Quantitative Morphological Analysis of Filamentous Microorganisms in Cocultures and Monocultures: Aspergillus terreus and Streptomyces rimosus Warfare in Bioreactors

The aim of this study was to quantitatively characterize the morphology of the filamentous microorganisms Aspergillus terreus ATCC 20542 and Streptomyces rimosus ATCC 10970, cocultivated in stirred tank bioreactors, and to characterize their mutual influence with the use of quantitative image analysis. Three distinct coculture initiation strategies were applied: preculture versus preculture, spores versus spores and preculture versus preculture with time delay for one of the species. Bioreactor cocultures were accompanied by parallel monoculture controls. The results recorded for the mono- and cocultures were compared in order to investigate the effect of cocultivation on the morphological evolution of A. terreus and S. rimosus. Morphology-related observations were also confronted with the analysis of secondary metabolism. The morphology of the two studied filamentous species strictly depended on the applied coculture initiation strategy. In the cocultures initiated by the simultaneous inoculation, S. rimosus gained domination or advance over A. terreus. The latter microorganism dominated only in these experiments in which S. rimosus was introduced with a delay.

Enhanced Oxytetracycline Production by Streptomyces rimosus in Submerged Co-Cultures with Streptomyces noursei

In the present study, Streptomyces rimosus was confronted with Streptomyces noursei, Penicillium rubens, Aspergillus niger, Chaetomium globosum, or Mucor racemosus in two-species submerged co-cultures in shake flasks with the goal of evaluating the oxytetracycline production and morphological development. The co-culture of S. rimosus with S. noursei exhibited stimulation in oxytetracycline biosynthesis compared with the S. rimosus monoculture, whereas the presence of M. racemosus resulted in a delay in antibiotic production. Different strategies of initiating the “S. rimosus + S. noursei” co-cultures were tested. The improvement in terms of oxytetracycline titers was recorded in the cases where S. noursei was co-inoculated with S. rimosus in the form of spores. As the observed morphological changes were not unique to the co-culture involving S. noursei, there was no evidence that the improvement of oxytetracycline levels could be attributed mainly to morphology-related characteristics.

“Microbial Wars” in a Stirred Tank Bioreactor: Investigating the Co-Cultures of Streptomyces rimosus and Aspergillus terreus, Filamentous Microorganisms Equipped With a Rich Arsenal of Secondary Metabolites

Microbial co-cultivation is an approach frequently used for the induction of secondary metabolic pathways and the discovery of novel molecules. The studies of this kind are typically focused on the chemical and ecological aspects of inter-species interactions rather than on the bioprocess characterization. In the present work, the co-cultivation of two textbook producers of secondary metabolites, namely Aspergillus terreus (a filamentous fungus used for the manufacturing of lovastatin, a cholesterol-lowering drug) and Streptomyces rimosus (an actinobacterial producer of an antibiotic oxytetracycline) in a 5.5-L stirred tank bioreactor was investigated in the context of metabolic production, utilization of carbon substrates and dissolved oxygen levels. The cultivation runs differed in terms of the applied co-culture initiation strategy and the composition of growth medium. All the experiments were performed in three bioreactors running in parallel (corresponding to a co-culture and two respective monoculture controls). The analysis based upon mass spectrometry and liquid chromatography revealed a broad spectrum of more than 40 secondary metabolites, including the molecules identified as the oxidized derivatives of rimocidin and milbemycin that were observed solely under the conditions of co-cultivation. S. rimosus showed a tendency to dominate over A. terreus, except for the runs where S. rimosus was inoculated into the already developed bioreactor cultures of A. terreus. Despite being dominated, the less aggressive strain still had an observable influence on the production of secondary metabolites and the utilization of substrates in co-culture. The monitoring of dissolved oxygen levels was evaluated as a fast approach of identifying the dominant microorganism during the co-cultivation process.

Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers

Background Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. Results In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. Conclusions This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.

Presence of the Weakly Pathogenic Fusarium poae in the Fusarium Head Blight Disease Complex Hampers Biocontrol and Chemical Control of the Virulent Fusarium graminearum Pathogen

Fusarium head blight (FHB) in wheat (Triticum aestivum L.) is caused by a consortium of mutually interacting Fusarium species. In the field, the weakly pathogenic F. poae often thrives on the infection sites of the virulent F. graminearum. In this ecological context, we investigated the efficacy of chemical and biocontrol agents against F. graminearum in wheat ears. For this purpose, one fungicide comprising prothioconazole + spiroxamine and two bacterial biocontrol strains, Streptomyces rimosus LMG 19352 and Rhodococcus sp. R-43120 were tested for their efficacy to reduce FHB symptoms and mycotoxin (deoxynivalenol, DON) production by F. graminearum in presence or absence of F. poae. Results showed that the fungicide and both actinobacterial strains reduced FHB symptoms and concomitant DON levels in wheat ears inoculated with F. graminearum. Where Streptomyces rimosus appeared to have direct antagonistic effects, Rhodococcus and the fungicide mediated suppression of F. graminearum was linked to the archetypal salicylic acid and jasmonic acid defense pathways that involve the activation of LOX1, LOX2 and ICS. Remarkably, this chemical- and biocontrol efficacy was significantly reduced when F. poae was co-inoculated with F. graminearum. This reduced efficacy was linked to a suppression of the plant’s intrinsic defense system and increased levels of DON. In conclusion, our study shows that control strategies against the virulent F. graminearum in the disease complex causing FHB are hampered by the presence of the weakly pathogenic F. poae. This study provides generic insights in the complexity of control strategies against plant diseases caused by multiple pathogens.

Production and statistical optimization of Paromomycin by Streptomyces rimosus NRRL 2455 in solid state fermentation

Background Paromomycin is a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation. Results Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. Optimization of environmental conditions using D-optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C. Conclusion Compared to submerged liquid fermentation, solid state fermentation resulted in comparable paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, solid state fermentation is a promising alternative to submerged liquid fermentation for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.

Production and Statistical Optimization of Paromomycin by Streptomyces rimosus NRRL 2455 in Solid State Fermentation

Background: Paromomycin is a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation.Results: Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. This value was higher than that obtained using submerged liquid fermentation using the same conditions. Optimization of environmental conditions using D optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C. Conclusion: Compared to submerged liquid fermentation, solid state fermentation resulted in higher paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, SSF is a superior alternative to SLF for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.