternating units of N-methyl amino and α-hydroxy acids. CDPs exhibit antibacterial, antifungal, insecticidal, anthelmintic or even anticancer activities. Two of these compounds are commercialized drugs: fusafungine (a mixture of enniatins) is applied as antibacterial compound for treating throat infections in humans and emodepside (a semisynthetic derivative of PF1022A) used as anthelmintic compound in the veterinary medicine . We showed recently that heterologous expression of the NRPS encoding gene esyn1 from Fusarium oxysporum in A. niger results in enniatin B production in multigram scale per litre during a fed-batch cultivation . A prerequisite to achieve such titres is controlled expression of the ESYN encoding gene esyn1 under the Tet-on system  and feeding of the strain with the enniatin B precursors d-2-hydroxyvaleric acid (d-Hiv) and l-valine during cultivation. We also showed that this esyn1 expressing strain DS3.1 became independent from d-Hiv feeding after introduction of the kivR gene from F. oxysporum into its genome (resulting in strain ÖV4.10). Constitutive expression of multiple copies of the kivR gene made ÖV4.10 competent to use its intracellular α-ketovaleric acid pool for d-Hiv generation. A direct comparison of both strains in 20 mL shake flask cultures revealed that the enniatin B titres achieved with strain ÖV4.10 are about 75% of the enniatin B titres of strain DS3.1 . Polycistronic expression of both esyn1 and kivR under control of the Tet-on expression system resulted in 40% of the product titres that were achieved with the strain DS3.1 in shake flask cultures. This finding proved that polycistronic secondary metabolite biosynthesis is possible in A. niger and suggested that the KivR enzyme catalyses the rate-limiting step in enniatin B biosynthesis . It was furthermore demonstrated that A. niger is (i) a superior expression host not only for enniatin B, but also for beauvericin and bassianolide, by producing the highest titres ever reported for heterologous hosts as well as for natural producing organisms ; and (ii) an ideal platform strain for the production of new-to-nature CDPs obtained by designing and expressing hybrid CDP synthetases in A. niger [13, 14]. Indeed, some of the novel CDPs displayed considerably higher bioactivities compared to their parental CDPs and reference drugs . The genetic (i.e. synthetic biology) tools to reprogram A. niger to overproduce bioactive non-ribosomal peptides at highest level have thus been successfully established and can be applied further to efficiently screen novel secondary metabolites from any fungal resource for desired bioactivities via heterologous expression of their encoding gene clusters in A. niger.
Apart from the necessity to discover and express novel bioactive compounds, another important technological development for their (industrial) production is the application of single-use bioreactors. These offer many opportunities for filamentous fungi as cell factories in pharmaceutical and cosmetics industries as recently highlighted in a White Paper of the EUROFUNG consortium . Single-use bioreactors provide a platform technology, “which is safer (decreased risk of microbial contamination and cross-contamination), greener (reduced requirements for sterilization and cleaning), faster and more flexible (easy process and product changes), and cheaper (saving of time and costs)” . In general, single-use bioreactors are reactor platforms that comprise of a non-disposable part of controllers, motor and housing, and a disposable bag, in which the cultivation is conducted. Several designs of single-use bioreactors were commercialized during the last decade. An increased process flexibility, high turnover and lower risk of cross contaminations led eventually to the penetration of this technology into the pharmaceutical industry [15, 16]. Several reactor types have been described and were compared with conventional non-disposable stirred tank bioreactors based on engineering parameters and process performances [17–22]. Single-use wave-mixed bioreactors are nowadays of interest for the cultivation of shear-sensitive cells, such as mammalian cell lines, insect cell lines , phototrophic algae  and plant hairy roots . Also, successful cultivations with wave-induced agitation have been reported for the filamentous basidiomycetes Flammmulina velutipes and Pleurotus sapidus  as recently reviewed in .
