Microparticle-enhanced Cultivation Research Articles

Morphological-metabolic analysis in Streptomyces rimosus microparticle-enhanced cultivations (MPEC)

Streptomyces produce a broad spectrum of biologically active molecules such as oxytetracycline and rimocidin, which are widely used in human and animal treatments. microparticle-enhanced cultivation (MPEC) is one of the tools used for Streptomyces bioprocesses intensification by the control of mycelial morphology. In the present work, morphological changes of Streptomyces rimosus caused by the addition of 10 µm talc microparticles in MPEC were correlated with the biosynthetic activity of the microorganism. Comparing the runs with and without microparticles, major morphological changes were observed in MPEC, including the deformation of pellets, variation of their size, appearance of hyphae and clumps as well as the aggregation of mycelial objects. The presence of talc microparticles also influenced the levels of the studied secondary metabolites produced by S. rimosus. Comparing control and MPEC runs, the addition of talc microparticles increased the amounts of oxytetracycline (9-fold), 2-acetyl-2-decarboxamido-oxytetracycline (7-fold), milbemycin A3+4[O] (3-fold) and CE 108 (1.5-fold), while rimocidin (27-ethyl) and milbemycin β11+4[O] production was reduced. In summary, the addition of talc microparticles to S. rimosus cultivations led to the development of smaller morphological forms like hyphae and clumps as well as to the changes in the amounts of secondary metabolites.Graphical abstract

Exopolysaccharides produced by Antrodia cinnamomea using microparticle-enhanced cultivation: Optimization, primary structure and antibacterial property

Microparticle-enhanced cultivation was used to enhance the production of exopolysaccharides (EPSs) from Antrodia cinnamomea. The structure and antibacterial activity of two EPSs produced by A. cinnamomea treated with Al2O3 [EPS-Al (crude) and EPS-Al-p (purified)] and without Al2O3 [EPS-C (crude) and EPS-C-p (purified)] were compared. It was observed that the addition of 4 g/L Al2O3 at 0 h resulted in the highest EPS yield of 1.46 g/L, possible attributed to the enhanced permeability of the cell membrane. The structural analysis revealed that EPS-C-p and EPS-Al-p had different structures. EPS-C-p was hyperbranched and spherical with a Mw of 10.8 kDa, while EPS-Al-p was irregular and linear with a Mw of 12.5 kDa. The proportion of Man in EPS-Al-p decreased, while those of Gal and Glc increased when compared to EPS-C-p. The total molar ratios of 6-Glcp and 4-Glcp in EPS-Al-p are 1.45 times that of EPS-C-p. Moreover, EPSs could alter bacterial cell morphology, causing intracellular substance leakage and growth inhibition, with EPS-Al having a stronger antibacterial activity than EPS-C. In conclusion, A. cinnamomea treated with Al2O3 could produce more EPSs, changing monosaccharide composition and glycosidic linkage profile, which could exert stronger antibacterial activity than that produced by untreated A. cinnamomea.

Morphology engineering for novel antibiotics: Effect of glass microparticles and soy lecithin on rebeccamycin production and cellular morphology of filamentous actinomycete Lentzea aerocolonigenes.

Lentzea aerocolonigenes, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10g·L-1) with a median diameter of 7.9µm, for instance, induced an up to fourfold increase in product synthesis accompanied by overall enhanced viability of biomass. Furthermore, structural elucidations showed that biopellets isolated from MPEC tend to have lower hyphal density than unsupplemented control pellets. In this context, oxygen microprofiling was conducted to better understand how internal structural changes interwind with oxygen supply into the pellets. Here, the resulting oxygen profiles are of a contradictive trend of steeper oxygen consumption with increasing glass microparticle supplementation. Eventually, MPEC was combined with another promising cultivation strategy, the supplementation of soy lecithin (7.5g·L-1), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213mg·L-1 after 10days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of L. aerocolonigenes.

