Biotechnology Letters

, Volume 34, Issue 11, pp 1975–1982 | Cite as

Microparticle based morphology engineering of filamentous microorganisms for industrial bio-production

  • Robert Walisko
  • Rainer Krull
  • Jens Schrader
  • Christoph Wittmann
Review

Abstract

Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.

Keywords

Aspergillus Core–shell pellet Filamentous microorganisms Microparticle enhanced cultivation Morphology engineering Mycelium Pellet Penicillium Streptomyces 

Biotechnological importance of filamentous microorganisms

Filamentous microorganisms, including eukaryotic filamentous fungi as well as prokaryotic actinomycetes provide important biotechnological products such as enzymes, pharmaceuticals, bulk and fine chemicals. Among the chemicals supplied annually by different species of the filamentous fungus Aspergillus are 1.6 million tons of citric acid (Bennett 2010; Dodds and Gross 2007), 100,000 tons of gluconic acid (Singh and Kumar 2007) and 4,000 tons of itaconic acid (Willke and Vorlop 2001). Strains of Aspergillus terreus or Penicillium citrinum supply high-value nutraceuticals and pharmaceuticals such as statins (Barrios-González and Miranda 2010). Moreover, filamentous fungi have been found particularly efficient in recombinant protein and enzyme synthesis, comprising pectinases, proteases, amyloglucosidases, cellulases, hemicellulases, lipases (Jones 2007), β-galactosidases (O’Connell and Walsh 2008), chymosin for cheese production (Kumar et al. 2010), and fructosyltransferases (Maiorano et al. 2008). Filamentous bacteria such as species of Streptomyces, Amycolatopsis and Nocardia are widely applied producers of anti-viral and anti-cancer drugs, modulators of immune response, enzyme inhibitors, herbicides insecticides, and anti-parasitic compounds (Thompson et al. 2002).

Link between growth morphology and production performance

In contrast to unicellular microorganisms, filamentous species grow highly heteromorphously and are therefore less favorable for production (van Wezel et al. 2006; Sohoni et al. 2012). Fungal conidia, when used as inocula, germinate into single hyphae during the initial cultivation phase, a process that is often accompanied by spore adhesion leading to larger aggregates (Kelly et al. 2006). Other fungal strains, poor in producing conidia are directly inoculated as hyphal mycelium. The same also holds for filamentous bacteria. Depending on strain physiology, culture conditions and process parameters the cells later form macroscopic mycelia or also pellets. The consequential problems related to filamentous growth include (i) slow growth rates and highly viscous cultures, (ii) large mycelial clumps with significant 2 and nutrient transfer problems towards the centre, (iii) mixing requirements that necessitate high stirrer speeds, resulting in uncontrolled fragmentation and lysis of the mycelium, and (iv) complex and therefore expensive downstream processing (van Wezel et al. 2006).

Due to this, production performance is largely determined by the underlying morphological shape (Gibbs et al. 2000; Papagianni and Mattey 2006; Zhang et al. 2007). Since many decades this has stimulated efforts to influence and control the morphology. Classically, approaches have relied on empirical variation of process parameters. As an example, the addition of surfactant compounds proved to influence morphology and formation of geldanamycin in Streptomyces hygroscopicus in a positive way (Dobson et al. 2008). In addition, also inoculum concentration, type and concentration of carbon, nitrogen and phosphate sources, as well as sources of metals and other ions, dissolved 2 and dissolved carbon dioxide tension, pH, temperature and increased stirring rates have been applied as reviewed comprehensively (Papagianni 2004).

However, despite much effort, none of these methods allows a precise adjustment of morphology, but rather leads to mixtures of different growth forms. In addition, such variation of process parameters is limited to the operational process window. As example, pH 3.0 applied throughout the process to induce mycelia might be incompatible with the stability of recombinant protein products having an optimum at higher pH (Driouch et al. 2010b). Likewise, increasing power input to obtain smaller pellets will cause increased energy costs. Genetic manipulation is an interesting alternative way to influence morphology. The morphogenesis of fungi involves conserved signal transduction cascades to sense environmental changes and control a complex cellular machinery of enzymes synthesizing the fungal cell wall (Shapiro et al. 2011). Genetic perturbations, interfering with these morphological programs, have shown value in biotechnology applications. As example with bacteria, the enhanced expression of the morphogene ssgA, involved in peptidoglycan maintenance, led to fragmented growth in several species of Streptomyces otherwise growing as pellets (van Wezel et al. 2006). Implemented in Streptomyces coelicolor, the ssgA mutation allowed faster growth and improved production of the antibiotic undecylprodigiosin. Similarly, positive effects could be shown for the ssgA mutant of Streptomyces lividans, producing tyrosinase. Despite these promising case studies, a broader application of genetic control is currently limited by our still poor understanding of the underlying complex regulatory circuits and negative side effects such as poor growth and stress tolerance that might occur upon genetic perturbation.