So far, no reports on submerged cultivations of A. niger in single-use wave-mixed bioreactor systems have been published. A. niger and in general filamentous fungi adopt different microscopic and macroscopic morphological forms during submerged cultivations, the latter varying from a freely dispersed mycelium over loose mycelial clumps to dense pellets [28, 29]. Whereas dispersed mycelia increase medium viscosity and are sensitive to shear stress, oxygen and nutrient transfer is impaired in the core of pellets [30–32]. Process parameters, which affect the development of different macromorphologies range from inoculum concentration  and medium composition [31, 34] over reactor shape, stirrer geometry and stirrer speed (generating different shear forces) , aeration rate  to pH and temperature . It was shown that macroscopic morphologies affect protein secretion rates in aspergilli in many, but not all cases [36, 38–47]. Unfortunately, the adequate macroscopic fungal morphology for given target products including organic acids, secreted proteins or secondary metabolites varies, cannot be generalized, and the advantages and disadvantages of mycelial or pellet cultivation have to be carefully adjusted for a given process and the final production scale [32, 35, 41, 48–57].
The main objective of this study was thus to determine whether a two-dimensional (horizontal and vertical) rocking-motion wave-mixed bioreactor is a viable alternative to well-established conventional STRs for the cultivation of A. niger. We therefore systematically analyzed for the first time the impact of controlled low shear stress cultivation on the growth of A. niger, the evolution of its macroscopic morphology and its product formation by cultivating the fungus in the wave-mixed bioreactor CELL-tainer. The two enniatin B-expressing strains DS3.1 and ÖV4.10 described above were used for this survey. They were cultivated under nutrient-limited fed-batch cultivation conditions with identical medium compositions, feeding schemes, temperature and pH control as described in our previously published STR cultivations  to allow a direct comparison. Naturally, we induced synthesis of enniatin B as model product via induction of the Tet-on expression system using the inductor doxycycline as previously described for STR cultivations .
Growth and morphological parameters obtained from A. niger during submerged fed-batch cultivations
Strain A. niger
Specific growth rate µmax (h−1)
Cbiomass,max (gdryweight kg−1)
+ (~ 15%)
+++ (~ 90%)
+++ (~ 90%)
Fed-batch + talcum
+ (~ 15%)
Fed-batch + talcum
+ (~ 15%)
Fed-batch + talcum
+ (~ 15%)
Fed-batch + talcum
+ (~ 15%)
Increased process flexibility, high turnover and lower risk of cross-contaminations in single-use bioreactor systems underpin the importance of this technology for the pharmaceutical industry [15, 16]. Another advantage is the design flexibility that made different geometries available. The aim of this study was to investigate the impact of low shear forces generated in the single-use wave-mixed CELL-tainer on growth, development of macroscopic morphologies and enniatin B product formation in A. niger and to compare the data with physiological and morphological data obtained for A. niger STR cultivations in a BioFlo3000 STR.