Efficient production of Antrodin C by microparticle-enhanced cultivation of medicinal mushroom Antrodia cinnamomea

The microparticle-enhanced cultivation (MPEC) was used to enhance the production of Antrodin C by submerged fermentation of medicinal mushroom Antrodia cinnamomea. The crucial factors such as types, sizes, concentrations, and addition time of microparticles were optimized. The mechanism of MPEC on the membrane permeability and fluidity of A.cinnamomea and the expression of key genes in Antrodin C were investigated. When talc (18μm, 2g/L) was added into the fermentation liquid at 0h, the promoting effect on Antrodin C was the best. The maximum yield of Antrodin C was 1615.7mg/L, which was about 2.98 times of the control (541.7mg/L). Talc slightly damaged the mycelia of A.cinnamomea, increased the release of intracellular constituents, and enhanced the index of unsaturated fatty acid. In addition, the key genes (IDI, E2.3.3.10, HMGCR, atoB) that might play an important role in the synthesis of the triquine-type sesquiterpene Antrodin C, were upregulated. In conclusion, talc increased the permeability and fluidity of cell membrane, upregulated the key genes and improved the biosynthesis process to enhance the yield of Antrodin C in the submerged fermentation of A.cinnamomea.

Co-cultivation of filamentous microorganisms in the presence of aluminum oxide microparticles

In the present work, the approaches of submerged co-cultivation and microparticle-enhanced cultivation (MPEC) were combined and evaluated over the course of three case studies. The filamentous fungus Aspergillus terreus was co-cultivated with Penicillium rubens, Streptomyces rimosus, or Cerrena unicolor in shake flasks with or without the addition of aluminum oxide microparticles. The influence of microparticles on the production of lovastatin, penicillin G, oxytetracycline, and laccase in co-cultures was compared with the effects recorded for the corresponding monocultures. In addition, the quantitative analyses of morphological parameters, sugars consumption, and by-products formation were performed. The study demonstrated that the influence of microparticles on the production of a given molecule in mono- and co-culture may differ considerably, e.g., the biosynthesis of oxytetracycline was shown to be inhibited due to the presence of aluminum oxide in “A. terreus vs. S. rimosus” co-cultivation variants but not in S. rimosus monocultures. The differences were also observed regarding the morphological characteristics, e.g., the microparticles-induced changes of projected area in the co-cultures and the corresponding monocultures were not always comparable. In addition, the study showed the importance of medium composition on the outcomes of MPEC, as exemplified by lovastatin production in A. terreus monocultures. Finally, the co-cultures of A. terreus with a white-rot fungus C. unicolor were described here for the first time.Key points• Aluminum oxide affects secondary metabolites production in submerged co-cultures.• Mono- and co-cultures are differently impacted by the addition of aluminum oxide.• Effect of aluminum oxide on metabolites production depends on medium composition.

Effects and interactions of metal oxides in microparticle-enhanced cultivation of filamentous microorganisms.

Filamentous microorganisms are used as molecular factories in industrial biotechnology. In 2007, a new approach to improve productivity in submerged cultivation was introduced: microparticle-enhanced cultivation (MPEC). Since then, numerous studies have investigated the influence of microparticles on the cultivation. Most studies considered MPEC a morphology engineering approach, in which altered morphology results in increased productivity. But sometimes similar morphological changes lead to decreased productivity, suggesting that this hypothesis is not a sufficient explanation for the effects of microparticles. Effects of surface chemistry on particles were paid little attention, as particles were often considered chemically-inert and bioinert. However, metal oxide particles strongly interact with their environment. This review links morphological, physical, and chemical properties of microparticles with effects on culture broth, filamentous morphology, and molecular biology. More precisely, surface chemistry effects of metal oxide particles lead to ion leaching, adsorption of enzymes, and generation of reactive oxygen species. Therefore, microparticles interfere with gene regulation, metabolism, and activity of enzymes. To enhance the understanding of microparticle-based morphology engineering, further interactions between particles and cells are elaborated. The presented description of phenomena occurring in MPEC eases the targeted choice of microparticles, and thus, contributes to improving the productivity of microbial cultivationtechnology.

Sodium starch octenyl succinate facilitated the production of water-soluble yellow pigments in Monascus ruber fermentation.