Impact of microparticles on growth morphology and production behavior

As novel technology, inorganic microparticles with a diameter of a few μm to mm were recently found to have a large impact on growth morphology of filamentous microorganisms. In a pioneering work, microparticles were applied to influence the morphology of the filamentous fungus Caldariomyces fumago, a natural producer of a heme-containing chloroperoxidase, a biocatalyst of interest for the production of chiral chemicals (Kaup et al. 2008). It was observed by the authors that two different kinds of microparticles (talc powder, 3MgO·4SiO2·H2O and aluminum oxide, Al2O3, both with a diameter below about 40 μm), added to the cultivation medium, influenced growth as well as product formation. For different concentrations of the micro material varied from 0.05 g l−1 to 25 g l−1, the enzyme titer could be increased up to tenfold. This corresponded to a fivefold enhancement of specific productivity to 10,000 U (g cell dry mass)−1 day−1. Supported by the stimulating effects, the new technique was named microparticle enhanced cultivation (MPEC). Microscopic analysis revealed that microparticles caused a change of growth morphology from pellets to single hyphae. Improved supply of O2 and nutrients to the dispersed cells was regarded as the reason for the improved enzymatic activity and productivity of the biomass, since their availability in the pellet interior is typically limited as shown, for example, by direct in situ measurement using micro electrodes (Gibbs et al. 2000; Rawool et al. 2001; Hille et al. 2005; Kelly et al. 2006; van Wezel et al. 2006; Dobson et al. 2008). Stimulating effects were also demonstrated by using larger particles. In a microtiter plate-based approach, glass beads with diameters of 3–4 mm enabled dispersed growth morphology for Streptomyces and allowed a much more reproducible cultivation as compared to control experiments (Sohoni et al. 2012). Similarly, glass beads added to submerged shake-flask cultures of S. hygroscopicus reduced pellet size by 70 %, which was accompanied by simultaneous increase of the geldanamycin yield by 88 % (Dobson et al. 2008). Overall, favorable microparticle effects are observed for a range of filamentous microorganisms. For different species tested so far, a reduction of pellet size up to single hyphae growth was noticed (Table 1).
Table 1

Impact of microparticles on different filamentous microorganisms as adapted from Kaup et al. (2008) and Sohoni et al. (2012)

Organism

DSMZ number

Division

Pellet sizea-particles (mm)

Pellet sizea + particles (mm)

Single hyphae/cellsb

Exemplary products

Penicillium digitatum

62840

Ascomycota

5–60

2.2–1.2

+

Enzymes

Penicillium chrysogenum

848

Ascomycota

2–60

0.1–3

++

Penicillium

Emericella nidulans

820

Ascomycota

1–3

0.05–0.2

++

Cholic acid, conversion of steroids

Aspergillus niger

821

Ascomycota

3–8

0.1–1.5

+

Citric acid, oxalic acid, enzymes

Acremonium chrysogenum

880

Ascomycota

1–10

0.1–0.7

+

Proteases, cephalosporins

Pleurotus sapidus

8266

Basidiomycota

30

0.1–6

+

Enzymes

Rhizopus oryzae

907

Zygomycota

80

1–5

+

Steroids

Chaetomium globosum

1962

Ascomycota

1–5

0.2–3.5

+

Cellulase

Streptomyces aureofaciens

40127

Eubacteria

0.9–2.1

0.06–0.50

+

Chlor–tetracycline

Streptomyces coelicolor A3(2)

−c

Eubacteria

0.26–0.33

0.058

+

Antibiotics

aThe pellet size was estimated by microscopy

bSingle hyphae/cells: ++ formed predominantly, + formed significantly

cClassification number not provided

Microparticles for tailor-made morphology engineering––defined pellets and mycelia