A critical parameter for any process design for aerobic cultivations is the volumetric oxygen transfer coefficient kLa of the reactor. The kLa value describes the capacity of a given system to introduce oxygen into the liquid phase. To increase the kLa, vigorous stirring or shaking is necessary to break up air bubbles, thereby increasing the area of the liquid–gas interface. Stirring is the main reason for the high shear stress in STRs. While one-dimensional rocking reactors exhibit relatively low kLa values of 1 to 5 h−1, kLa-values beyond 400 h−1 were reported for wave-mixed bioreactors [17, 27], similar to values obtained in lab-scale stirred tank reactors with stirring rates as applied in this study. The CELL-tainer concept was thus successfully applied for aerobic fed-batch cultivations of Escherichia coli, in which typical growth rates and recombinant protein expression levels were achieved . Notably, specific growth rates for A. niger cultivated under identical nutrient-limited fed-batch cultivations under wave-mixed conditions are considerably higher (0.24 h−1) in comparison to STR cultivations (0.15 h−1), indicating that high shear forces under stirred agitations indeed limit the growth of A. niger. It is conceivable that reduced shear stress under wave-mixed conditions allows self-assembly of A. niger growth units, which generate the natural architecture for a very heterogeneous pellet population, which is eventually important to ensure fast nutrient uptake, and thus biomass accumulation. This data also suggests that macromorphological pellet heterogeneity of A. niger can be beneficial to the growing population to grow as fast as possible. This hypothesis is highly speculative and has not been studied yet in filamentous fungi; however, many studies, which investigated bacterial population heterogeneities indeed propose that even single cells follow a population-based strategy to allocate different tasks to different cells (e.g. to specialise on different metabolic pathways) in order to maximise population growth [61–63]. Given that macromorphological pellet heterogeneity provides a fitness advantage to A. niger, one would expect that this advantage will get lost when the pellet size becomes homogeneous. This is exactly what we have observed after the addition of talcum to wave-mixed cultures: the specific growth rate dropped to 0.11 h−1 and glucose was metabolised much slower (Figs. 1, 2 and Table 1). On the one hand, talcum microparticles could probably interfere with important physical factors that control the self-organisation of growth units, especially in early growth phases (note that RQ variations during the first 10 h were only evident for wave-mixed cultivations lacking talcum; Fig. 3), on the other hand, talcum microparticles might cause additional friction stress to A. niger. We are currently studying this hypothesis further on the molecular level.
In conclusion, CELL-tainer cultivations seem to be superior for fast biomass accumulation of A. niger and could thus be interesting for growth-coupled product formation such as primary metabolites (organic acids) or even secreted proteins. The synthesis of enniatin B, however, is linked to secondary metabolism of A. niger. We showed previously that reduced growth rates and even carbon starvation are beneficial for higher enniatin B yields, likely because it generally activates secondary metabolism of A. niger [9, 58, 64]. Hence, one would expect lower enniatin B yield in fast-growing cultures like in wave-mixed cultivations of DS3.1 lacking talcum compared with slow-growing wave-mixed cultures of DS3.1 with talcum. This is indeed what we have observed in this study (Fig. 4).
In this study, we present a single-use wave-mixed bioreactor concept as an alternative to well-established conventional STR cultivations for A. niger for the first time. The CELL-tainer system can ensure higher specific growth rates than in the STR cultivations and, interestingly, does not require the addition of antifoam agents as no foam formation was observed (data not shown). It thus makes the addition of antifoaming agents needless, as previously reported for wave-mixed bioreactor systems . We thus conclude that this reactor system has a very high potential for growth-coupled production processes for A. niger, in which the formation of macromorphologies should remain unrestricted. For secondary metabolite production, however, wave-mixed cultivation needs to be further optimized with respect to controllable pellet morphologies.
Strains and media
Submerged cultivations were performed with 6.6 L BioFlo3000 bioreactors (New Brunswick Scientific, NJ, USA) and in the 20 L disposable wave-mixed bioreactor CELL-tainer®. The main fermentation settings were already described in Refs. [9, 58, 64]. In brief, fed-batch cultivations were inoculated with spore suspension of A. niger strains with a spore titre of 109 conidia L−1 and 0.003% yeast extract to improve germination. Initial batch cultivations were conducted with 4 L of cultivation medium, containing 0.8% glucose (w v−1). When reaching the late exponential growth phase, the Tet-on system was induced by addition of 20 µg mL−1 doxycycline (Dox). At the same time, the feed was started with a feeding rate of F = 0.046 L h−1. The feed medium (1.5 L) contained 5% glucose, 0.5% yeast extract, 0.1% casamino acids, 20 mM d-2-hydoxyvaleric acid (d-Hiv) and 20 mM l-valine for DS3.1 and 40 mM l-valine for ÖV4.10 cultivations additionally to the initial fermentation medium. In total, 100 µg mL−1 of Dox were added to the culture (20 µg mL−1 of Dox addition every 4–7 h). A temperature of 30 °C was kept constant throughout the whole cultivation process and a pH-value of 3.0 was maintained by addition of 2 M NaOH or 1 M HCl, respectively. The aeration rate was kept constant at 1 L min−1.
