SParticle, an algorithm for the analysis of filamentous microorganisms in submerged cultures

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favouring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.

[1]  D J Barry,et al.  Microscopic characterisation of filamentous microbes: towards fully automated morphological quantification through image analysis , 2011, Journal of microscopy.

[2]  Dennis Claessen,et al.  Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies , 2014, Nature Reviews Microbiology.

[3]  J. Worrall,et al.  GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans. , 2015, The Biochemical journal.

[4]  A. Nienow,et al.  Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus , 2017, Journal of microscopy.

[5]  J. Prosser,et al.  Experimental verification of a mathematical model for pelleted growth of Streptomyces coelicolor A3(2) in submerged batch culture. , 1996, Microbiology.

[6]  S. Stocks,et al.  Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production. , 2002, Biotechnology and bioengineering.

[7]  K. Luyben,et al.  Modeling and measurements of fungal growth and morphology in submerged fermentations. , 1998, Biotechnology and bioengineering.

[8]  Satoshi Omura,et al.  Trends in the search for bioactive microbial metabolites , 1992, Journal of Industrial Microbiology.

[9]  Preben Krabben,et al.  Unlocking Streptomyces spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering , 2006, Applied and Environmental Microbiology.

[10]  J. Willemse,et al.  Aggregation of germlings is a major contributing factor towards mycelial heterogeneity of Streptomyces , 2016, Scientific Reports.

[11]  G. V. van Wezel,et al.  Cell division and DNA segregation in Streptomyces: how to build a septum in the middle of nowhere? , 2012, Molecular microbiology.

[12]  Amiya Nayak,et al.  Fast iris detection via shape based circularity , 2013, 2013 IEEE 8th Conference on Industrial Electronics and Applications (ICIEA).

[13]  Daniel J. Brat,et al.  Novel genotype-phenotype associations in human cancers enabled by advanced molecular platforms and computational analysis of whole slide images , 2014, Laboratory Investigation.

[14]  J. Büchs,et al.  Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks , 2011, Biotechnology and bioengineering.

[15]  C. R. Thomas,et al.  Morphological measurements on filamentous microorganisms by fully automatic image analysis , 1990, Biotechnology and bioengineering.

[16]  T. Kieser Practical streptomyces genetics , 2000 .

[17]  J. Willemse,et al.  Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor , 2012, Journal of bacteriology.

[18]  U. Reichl,et al.  Study of the early growth and branching of Streptomyces tendae by means of an image processing system , 1990, Journal of microscopy.

[19]  D. Hopwood,et al.  Streptomyces in nature and medicine : the antibiotic makers , 2007 .

[20]  K. Bellgardt,et al.  Two mathematical models for the development of a single microbial pellet , 1995 .

[21]  J. D. Webster,et al.  Whole-Slide Imaging and Automated Image Analysis , 2014, Veterinary pathology.

[22]  R. Krull,et al.  Comprehension of viscous morphology--evaluation of fractal and conventional parameters for rheological characterization of Aspergillus niger culture broth. , 2013, Journal of biotechnology.

[23]  C. A. van den Hondel,et al.  GTP-binding protein Era: a novel gene target for biofuel production , 2015, BMC Biotechnology.

[24]  G. V. van Wezel,et al.  Chapter 5. Applying the genetics of secondary metabolism in model actinomycetes to the discovery of new antibiotics. , 2009, Methods in enzymology.

[25]  János Bérdy,et al.  Bioactive microbial metabolites. , 2005, The Journal of antibiotics.

[26]  D. Ryoo Fungal fractal morphology of pellet formation in Aspergillus niger , 1999 .

[27]  H. D. Tresner,et al.  Morphology of submerged growth of streptomycetes as a taxonomic aid. I. Morphological development of Streptomyces aureofaciens in agitated liquid media. , 1967, Applied microbiology.

[28]  Dennis Claessen,et al.  Morphogenesis of Streptomyces in submerged cultures. , 2014, Advances in applied microbiology.

[29]  P. Pfeifer,et al.  Microbial growth patterns described by fractal geometry , 1990, Journal of bacteriology.

[30]  U. Reichl,et al.  Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis , 1992, Biotechnology and bioengineering.

[31]  R. Krull,et al.  Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity , 2011, Microbial cell factories.

[32]  H. Wösten,et al.  Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter , 2014, Journal of visualized experiments : JoVE.

[33]  B. Barrell,et al.  Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2) , 2002, Nature.

[34]  A. Ram,et al.  Hyphal differentiation in the exploring mycelium of Aspergillus niger , 2005, Molecular microbiology.

[35]  M. V. van Loosdrecht,et al.  Structured morphological modeling as a framework for rational strain design of Streptomyces species , 2012, Antonie van Leeuwenhoek.

[36]  K. Gernaey,et al.  Comparison of laser diffraction and image analysis for measurement of Streptomyces coelicolor cell clumps and pellets , 2012, Biotechnology Letters.

[37]  J. Willemse,et al.  Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization , 2009, PloS one.

[38]  G. V. van Wezel,et al.  The SsgA-like proteins in actinomycetes: small proteins up to a big task , 2008, Antonie van Leeuwenhoek.

[39]  Mike Hale,et al.  Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control , 2014, Journal of Clinical Pathology.

[40]  S E Vecht-Lifshitz,et al.  Pellet formation and cellular aggregation in Streptomyces tendae , 1990, Biotechnology and bioengineering.

[41]  Z. Deng,et al.  A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces , 2008, Journal of bacteriology.

[42]  Philipe A. Dias,et al.  Image processing for identification and quantification of filamentous bacteria in in situ acquired images , 2016, BioMedical Engineering OnLine.

[43]  L. Vining,et al.  Sporulation of Streptomyces venezuelae in submerged cultures. , 1990, Journal of general microbiology.

[44]  Maria Papagianni,et al.  Characterization of Fungal Morphology using Digital Image AnalysisTechniques , 2014 .

[45]  P. Cox,et al.  Classification and measurement of fungal pellets by automated image analysis , 1992, Biotechnology and bioengineering.

[46]  G. V. van Wezel,et al.  A novel taxonomic marker that discriminates between morphologically complex actinomycetes , 2013, Open Biology.

[47]  E. Gilles,et al.  Morphological characterization of filamentous microorganisms in submerged cultures by on-line digital image analysis and pattern recognition. , 1997, Biotechnology and bioengineering.

[48]  J. Menezes,et al.  Morphology and viability analysis of Streptomyces clavuligerus in industrial cultivation systems , 2004, Bioprocess and biosystems engineering.

[49]  K. Kendrick,et al.  Sporulation of Streptomyces griseus in submerged culture , 1983, Journal of bacteriology.

[50]  G. V. van Wezel,et al.  Taxonomy, Physiology, and Natural Products of Actinobacteria , 2015, Microbiology and Molecular Reviews.

[51]  Dennis Claessen,et al.  A novel locus for mycelial aggregation forms a gateway to improved Streptomyces cell factories , 2015, Microbial Cell Factories.

[52]  Andreas E. Posch,et al.  A novel method for fast and statistically verified morphological characterization of filamentous fungi. , 2012, Fungal genetics and biology : FG & B.