More recently, the use of microparticles could be extended by fine adjustment of particle material, size and concentration into a concept of rational morphology engineering (Driouch et al. 2010b, Sohoni et al. 2012). This allows specific engineering of pellet size by variation of the particle concentration, as demonstrated for Aspergillus niger (Fig. 1) where the growth morphology could be precisely adjusted into pellets of distinct size in the range of 100–1,000 μm as well as into free mycelia of different fragmentation degree, simply by increasing the amount of talc particles added to the culture prior to inoculation. It turned out that certain conditions favor the production of homologous and heterologous enzymes in different strains of A. niger. The addition of talc particles (6 μm in diam.) at 10 g l−1 resulted in reproducible, mycelial growth of different strains, otherwise growing as clumps. In line with this, the amount, activity and specific activity of secreted fructofuranosidase and glucoamylase, respectively, was enhanced. Interestingly, the formation of the undesired by-product oxalate was reduced almost completely in cultivations with microparticles. This effect might be explained by an improved O2 supply of the cells during dispersed growth, as compared to O2 limitation typically observed for dense pellets (Hille et al. 2005). Under O2 limited conditions (in pellets) pyruvate cannot efficiently be metabolized in the TCA cycle. Aspergillus then converts excess pyruvate into oxalate, an undesired by-product with regard to carbon loss (Kubicek et al. 1988).
Fig. 1

Influence of microparticles on morphological development of A. niger SKAn1015. The resulting morphology is shown for different time points without microparticles (a–h) and with 10 g l−1 3·MgO·4SiO2·H2O (talc) microparticles (6 μm particle size) added to the culture prior to inoculation (ip). Figures h and p display detailed views with higher magnification from the corresponding time point of 6 h of the cultivation. The arrows in subfigure j mark single spores which have partly germinated. The figure has been adapted from Driouch et al. (2010b)

Further analysis investigated the effect of particle size. Whereas highly productive hyphal growth was accomplished by addition of 4 g talc (6 μm diam.) l−1, concentrations higher than 25 g l−1 were needed in case of larger aluminum oxide particles (15 μm diam.) to achieve the same effect.

Using a reporter strain of A. niger which co-expressed the target enzyme glucoamylase with green fluorescent protein (GFP2), the influence of the microparticles was visualized on a cellular level (Driouch et al. 2010b). It could be demonstrated for the pellet-growing control that protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior (Fig. 2a, b), whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production (Fig. 2e, f). Thus, one of the key effects of microparticles seems to be the release from diffusional limitations within the fungal aggregates. Protein production in the control process stopped before the nutrients from the process were depleted. In contrast, the mycelial biomass created by the added microparticles was found active throughout the whole process. The presence of local differences in protein expression levels within fungal pellets is supported by recent data from micro colonies of A. niger in liquid cultures (de Bekker et al. 2011). Using laser micro dissection and pressure catapulting the authors could isolate central and peripheral parts of the mycelium from such colonies. Quantitative RT-PCR analysis then revealed that levels of mRNA, encoding for the glucoamylase gene glaA and for the feruoyl esterase gene faeA, both relevant industrial enzymes, were up to 45-fold higher in the peripheral areas of the microcolonies compared to the central parts. The size and open structure of the investigated microcolonies suggested that the center was not affected in the uptake of nutrients and transfer of gases as concluded by the authors so that the underlying effects remain to be elucidated (de Bekker et al. 2011).
Fig. 2

Confocal laser scanning microscopic analysis of growth morphology of A. niger engineered by microparticle addition. The recombinant strain expresses the fluorescent protein GFP2 under control of the glucoamylase promoter which allows visualizing and resolving the production performance within the aggregate. The addition of titanate microparticles results in highly active core–shell pellets (c+d) and the addition of talc microparticles creates highly active dispersed hyphal growth (e+f), whereas the pellet formed in the control experiment reflects only weak production on the pellet surface (a+b). The images refer to the experimental conditions and strains as described previously (Driouch et al. 2010a; Driouch et al. 2011a), where similar pictures are shown