The rocking motion of the CELL-tainer was initially set to 5 rpm during the germination phase of 5–6 h. The rocking speed was controlled between 15 and 45 rpm after germination, depending on the dissolved oxygen (DO) value output (DO-controlled rpm mode).
Metabolite and gas analysis
Metabolite analysis of the supernatant samples (i.e. carbohydrates and short chain carboxylic acids) was conducted with a refractometric detector on HPLC systems as previously described . Enniatin extraction and measurements were performed as previously described . In brief, defined amounts of biomass were subjected to ethyl acetate extraction, centrifuged and evaporated. Samples were dissolved in 40% isopropanol, diluted 10- to 10,000-fold and enniatin concentration quantified by ESI-Triple-Quadrupol-MS (6460 Series, Agilent Technologies) analysis in the multiple reaction monitoring mode . Enniatin isolated from F. oxysporum was used as an external standard to generate a calibration curve out of the manually integrated peak areas for each measurement. The O2 and CO2 content in the off-gas was measured with the Blue-In-One analyser (Bluesens).
Microscopy and determination of pellet size
Mycelia and pellets were stained with lactophenol blue and analysed with differential interference contrast (DIC) microscopy using Leica DM 5000 CS and evaluated using Leica’s microscope software LAS V4.3. Image analysis of the pellet width and length was performed using ImageJ 1.51p. Disperse mycelia, loose clumps or dense pellets were each defined as macromorphological unit given that they were individually distinguishable by microscopy from each other. More than hundred macromorphological units were analysed per sample.
Off-gas data were fitted with the fitting toolbox of SigmaPlot TableCurve 2D v5.01 (Systat Software Inc.).
VM and SJ conceived of the study. TK, AMMA, ZT conducted experiments. TK, VM and SJ analysed and interpreted data and generated figures. TK, SJ and VM wrote the manuscript. PN contributed with discussions. All authors read and approved the final manuscript.
The authors wish to thank Simon Boecker and Prof. Roderich Süssmuth (TU Berlin) for support with enniatin B measurements.
The authors declare that they have no competing interests.
Availability of data and materials
Data and materials generated during and/or analysed in the current study are available from the corresponding author on request.
Consent for publication
Ethics approval and consent to participate
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Meyer V, Wu B, Ram AFJ. Aspergillus as a multi-purpose cell factory: current status and perspectives. Biotechnol Lett. 2011;33:469–76.View ArticleGoogle Scholar
- Cairns TC, Nai C, Meyer V. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol Biotechnol. 2018;5:13.View ArticleGoogle Scholar
- Meyer V. Genetic engineering of filamentous fungi-progress, obstacles and future trends. Biotechnol Adv. 2008;26:177–85.View ArticleGoogle Scholar
- Lubertozzi D, Keasling JD. Developing Aspergillus as a host for heterologous expression. Biotechnol Adv. 2009;27:53–75.View ArticleGoogle Scholar
- Meyer V, Andersen MR, Brakhage AA, Braus GH, Caddick MX, Cairns TC, et al. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biol Biotechnol. 2016;3:6.View ArticleGoogle Scholar
- Nielsen JC, Grijseels S, Prigent S, Ji B, Dainat J, Nielsen KF, et al. Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nat Microbiol. 2017;2:17044.View ArticleGoogle Scholar
