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Morphological differentiation of S. coelicolor A3(2) grown on glass beads. (A) Image a shows a young colony composed of only substrate mycelia. Later on, aerial mycelia started to stand up into the air (b) and elongate (c), coincident with the appearance of pigments. Later, coiled aerial mycelia started to form septa and spores (d). Images a, b, c, and d were taken at 6, 16, 19, and 36 h after transfer to glass beads, respectively. The magnified image in d shows an aerial mycelium septated to form spores. Panel B shows spores germinating on glass beads after 8 h (e) and primary network of mycelium formed after 24 h (f). One or two germ tubes coming from the spore were observed (arrows).
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A two-phase cultivation system was developed which will enable studies of streptomycete differentiation by molecular biological and global techniques such as transcriptomics and proteomics. The system is based on a solid phase formed by glass beads corresponding to particles in soil, clay, or sand natural habitats of streptomycetes. The beads are i...
Citations
... In addition to lipids, other nutrients in the substrate also play a pivotal role in AFB1 biosynthesis and mycelium growth (Liu et al. 2016). Glass beads as a solid support have shown utility not only in plating cell cultures and colony growth (Worthington et al. 2001;Prusokas et al. 2021), but also in the cultivation of filamentous fungi (Bottcher and Conn, 1942;Nguyen et al. 2005;Droce et al. 2013;Ali et al. 2016) showing advantages to both agar plate cultures and liquid cultures. ...
Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3–fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources.
Key points
• A glass bead system ensuring the flow of air when studying powders was developed.
• AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied.
• 3–fivefold higher mycotoxin levels on rice starch than on corn starch were detected.
... Liquid media provide many advantages compared to solid media: they guarantee a homogenous distribution of nutrients and oxygen, while also facilitating the manipulation of cultures. Aiming to combine the benefits of liquid media while meeting the requirements of microorganisms that live attached to surfaces, liquid media can be improved by adding a small amount of gelling agents such as gellan gum, xanthan gum, or carrageenan (Das et al., 2015), glass beads (Nguyen et al., 2005;Droce et al., 2013), or sand (Suman et al., 2019). Adding these supplementary solid agents can help the microorganisms to attach to the surface but still live and divide in the liquid or semiliquid medium. ...
In terms of the number and diversity of living units, the prokaryotic empire is the most represented form of life on Earth, and yet it is still to a significant degree shrouded in darkness. This microbial “dark matter” hides a great deal of potential in terms of phylogenetically or metabolically diverse microorganisms, and thus it is important to acquire them in pure culture. However, do we know what microorganisms really need for their growth, and what the obstacles are to the cultivation of previously unidentified taxa? Here we review common and sometimes unexpected requirements of environmental microorganisms, especially soil-harbored bacteria, needed for their replication and cultivation. These requirements include resuscitation stimuli, physical and chemical factors aiding cultivation, growth factors, and co-cultivation in a laboratory and natural microbial neighborhood.
... Furthermore, we demonstrated that individual glass beads could be inoculated with yeast, exposed to a stressor (lead nitrate or heat in this study), and then subsequently recovered and cells grown out to determine relative survival. Glass beads inoculated with bacteria have previously been used to study denitrification in porous environments (7), and beads have also been used as a solid interface to cultivate bacteria and study differentiation on solid surfaces (23). This report presents a simple method for making and incorporating organisms into three-dimensional (3D) structures of glass beads, enabling investigation of interrelationships between microbial activity and environmental structure. ...
The physical environments in which microorganisms naturally reside rarely have homogeneous structure, and changes in their porous architecture can have a profound effect on microbial activities – effects that are not typically captured in conventional laboratory studies. Here, to investigate the influence of environmental structure on microbial responses to stress, we constructed structured environments with different pore properties (determined by X-ray Computed Tomography). First, using glass beads in different arrangements and inoculated with the soil yeast Saitozyma podzolica , increases in the average equivalent spherical diameters (ESD) of a structure’s porous architecture led to decreased survival of the yeast under a toxic metal challenge. This relationship was reproduced when yeasts were introduced into additively-manufactured lattice structures, comprising regular arrays with ESDs comparable to those of the bead structures. The pore ESD-dependency of metal resistance was not attributable to differences in cell density in micro-environments delimited by different pore sizes, supporting the inference that pore size specifically is the important parameter here in determining microbial survival of stress. These findings highlight the importance of the physical architecture of an organism’s immediate environment for its response to environmental perturbation, while offering new tools for investigating these interactions in the laboratory.
IMPORTANCE
Interactions between cells and their structured environments are poorly understood but have significant implications for organismal success in both natural and non-natural settings. This work uses a multidisciplinary approach to develop laboratory models with which the influence of a key parameter of environmental structure – pore size – on cell activities can be dissected. Using these new methods in tandem with additive manufacturing, we demonstrate that resistance of yeast soil-isolates to stress (from a common metal pollutant) is inversely related to pore size of their environment. This has important ramifications for understanding how microorganisms respond to stress in different environments. The findings also establish new pathways for resolving the effects of physical environment on microbial activity, enabling important understanding that is not readily attainable with traditional bulk-sampling and analysis approaches.