Creation of core–shell bio-pellets by titanate microparticles

Interestingly, the use of titanate (titanium silicate oxide, TiSiO4, mean diam. 8 μm) has a strongly different effect on growth morphology as compared to other micro materials (Driouch et al. 2011a). Increasing concentrations of titanate based microparticles resulted in decreasing pellet size of A. niger down to a diameter of about 300 μm. Image analysis revealed a different form of interaction between cells and microparticles as compared to previously described talc and aluminum oxide. Here pellets were formed which consisted of an inner titanate core surrounded by a layer of cells (Fig. 2c, d). For shake flask cultivations a fourfold (fructofuranosidase, 150 U ml−1) and tenfold improvement of activity (glucoamylase, 190 U ml−1) was gained. Transferred to fed-batch production, a volumetric fructofuranosidase activity of 1,080 U ml−1 was achieved which was sevenfold higher as compared to the process without particles. The use of titanate microparticles opens possibilities for processes where growth as pellet is desired from the product point of view. In addition, high productivity seems attainable with lower viscosity of the culture, avoiding diffusional limitations within the broth, a problem often observed in processes with filamentous microorganisms (Wucherpfennig et al. 2010). Upon addition of titanate microparticles, viscosity reached only about 30 % of the viscosity with mycelial growth accomplished by addition of corresponding amounts of talc particles (8.7 mPa s compared to 27.6 mPa s, Driouch et al. 2011a).

Industrial relevance––microparticle enhanced production processes

The concept of microparticles was successfully transferred into bioreactor based production processes. Exemplified for batch cultivation in a 3 l stirred tank bioreactor, the addition of 10 g talc particles (4 μm) l−1 increased the absolute fructofuranosidase activity by recombinant A niger twofold to 160 U ml−1 in comparison to the negative control without microparticles (80 U ml−1) (Driouch et al. 2010a). Combined with medium design and process layout, the microparticle based enzyme production was then transferred to fed-batch operation (Driouch et al. 2010a). This provided a volumetric fructofuranosidase activity of 2,800 U ml−1 which is about tenfold higher than that of any other process reported for this enzyme to date. It is interesting to note that the use of microparticles did not interfere with the product quality, i.e. the catalytic properties of the obtained enzyme. With a simple one-step separation of cells and particles from the broth, a highly active enzyme containing supernatant was obtained that––without further treatment––enabled the biosynthesis of 450 g l−1 of rare oligosugars from sucrose within only 10 min (Driouch et al. 2011b). Similarly, purification of chloroperoxidase, the enzyme of the pioneering study, was not affected by the use of microparticles (Kaup et al. 2008). This suggests straightforward implementation of the use of microparticles into existing process environments. Questions to be addressed with regard to process costs are the search for cheap materials and concepts for their recycling.

Conclusions and outlook

As indicated by first promising examples, the addition of microparticles seems to be a useful new approach to engineer the morphology of filamentous microorganisms and enhance their productivity. Obviously, the range of microorganisms accessible by this technology is broad. Different production processes obviously benefit from the addition of microparticles (Table 2). Due to that we can expect a high potential to improve existing and future biotechnological processes (Fig. 3). Remaining basically unanswered at this stage are the underlying mechanisms of interactions between particles and cells. Therefore, fundamental systematic investigations regarding the influence of the microparticles on the cells are strongly required. This might involve a broader screening of various organic and inorganic microparticle materials available with defined size distributions, promising a better understanding of the microparticle mode of action, i.e. the influence of particle size and form, density, hardness, surface chemistry and particularly the resulting mechanical forces imposed on the microorganisms. The integration of dimensionless morphology numbers and automated image analysis seems quite relevant to support such systematic studies (Wucherpfennig et al. 2011). Clearly, also systems biology studies applying transcriptomics, proteomics, metabolomics and fluxomics appear very relevant to better understand the impact of the particles on the metabolic level. Vice versa, the new concept provides, for the first time, the possibility to fine tune morphology with rather high precision and generate defined populations for systems biology investigations.
Table 2

Impact of microparticles on production performance of filamentous microorganisms

Organism

Scale of cultivation

Microparticle material

Product

Product activitya (U ml−1)

Enhancement factorb

Reference

C. fumago

Shaking flask

3·MgO·4SiO2·H2O

Chloroperoxidase

1,000 

10

Kaup et al. (2008)

C. fumago

Shaking flask

Al2O3

Chloroperoxidase

600

6

Kaup et al. (2008)

A. niger

Shaking flask

3·MgO·4SiO2·H2O

Fructofuranosidase

>80

2

Driouch et al. (2010b)

A. niger

Shaking flask

3·MgO·4SiO2·H2O

Glucoamylase

61

4

Driouch et al. (2010b)