Natural water-soluble Monascus pigments (WSMPs) have been in increasing demand but have not been able to achieve industrial production due to the low production rate. This study aimed to improve the biosynthesis and secretion of extracellular yellow pigments (EYPs) through submerged fermentation with Monascus ruber CGMCC 10,910 supplemented with sodium starch octenyl succinate (OSA-SNa). The results demonstrated that the yield was 69.68% and 48.89% higher than that without OSA-SNa in conventional fermentation (CF) and extractive fermentation (EF), respectively. The mainly increased EYP components were Y3 and Y4 in CF, but they were mainly Y1 and Y2 as well as secreted intracellular pigments, including Y5, Y6, O1, and O2, in EF. Scanning electron microscopy analysis revealed that the mycelium presented an uneven surface profile with obvious wrinkles and small fragments with OSA-SNa. It was found that a higher unsaturated/saturated fatty acids ratio in the cell membrane resulted in increased permeability and facilitated the export of intracellular yellow pigments into the broth with OSA-SNa treatment. In addition, a higher NAD+/NADH ratio and glucose-6-phosphate dehydrogenase activity provided a reducing condition for yellow pigment biosynthesis. Gene expression analysis showed that the expression levels of the key genes for yellow pigment biosynthesis were significantly upregulated by OSA-SNa. This study provides an effective strategy to promote the production of WSMPs by microparticle-enhanced cultivation using OSA-SNa. KEY POINTS: • OSA-SNa addition facilitated the production of Monascus yellow pigments. • Mycelial morphology and membrane permeability were affected by OSA-SNa. • The key gene expression of yellow pigments was upregulated.

Enhancement of schizophyllan production in Schizophyllum commune using microparticles in medium.

Schizophyllum commune is a wood-rotting filamentous fungus that secrets a homopolysaccharide called as schizophyllan. Schizophyllan has several applications such as enhanced oil recovery, pharmaceutical materials and an anti-cancer drug carrier. Biomass growth and schizophyllan production increase the viscosity of the cultivation medium, thus resulting in mass transfer limitation for the substrate. In this study, adding talc and aluminium oxide microparticles into the cultivation medium was studied to improve the fungal growth and morphology. The response surface methodology and one factor at a time were applied to find the effects of microparticles with different sizes and concentrations on the schizophyllan production. The optimum concentration and size of aluminium oxide microparticles were obtained as 20gL-1 and < 30µm, respectively. Aluminium oxide microparticles in shake flask culture caused to increase the schizophyllan production from 10 to 15gL-1 and decrease the cultivation time from 10 to 7days. The production yield also increased from 0.11 to 0.30g of schizophyllan/g glucose. Bioreactor cultivation showed a twofold increase in schizophyllan production from 1.5 to 3gL-1. The results of this study suggested a significant increase in the production of schizophyllan using a low-cost "microparticle-enhanced cultivation" without any further optimization of the culture medium.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275mg/L at 168h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20g/L, diameter of 45µm) could make the maximum titer of echinocandin B reach 3148 ± 100mg/L.

Adding bamboo charcoal powder to Shiraia bambusicola preculture improves hypocrellin A production

Hypocrellin A (HA), a photodynamic therapy (PDT) photosensitizer for cancers and viruses, is a perylenequinone pigment from the fruiting bodies of a bamboo pathogenic Shiraia bambusicola. Due to the limitation of the supply of wild Shiraia fruiting bodies and the complex chemosynthesis of HA, mycelium culture is becoming a biotechnological alternative for HA production. In this study, the novel approach recruited the addition of bamboo charcoal powder (BCP) (Ø2.3-5.5 μm) in preculture of S. bambusicola to stimulate HA production. BCP decreased the size of S. bambusicola pellets, enhanced the oxygen and sugar consumption, and increased the pH value of the broth of the production culture without any fungal growth retardation. The highest HA production (604.81 mg/L) was achieved when BCP at 2.0 g/L was added in the preculture. BCP treatment not only increased HA content in mycelia by 44.9-265.5%, but promoted its exudation to the medium by 57.0-160.5%, compared with the control respectively. Further analysis revealed that BCP treatment could induce the generation of reactive oxygen species (ROS) and occurrence of cell apoptosis in the pellets. The BCP addition up-regulated the gene expressions for HA biosynthesis and its exudation. Our study provides BCP as an inexpensive, bio-friendly and less toxic microparticle used in microparticle-enhanced cultivation (MPEC) to enhance the sustainable production of pharmaceutical important metabolites in mycelium cultures.

Kinetic model to describe the morphological evolution of filamentous fungi during their early stages of growth in the standard submerged and microparticle-enhanced cultivations.