- Weinhold M, Mast-Gerlach E, Meyer V. Vita activa in biotechnology: what we do with fungi and what fungi do with us. Fungal Biol Biotechnol. 2017;4:14.View ArticleGoogle Scholar
- Dang T, Süssmuth RD. Bioactive peptide natural products as lead structures for medicinal use. Acc Chem Res. 2017;50:1566–76.View ArticleGoogle Scholar
- Richter L, Wanka F, Boecker S, Storm D, Kurt T, Vural Ö, et al. Engineering of Aspergillus niger for the production of secondary metabolites. Fungal Biol Biotechnol. 2014;1:4.View ArticleGoogle Scholar
- Meyer V, Wanka F, Van Gent J, Arentshorst M, Den Van, Hondel CAMJJ, Ram AFJ. Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger. Appl Environ Microbiol. 2011;77:2975–83.View ArticleGoogle Scholar
- Schuetze T, Meyer V. Polycistronic gene expression in Aspergillus niger. Microb Cell Fact. 2017;16:162.View ArticleGoogle Scholar
- Boecker S, Grätz S, Kerwat D, Adam L, Schirmer D, Richter L, et al. Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides. Fungal Biol Biotechnol. 2018;5:4.View ArticleGoogle Scholar
- Zobel S, Boecker S, Kulke D, Heimbach D, Meyer V, Süssmuth RD. Reprogramming the biosynthesis of cyclodepsipeptide synthetases to obtain new enniatins and beauvericins. ChemBioChem. 2016;17:283–7.View ArticleGoogle Scholar
- Steiniger C, Hoffmann S, Mainz A, Kaiser M, Voigt K, Meyer V, et al. Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering. Chem Sci. 2017;8:7834–43.View ArticleGoogle Scholar
- Langer ES. Single-use bioreactors get nod. Genet Eng Biotechnol News. 2012;32:16.View ArticleGoogle Scholar
- Jacquemart R, Vandersluis M, Zhao M, Sukhija K, Sidhu N, Stout J. A single-use strategy to enable manufacturing of affordable biologics. Comput Struct Biotechnol J. 2016;14:309–18.View ArticleGoogle Scholar
- Oosterhuis NM, Neubauer P, Junne S. Single-use bioreactors for microbial cultivation. Pharm. Bioprocess. 2013;1:167–77.View ArticleGoogle Scholar
- Eibl R, Löffelholz C, Eibl D. Single-use bioreactors-an overview. Single-use technology in biopharmaceutical manufacture. Hoboken: Wiley; 2011. p. 33–51.View ArticleGoogle Scholar
- Klöckner W, Diederichs S, Büchs J. Orbitally shaken single-use bioreactors. New York: Springer; 2013. p. 45–60.Google Scholar
- Eibl R, Kaiser S, Lombriser R, Eibl D. Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl Microbiol Biotechnol. 2010;86:41–9.View ArticleGoogle Scholar
- Neubauer P, Cruz N, Glauche F, Junne S, Knepper A, Raven M. Consistent development of bioprocesses from microliter cultures to the industrial scale. Eng Life Sci. 2013;13:224–38.View ArticleGoogle Scholar
- Hillig F, Pilarek M, Junne S, Neubauer P. Cultivation of marine microorganism in single-use systems. Adv Biochem Eng Biotechnol. 2014;138:179–206.Google Scholar
- Singh V. Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology. 1999;30:149–58.View ArticleGoogle Scholar
- Lehmann N, Rischer H, Eibl D, Eibl R. Wave-mixed and orbitally shaken single-use photobioreactors for diatom algae propagation. Chem Ing Tech. 2013;85:197–201.View ArticleGoogle Scholar
- Eibl R, Werner S, Eibl D. Bag bioreactor based on wave-induced motion: characteristics and applications. Berlin: Springer; 2010. p. 55–87.Google Scholar
- Jonczyk P, Takenberg M, Hartwig S, Beutel S, Berger RG, Scheper T. Cultivation of shear stress sensitive microorganisms in disposable bag reactor systems. J Biotechnol. 2013;167:370–6.View ArticleGoogle Scholar
- Junne S, Neubauer P. How scalable and suitable are single-use bioreactors? Curr Opin Biotechnol. 2018;53:240–7.View ArticleGoogle Scholar