A. niger

Bioreactor (3l, batch)

3·MgO·4SiO2·H2O

Fructofuranosidase

160

2

Driouch et al. (2010b)

A. niger

Bioreactor (3l, fed-batch)

3·MgO·4SiO2·H2O

Fructofuranosidase

2,800

4

Driouch et al. (2010a)

A. niger

Shaking flask

TiSiO4

Fructofuranosidase

150

3

Driouch et al. (2011a)

A. niger

Shaking flask

TiSiO4

Glucoamylase

190

9

Driouch et al. (2011a)

A. niger

Bioreactor (3l, batch)

TiSiO4

Glucoamylase

320

6

Driouch et al. (2011a)

A. niger

Bioreactor (3l, fed-batch)

TiSiO4

Glucoamylase

1,080

7

Driouch et al. (2011a)

aWith particles

bCompared to cultivation without microparticles

Fig. 3

Sketch of microparticle enhanced cultivation as platform technology for different filamentous microorganisms and biotechnological processes. Vertical direction indicates existing and potential biotechnological processes with a variety of products manufactured by several established microorganisms. Horizontal direction indicates the versatility of microparticles and the corresponding influence on essential process parameters

Notes

Acknowledgments

All authors acknowledge financial support by the Allianz Industrie Forschung in the project “Microparticle based cultivation of filamentous fungi” (IGF: 16926 N/2). Christoph Wittmann and Rainer Krull additionally acknowledge support by the Collaborative Research Center SFB 578 “From gene to product” at Technische Universität Braunschweig, Germany, funded by the German Research Foundation.