Biosynthesis of metabolites and enzymes by filamentous fungi depends on their morphological form in submerged cultures. However, their early stages of growth lasting approximately 24h, from the introduction of spores to the medium until the formation of stable morphological forms, such as clumps or pellets, have rarely been the objects of experimental and modeling studies. Microparticle-enhanced cultivation (MPEC) has been applied only to a few fungal species, mainly Aspergilli. Therefore, the objective of this work was to formulate the kinetic model to describe the early stages of the fungal evolution in the standard cultivation and MPEC for Aspergillus terreus, Chaetomium globosum, Penicillium rubens, and Mucor racemosus. These fungi exhibit various mechanisms of agglomerates formation in submerged cultures. The experiments were performed in batch shake flasks (parameters identification) and a stirred tank bioreactor (model verification). In the balance equation for fungal cells, the mean projected area of hyphal objects measured by the digital analysis of microscopic images was used as the dependent variable. The analysis of the experimental data and model solution revealed that the effect of the microparticles (aluminum oxide at 6gL-1) in MPEC toward the studied filamentous fungi was to the high extent species dependent. This effect was most evident in the case of spore coagulative A. terreus and noncoagulative M. racemosus.

Application of Aluminum Oxide Nanoparticles in Aspergillus terreus Cultivations: Evaluating the Effects on Lovastatin Production and Fungal Morphology.

Aluminum oxide nanoparticles were supplemented to Aspergillus terreus ATCC 20542 precultures and the outcomes of the process were evaluated relative to the results of microparticle-enhanced and standard cultivations. The selected morphological parameters of fungal pellets (projected area, elongation, convexity, and shape factor) were monitored throughout the experiment, together with biomass, lactose, and lovastatin concentration. The qualitative and quantitative chemical analysis was performed with the use of liquid chromatography coupled with high resolution mass spectrometry. The results of the study indicated that the application of nanoparticles was indeed associated with morphological consequences, most notably the decreased pellet size. However, it turned out that the term “nanoparticle-enhanced cultivation” could not be used in the context of lovastatin production, as no marked increase of product titer was observed in nanoparticle-influenced variants relative to standard and microparticle-enhanced cultivation. In addition, the concentration of biomass in the nanoparticle-influenced runs was relatively low. Comparative analysis of total ion chromatograms revealed the presence of a molecule of unknown structure that could be detected solely in broths from standard and microparticle-containing cultures. This study represents the first evaluation of nanoparticles as the tools of morphological engineering aimed at enhanced lovastatin biosynthesis in A. terreus cultures.

Microparticle-enhanced cultivation of filamentous fungi – insight into the action of microparticles towards various fungal species

Research science is potentially dangerous. For almost thirty years, the federal government has mandated laboratory safety and health programs to protect scientists, others and the environment. The effectiveness of any safety program depends, in part, on understanding the type, frequency and severity of potential injuries and employing appropriate prevention strategies. A mismatch between understanding and prevention could mean that otherwise avoidable laboratory injuries will occur, individuals will be hurt and costs incurred.This study is a retrospective look at laboratory-associated injuries (called incidents) at Northwestern University, Evanston, IL between 2010-2017. Compensable injuries of scientists were analyzed by type and potential severity (assumed by cost), then further examined by job classification and time of year.Cuts, lacerations and punctures accounted for the largest number of laboratory incidents—thirty-two percent (32%). Chemical exposure(s) accounted for another nineteen-percent (19%). Needle-stick(s), animal bite(s) and exposure to blood and body fluids were another twenty-five percent (25%).Insights from this study led to the implementation of a hand injury prevention program in the Fall of 2017. The intended effectiveness of this program in reducing the frequency and severity of cuts, lacerations and punctures in research laboratories will be evaluated over time. Our hypothesis is that targeted prevention strategies are likely to reduce the frequency and cost of future incidents involving scientists.

Morphological evolution of various fungal species in the presence and absence of aluminum oxide microparticles: Comparative and quantitative insights into microparticle-enhanced cultivation (MPEC).