... The notion of MOI loses a lot of its significance once mycelium has formed, as the network structure originating from one spore has greatly increased phage adsorption but would still be counted as one CFU [14,50]. Furthermore, the formation of clumps, although mitigated by the addition of glass beads or increase of osmotic pressure [51], makes accurate monitoring of cell growth (based on optical density or backscatter) difficult. ...
Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.
... [36] These findings indicate that cultivation systems supported by organic fibers allow the biosynthetic potential of filamentous microorganisms to be stimulated in vitro, opening a new field of application for electrospun polymers. A biphasic cultivation [37] in presence of nanofibers might support bacterial attachment and growth mimicking surface dehydration and particle microenvironments of soil in natural habitats. Importantly, electrospun fibers are realized cheaply, thus being well-suited to be used in relatively low-cost antibiotic production process. ...
Streptomycetes are exploited for the production of a wide range of secondary metabolites, including antibiotics. Therefore, both academic and industrial research efforts are focused on enhancing production of these precious metabolites. So far, this has been mostly achieved by classical or recombinant genetic techniques, in association with process optimization for either submerged or solid state fermentation. New cultivation approaches addressing the natural mycelial growth and life cycle would allow the biosynthetic potential of filamentous strains to be much better exploited. We developed a cultivation system for antibiotic-producing microorganisms which involves electrospun organic nanofibers deposited onto agar plates or immersed in liquid media. Dense filamentous networks of branched hyphae formed by bacterial colonies were found to wrapped around the fibers. We analyzed the effects of fibers on growth and antibiotic production in Streptomyces lividans, and found that the actinorhodin, undecylprodigiosin and calcium dependent antibiotic productions were positively modulated, with a 2 to 6-fold enhancement compared to standard culture conditions. Highlighting the secondary metabolism-promoting role of nanofibers in bacterial cultures, these results open a route to the design of improved culture systems for microorganisms based on organic nanostructures.
... In particular, a stimulation of cell division and growth of starved bacteria by solid surfaces was observed in early experiments (Kjelleberg et al., 1982) and the adhesion of bacteria onto glass surfaces resulted in enhanced metabolic activity (Hong and Brown, 2009). However, nutrient depletion of Vibrio cholerae can induce a switch from the sessile to the planktonic state (Kierek and Watnick, 2003) and glass beads promote the differentiation of streptomycetes (Nguyen et al., 2005). So far it is unknown to which extent the specific adhesion and the different physiological states of cells in biofilms could be exploited for the selective enrichment of bacteria. ...
The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm-forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so-far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm-enrichment approach was exemplified by the successful isolation of a not-yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.
... The system allowed for both good microbial growth confined to the liquid medium and glass beads matrix and suitable and rapid PAH quantification with a PAHs recovery yield of up to 95%. Glass beads also have been used to simulate the soil particles and monitor the growth of the mycorrhizal fungus Glomus intraradices (Rilling and Steinberg, 2002) and the actinobacterium Streptomyces (Nguyen et al, 2005). Ehlers and Rose (2005) evaluated the immobilization of three white-rot fungal cultures in trickling packed-bed reactors employing the glass beads as support in the biodegradation of phenol and 2,4,6trichlorophenol-contaminated liquid. ...
In this study, different growth conditions of Anthracophyllum discolor Sp4 including the effect of agitation, additions of lignocellulosic support, inducer and surfactant were evaluated on the MnP production in Kirk medium using a culture system made up of the tubes containing the glass bead . The highest MnP production (1,354 U/L on day 13) was obtained when the medium was supplemented with wheat grain and 0.25 mM MnSO4 as inducer, under static conditions at 30°C. Two isoenzymes were purified (35 and 38 kDa respectively). MnP presented a maximal activity in the pH range between 4.5 and 5.5, a relatively high temperature tolerance (50ºC) and a high catalytic activity for 2,6-dimethoxyphenol and hydrogen peroxide.
... Augmentation of the rate of sporulation maybe required as antibiotic synthesis would not be expected when spores develop dormancy. This model will allow investigation into the biochemistry of cell signalling, differentiation and antibiotic production in this industrially important organism in a liquid environment which affords the advantage of a tightly controlled system allowing addition or analysis of nutrients, growth factors and labelled substrates (Nguyen et al., 2005). ...
Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free-dwelling population. These organisms sporulated with reduced antibiotic production compared with free-dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid-liquid interface.
This review deals with recent developments in complex molecular biology, which is moving from descriptive towards dynamic or quantitative proteomics, and its use as a tool in the studies of microbial physiology. It describes basic methodological strategies as well as examples of several projects where this approach is used in the studies of gene expression regulation in primary and secondary metabolism of industrially important bacteria.