References

  1. Barrios-González J, Miranda RU (2010) Biotechnological production and applications of statins. Appl Microbiol Biotechnol 85:869–883PubMedCrossRefGoogle Scholar
  2. Bennett JW (2010) An overview of the genus Aspergillus. In: Machida M, Gomi K (eds) Aspergillus: molecular biology and genomics. Caister Academic, Wymondham, pp 1–17Google Scholar
  3. de Bekker C, van Veluw GJ, Vinck A, Wiebenga LA, Wosten HAB (2011) Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures. Appl Environ Microbiol 77(4):1263–1267PubMedCrossRefGoogle Scholar
  4. Dobson LF, O’Cleirigh CC, O’Shea DG (2008) The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus. Appl Microbiol Biotechnol 79(5):859–866PubMedCrossRefGoogle Scholar
  5. Dodds DR, Gross RA (2007) Chemicals from biomass. Science 318:1250–1251PubMedCrossRefGoogle Scholar
  6. Driouch H, Roth A, Dersch P, Wittmann C (2010a) Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger. Appl Microbiol Biotechnol 87:2011–2024PubMedCrossRefGoogle Scholar
  7. Driouch H, Sommer B, Wittmann C (2010b) Morphology engineering of Aspergillus niger for improved enzyme production. Biotechnol Bioeng 105:1058–1068PubMedGoogle Scholar
  8. Driouch H, Hänsch R, Wucherpfennig T, Krull R, Wittmann C (2011a) Improved enzyme production by bio-pellets of Aspergillus niger––targeted morphology engineering using titanate microparticles. Biotechnol Bioeng 109:462–471PubMedCrossRefGoogle Scholar
  9. Driouch H, Roth A, Dersch P, Wittmann C (2011b) Filamentous fungi in good shape: microparticles for tailor-made fungal morphology and enhanced enzyme production. Bioeng Bugs 2:100–104PubMedCrossRefGoogle Scholar
  10. Gibbs PA, Seviour RJ, Schmid F (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit Rev Biotechnol 20(1):17–48PubMedCrossRefGoogle Scholar
  11. Grimm LH, Kelly S, Krull R, Hempel DC (2005) Morphology and productivity of filamentous fungi. Appl Microbiol Biotechnol 69:375–384PubMedCrossRefGoogle Scholar
  12. Hille A, Neu TR, Hempel DC, Horn H (2005) 2 profiles and biomass distribution in biopellets of Aspergillus niger. Biotechnol Bioeng 92(5):614–623PubMedCrossRefGoogle Scholar
  13. Jones MG (2007) The first filamentous fungal genome sequences: Aspergillus leads the way for essential everyday resources or dusty museum specimens? Microbiology 153:1–6PubMedCrossRefGoogle Scholar
  14. Kaup B, Ehrich K, Pescheck M, Schrader J (2008) Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example. Biotechnol Bioeng 99:491–498PubMedCrossRefGoogle Scholar
  15. Kelly S, Grimm LH, Jonas R, Hempel DC, Krull R (2006) Investigations of the morphogenesis of filamentous microorganisms. Eng Life Sci 6(5):475–480CrossRefGoogle Scholar
  16. Kubicek CP, Schreferl-Kunar G, Wohrer W, Rohr M (1988) Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger. Appl Environ Microbiol 54(3):633–637PubMedGoogle Scholar
  17. Kumar A, Grover S, Sharma J, Batish VK (2010) Chymosin and other milk coagulants: sources and biotechnological interventions. Crit Rev Biotechnol 30:243–258PubMedCrossRefGoogle Scholar
  18. Maiorano AE, Piccoli RM, da Silva ES, de Andrade Rodrigues MF (2008) Microbial production of fructosyltransferases for synthesis of pre-biotics. Biotechnol Lett 30:1867–1877PubMedCrossRefGoogle Scholar
  19. McIntyre M, Müller C, Dynesen J, Nielsen J (2001) Metabolic engineering of the morphology of Aspergillus. In: Nielsen J, Eggeling L et al (eds) Metabolic engineering, vol 73. Springer, Berlin, pp 103–128CrossRefGoogle Scholar
  20. O’Connell S, Walsh G (2008) Application relevant studies of fungal β-galactosidases with potential application in the alleviation of lactose intolerance. Appl Biochem Biotechnol 149:129–138PubMedCrossRefGoogle Scholar
  21. Papagianni M (2004) Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22:189–259PubMedCrossRefGoogle Scholar
  22. Papagianni M, Mattey M (2006) 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 5:3PubMedCrossRefGoogle Scholar
  23. Rawool SB, Sahoo S, Rao KK, Sureshkumar GK (2001) Improvement in enzyme productivities from mold cultivations using the liquid-phase 2 supply strategy. Biotechnol Prog 17(5):832–837PubMedCrossRefGoogle Scholar
  24. Shapiro RS, Robbins N, Cowen LE (2011) Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol Mol Biol Rev 75:213–267PubMedCrossRefGoogle Scholar
  25. Singh OV, Kumar R (2007) Biotechnological production of gluconic acid: future implications. Appl Microbiol Biotechnol 75:713–722PubMedCrossRefGoogle Scholar
  26. Sohoni SV, Bapat PM, Eliasson Lantz A (2012) Robust, small-scale cultivation platform for Streptomyces coelicolor. Microb Cell Fact 11:9PubMedCrossRefGoogle Scholar
  27. Thompson CJ, Fink D, Nguyen LD (2002) Principles of microbial alchemy: insights from the Streptomyces coelicolor genome sequence. Genome Biol 3:1–4CrossRefGoogle Scholar
  28. van Wezel GP, Krabben P, Traag BA, Keijser BJF, Kerste R, Vijgenboom E, Heijnen JJ, Kraal B (2006) Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering. Appl Environ Microbiol 72:5283–5288PubMedCrossRefGoogle Scholar
  29. Willke T, Vorlop K-D (2001) Biotechnological production of itaconic acid. Appl Microbiol Biotechnol 56:289–295PubMedCrossRefGoogle Scholar
  30. Wucherpfennig T, Kiep KA, Driouch H, Wittmann C, Krull R (2010) Chapter 4––morphology and rheology in filamentous cultivations. In: Allen I, Laskin SS (eds) Adv Appl Microbiol, vol 72. Academic Press, New York, pp 89–136Google Scholar
  31. Wucherpfennig T, Hestler T, Krull R (2011) Morphology engineering––osmolality and its effect on Aspergillus niger morphology and productivity. Microb Cell Fact 10:58PubMedCrossRefGoogle Scholar
  32. Zhang ZY, Jin B, Kelly JM (2007) Effects of cultivation parameters on the morphology of Rhizopus arrhizus and the lactic acid production in a bubble column reactor. Eng Life Sci 7:490–496CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Robert Walisko
    • 1
  • Rainer Krull
    • 1
  • Jens Schrader
    • 2
  • Christoph Wittmann
    • 1
  1. 1.Institute of Biochemical EngineeringTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Biochemical Engineering GroupDECHEMA Research InstituteFrankfurtGermany

Personalised recommendations