- Casas López JL, Sánchez Pérez JA, Fernández Sevilla JM, Rodríguez Porcel EM, Chisti Y. Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus. J Biotechnol. 2005;116:61–77.View ArticleGoogle Scholar
- Kim JH, Lebeault JM, Reuss M. Comparative study on rheological properties of mycelial broth in filamentous and pelleted forms. Eur J Appl Microbiol Biotechnol. 1983;18:11–6.View ArticleGoogle Scholar
- Gibbs PA, Seviour RJ, Schmid F. Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit Rev Biotechnol. 2000;20:17–48.View ArticleGoogle Scholar
- Kaup B-A, Ehrich K, Pescheck M, Schrader J. Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example. Biotechnol Bioeng. 2008;99:491–8.View ArticleGoogle Scholar
- Driouch H, Hänsch R, Wucherpfennig T, Krull R, Wittmann C. Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles. Biotechnol Bioeng. 2012;109:462–71.View ArticleGoogle Scholar
- Papagianni M, Mattey M. Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology. Microb Cell Fact. 2006;5:3.View ArticleGoogle Scholar
- Wucherpfennig T, Lakowitz A, Krull R. Comprehension of viscous morphology—evaluation of fractal and conventional parameters for rheological characterization of Aspergillus niger culture broth. J Biotechnol. 2013;163:124–32.View ArticleGoogle Scholar
- Jüsten P, Paul GC, Nienow AW, Thomas CR. Dependence of mycelial morphology on impeller type and agitation intensity. Biotechnol Bioeng. 1996;52:672–84.View ArticleGoogle Scholar
- Wongwicharn A, McNeil B, Harvey LM. Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D. Biotechnol Bioeng. 1999;65:416–24.View ArticleGoogle Scholar
- Das RK, Brar SK. Enhanced fumaric acid production from brewery wastewater and insight into the morphology of Rhizopus oryzae 1526. Appl Biochem Biotechnol. 2014;172:2974–88.View ArticleGoogle Scholar
- Krijgsheld P, Bleichrodt R, van Veluw GJ, Wang F, Müller WH, Dijksterhuis J, et al. Development in Aspergillus. Stud Mycol. 2013;74:1–29.View ArticleGoogle Scholar
- Amanullah A, Christensen LH, Hansen K, Nienow AW, Thomas CR. Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production. Biotechnol Bioeng. 2002;77:815–26.View ArticleGoogle Scholar
- Wucherpfennig T, Kiep KA, Driouch H, Wittmann C, Krull R. Morphology and rheology in filamentous cultivations. Adv Appl Microbiol. 2010;72:89–136.View ArticleGoogle Scholar
- Wucherpfennig T, Hestler T, Krull R. Morphology engineering—osmolality and its effect on Aspergillus niger morphology and productivity. Microb Cell Fact. 2010;10:58.View ArticleGoogle Scholar
- Driouch H, Sommer B, Wittmann C. Morphology engineering of Aspergillus niger for improved enzyme production. Biotechnol Bioeng. 2010;105:1058–68.Google Scholar
- Driouch H, Roth A, Dersch P, Wittmann C. Filamentous fungi in good shape: microparticles for tailor-made fungal morphology and enhanced enzyme production. Bioeng Bugs. 2011;2:100–4.View ArticleGoogle Scholar
- Sitanggang AB, Wu H-S, Wang SS, Ho Y-C. Effect of pellet size and stimulating factor on the glucosamine production using Aspergillus sp. BCRC 31742. Bioresour Technol. 2010;101:3595–601.View ArticleGoogle Scholar
- Choy V, Patel N, Thibault J. Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30. Biotechnol Prog. 2011;27:1544–53.View ArticleGoogle Scholar
- Tepwong P, Giri A, Ohshima T. Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes. Mycoscience. 2012;53:102–12.View ArticleGoogle Scholar
- Amanullah A, Blair R, Nienow AW, Thomas CR. Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae. Biotechnol Bioeng. 1999;62:434–46.View ArticleGoogle Scholar