The application of microparticle‐enhanced cultivation (MPEC) is an attractive method to control mycelial morphology, and thus enhance the production of metabolites and enzymes in the submerged cultivations of filamentous fungi. Unfortunately, most literature data deals with the spore‐agglomerating species like aspergilli. Therefore, the detailed quantitative study of the morphological evolution of four different fungal species (Aspergillus terreus, Penicillium rubens, Chaetomium globosum, and Mucor racemosus) based on the digital analysis of microscopic images was presented in this paper. In accordance with the current knowledge, these species exhibit different mechanisms of agglomerates formation. The standard submerged shake flask cultivations (as a reference) and MPEC involving 10 μm aluminum oxide microparticles (6 g·L−1) were performed. The morphological parameters, including mean projected area, elongation, roughness, and morphology number were determined for the mycelial objects within the first 24 hr of growth. It occurred that heretofore observed and widely discussed effect of microparticles on fungi, namely the decrease in pellet size, was not observed for the species whose pellet formation mechanism is different from spore agglomeration. In the MPEC, C. globosum developed core‐shell pellets, and M. racemosus, a nonagglomerative species, formed the relatively larger, compared to standard cultures, pellets with distinct cores.

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor.

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle-enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·108 L-1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sn at shear rate about 0.05 s-1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5-fold.

Modern morphological engineering techniques for improving productivity of filamentous fungi in submerged cultures.

Morphological engineering techniques have recently gained popularity as they are used for increasing the productivity of a variety of metabolites and enzymes in fungi growing in submerged cultures. Their action is mainly associated with the changes they evoke in fungal morphology. Traditional morphological engineering approaches include manipulation of spore concentration, pH-shifting and mechanical stress exerted by stirring and aeration. As the traditional methods proved to be insufficient, modern techniques such as changes of medium osmolality or addition of mineral microparticles to the media (microparticle-enhanced cultivation, MPEC) were proposed. Despite the fact that this area of knowledge is still being developed, there are a fair amount of scientific articles concerning the cultivations of filamentous fungi with the use of these techniques. It was described that in Ascomycetes fungi both MPEC or change of osmolality successfully led to the change of mycelial morphology, which appeared to be favorable for increased productivity of secondary metabolites and enzymes. There are also limited but very promising reports involving the successful application of MPEC with Basidiomycetes species. Despite the fact that the mineral microparticles behave differently for various microorganisms, being strain and particle specific, the low cost of its application is a great benefit. This paper reviews the application of the modern morphology engineering techniques. The authors critically assess the advantages, shortcomings, and future prospects of their application in the cultivation of fungi.

Application of microparticle-enhanced cultivation to increase the access of oxygen to Aspergillus terreus ATCC 20542 mycelium and intensify lovastatin biosynthesis in batch and continuous fed-batch stirred tank bioreactors

Microparticle-enhanced cultivation (MPEC) was used in the bioreactor cultivation of lovastatin-producing Aspergillus terreus ATCC 20542. Increasing oxygen saturation of the broth increased lovastatin titer but more important was pellet morphology, in that oxygen reached the fungal cells better in smaller and looser pellets. Thus, we changed pellet size adding 10μm talc microparticles to the preculture. This resulted in 3.5-fold higher lovastatin titer in the subsequent production culture, compared to the control runs (without microparticles), reaching 250mgl−1 in the best continuous fed-batch run. The significant increase of lovastatin titer and low cost of microparticles makes MPEC cultivation for lovastatin production an attractive alternative.

Morphology engineering of basidiomycetes for improved laccase biosynthesis.

ObjectiveThis work is the first application of a morphological engineering technique called microparticle-enhanced cultivation (MPEC) aimed at the facilitation of laccase production in the submerged cultures by two basidiomycetes species Cerrena unicolor and Pleurotus sapidus.ResultsThe positive effect of the applied 10 μm Al2O3 microparticles at concentrations from 5 to 30 g Al2O3 l−1 was shown. Laccase activity increased 3.5-fold for C. unicolor and 2-fold for P. sapidus at 15 g Al2O3 l−1 on 9 and 14 day of the cultivation, respectively, compared to the control culture without microparticles. The increase of laccase activity in the cultivation broths was caused by the action of Al2O3 microparticles on the agglomeration of hyphae. It led to the decrease of the size of the pellets, (on average by 2 mm for C. unicolor), the change of their shape (star-shaped pellets for C. unicolor) and the change of their structure (more compact pellets for P. sapidus).ConclusionsApplication of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.

Microparticle‐enhanced cultivation of filamentous microorganisms: Increased chloroperoxidase formation by Caldariomyces fumago as an example

Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of approximately 500 microm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of < or =42 microm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL(-1) was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/cell formation.