- Clark DS, Ito K, Horitsu H. Effect of manganese and other heavy metals on submerged citric acid fermentation of molasses. Biotechnol Bioeng. 1966;8:465–71.View ArticleGoogle Scholar
- Schügerl K, Wittler R, Lorenz T. The use of molds in pellet form. Trends Biotechnol. 1983;1:120–3.View ArticleGoogle Scholar
- Papagianni M. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv. 2004;22:189–259.View ArticleGoogle Scholar
- Pirt SJ, Callow DS. Continuous-flow culture of the filamentous mould Penicillium chrysogenum and the control of its morphology. Nature. 1959;184:307–10.View ArticleGoogle Scholar
- Rocha-Valadez JA, Galindo E, Serrano-Carreón L. The influence of circulation frequency on fungal morphology: a case study considering Kolmogorov microscale in constant specific energy dissipation rate cultures of Trichoderma harzianum. J Biotechnol. 2007;130:394–401.View ArticleGoogle Scholar
- Braun S, Vecht-Lifshitz SE. Mycelial morphology and metabolite production. Trends Biotechnol. 1991;9:63–8.View ArticleGoogle Scholar
- Metz B, Kossen NWF. The growth of molds in the form of pellets—a literature review. Biotechnol Bioeng. 1977;19:781–99.View ArticleGoogle Scholar
- Elmayergi H, Scharer JM, Moo-Young M. Effects of polymer additives on fermentation parameters in a culture of A. niger. Biotechnol Bioeng. 1973;15:845–59.View ArticleGoogle Scholar
- Ruohang W, Webb C. Effect of cell concentration on the rheology of glucoamylase fermentation broth. Biotechnol Tech. 1995;9:55–8.View ArticleGoogle Scholar
- Jørgensen TR, Goosen T, van den Hondel CA, Ram AF, Iversen JJ. Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway. BMC Genomics. 2009;10:44.View ArticleGoogle Scholar
- Nitsche BM, Jørgensen TR, Akeroyd M, Meyer V, Ram AF. The carbon starvation response of Aspergillus niger during submerged cultivation: insights from the transcriptome and secretome. BMC Genomics. 2012;13:380.View ArticleGoogle Scholar
- Hillig F. Impact of cultivation conditions and bioreactor design on docosahexaenoic acid production by a heterotrophic marine microalga. Technische Universität Berlin, Faculty III-Process Sciences; 2014.Google Scholar
- Hillig F, Annemüller S, Chmielewska M, Pilarek M, Junne S, Neubauer P. Bioprocess development in single-use systems for heterotrophic marine microalgae. Chem Ing Tech. 2013;85:153–61.View ArticleGoogle Scholar
- Wang X, Kang Y, Luo C, Zhao T, Liu L, Jiang X, et al. Heteroresistance at the single-cell level: adapting to antibiotic stress through a population-based strategy and growth-controlled interphenotypic coordination. MBio. 2014;5:e00942.Google Scholar
- Nikolic N, Schreiber F, Dal Co A, Kiviet DJ, Bergmiller T, Littmann S, et al. Cell-to-cell variation and specialization in sugar metabolism in clonal bacterial populations. PLoS Genet. 2017;13:e1007122.View ArticleGoogle Scholar
- Martins BM, Locke JC. Microbial individuality: how single-cell heterogeneity enables population level strategies. Curr Opin Microbiol. 2015;24:104–12.View ArticleGoogle Scholar
- Jørgensen TR, Nitsche BM, Lamers GE, Arentshorst M, Van Den Hondel CA, Ram AF. Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero. Appl Environ Microbiol. 2010;76:5344–55.View ArticleGoogle Scholar
- Lemoine A, Maya Martίnez-Iturralde N, Spann R, Neubauer P, Junne S. Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two- and a novel three-compartment scale-down bioreactor. Biotechnol Bioeng. 2015;112:1220–31.View ArticleGoogle Scholar