Mycelial Pellets Research Articles

Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology

Natural yellow pigments produced by submerged fermentation of Monascus purpureus have potential economic value and application in the food industry. In the present study, the relationships among fermentation conditions (in terms of pH and shaking/agitation speed), mycelial morphology and the production of Monascus yellow pigments were investigated in both shake-flask and scale-up bioreactor experiments. In the shake-flask fermentation, the highest yield of the Monascus yellow pigments was obtained at pH 5.0 and a shaking speed of 180rpm. Microscopic images revealed that these results were associated with the formation of freely dispersed small mycelial pellets with shorter, thicker and multi-branched hyphae. Further investigation indicated that the hyphal diameter was highly correlated with the biosynthesis of the Monascus yellow pigments. In a scaled-up fermentation experiment, the yield of yellow pigments (401U) was obtained in a 200-L bioreactor, which is the highest yield to the best of our knowledge. The present findings can advance our knowledge on the conditions used for enhancing the production of Monascus yellow pigments in submerged fermentation and facilitate large-scale production of these natural pigments.

Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations

Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O2-plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense “biofilm-like” mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O2-plasma treated PLA membranes specifically promoted higher ACT production than not treated membranes.

Removal of dyes from wastewater by growing fungal pellets in a semi-continuous mode

To increase the efficiency of dye removal from wastewater using mycelial pellets, a bubble column reactor with a simple structure was designed and efficiently used to remove dyes from solution containing dyes. The mycelial pellets were prepared by marine fungus Aspergillus niger ZJUBE-1. Eight dyes were tested as dye targets for the adsorption capacity of mycelial pellets and good removal results were obtained. Eriochrome black T was selected as a model dye for characterizing the adsorption processes in detail. The measurement results of Zeta potential and FT-IR analysis indicate that the electrostatic attraction may play a key role in the biosorption process. The bubble column reactor was utilized to study the batch dye-removal efficiency of mycelial pellets. A re-culture process between every two batches, which was under non-sterile condition, successfully enhanced the utilization of mycelium biomass. The dye removal rate is 96.4% after 12 h in the first batch and then decreases slowly in the following batches. This semi-continuous mode, which consists of commutative processes of dye-removal and re-culture, has some outstanding advantages, such as low power consumption, easy operation, high dye removal rate, and efficient biomass utilization.

Empirical and experimental determination of the kinetics of pellet growth in filamentous fungi: A case study using Neurospora intermedia

Pellet morphology formation by filamentous fungi has gained a lot of attention because of its multiple benefits such as the ease of separation and smaller bioreactor volume requirement. Most reported kinetics studies on fungal pellet growth are centred on aeration, despite the experimental results pointing to the importance of other factors such as pH, substrates and product concentration etc., influencing the pellet formation. Hence a kinetic study on the effect of multiple factors such as aeration, substrate and product concentration and pH was done in this paper using Neurospora intermedia as a model organism, whose ability to form mycelial pellets was recently reported. The maximum growth rate of the pellets under uninhibited conditions at its optimal growth pH was 0.318h−1. The pellets were found to be inhibited by high product (ethanol) concentration with no growth occurring at 70gL−1 and above. High substrate concentration favoured the formation of loose fur-like fluffy pellets. The specific oxygen uptake rate of the pellets was between 0.27–0.9 mmol-O2 g-biomass−1h−1 depending on the pellet average diameter. The results from this kinetic study can be used for bioreactor design, operations and optimization of fermentation processes utilizing N. intermedia.

Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris

In this study, an actinomycete Streptomyces sp. hsn06 with the ability to harvest Chlorella vulgaris biomass was used to investigate the flocculation mechanism. Streptomyces sp. hsn06 exhibited flocculation activity on algal cells through mycelial pellets with adding calcium. Calcium was determined to promote flocculation activity of mycelial pellets as a bridge binding with mycelial pellets and algal cells, which implied that calcium bridging is the main flocculation mechanism for mycelial pellets. Characteristics of flocculation activity confirmed proteins in mycelial pellets involved in flocculation procedure. The morphology and structure of mycelial pellets also caused dramatic effects on flocculation activity of mycelial pellets. According to the results, Streptomyces sp. hsn06 can be used as a novel flocculating microbial resource for high-efficiency harvesting of microalgae biomass.

A new bioreactor design for culturing basidiomycetes: Mycelial biomass production in submerged cultures of Ceriporiopsis subvermispora

Oxygen transfer in submerged cultures of basidiomycetes is a key factor for efficient fungal growth. However, conventional stirred tank and airlift reactors are not utterly suitable for basidiomycetes culturing because they promote high shear stress to the mycelial hyphae and favor rapid agglomeration of mycelial pellets, compromising the diffusion of oxygen to the inner side of mycelium. We describe an original reactor design that overcome some of the limitations of conventional bioreactors used in submerged cultures of basidiomycetes. The strategy was to use a mechanism that permits simultaneous axis rotation and air injection, being the agitation promoted by an L-shaped tube. Adherence of mycelium and shear stress is avoided once only rounded surfaces exist inside the bioreactor. Ceriporiopsis subvermispora was selected as a model basidiomycete. The most productive system employed sucrose/corn steep liquor as the culture medium and pulsed addition of sucrose during the culturing. This approach provided efficient mycelial growth (maximum of 14.1g·L–1), and avoided pH increase and pellet agglomeration throughout the fungal cultivation for 7days, resulting in a biomass productivity of 1.72g·L–1·day–1. Microscopic evaluation of chlamydospores accumulation in the grown mycelium confirmed that minimal fungal stress occurred in the cultures performed in the new designed bioreactor, contrasting with cultures carried out in conventional stirred tank bioreactors.

Effects of nonionic surfactants on pellet formation and the production of β-fructofuranosidases from Aspergillus oryzae KB.

Aspergillus oryzae KB produces two β-fructofuranosidases (F1 and F2). F1 has high transferring activity and produces fructooligosaccharides from sucrose. Mycelial growth pellets were altered by the addition of Tween 20, 40 and 80 (HLB=16.7, 15.6 and 15.0, respectively) in liquid medium cultures to form small spherical pellets. The particle size of the pellets decreased with the HLB value, which corresponds to an increase in surfactant hydrophobicity. Selective F1 production and pellet size were maximized using Tween 20. Adding polyoxyethylene oleyl ethers (POEs) with various degrees of polymerization (2, 7, 10, 20 and 50: HLB=7.7, 10.7, 14.7, 17.2 and 18.2, respectively) was investigated. A minimum mean particle size was obtained using a POE with DP=10, HLB=14.7. The POE surfactants had little effect on the selective production of F1. The formation of filamentous pellets depended on the surfactant HLB value, and F1 enzymes were produced most efficiently using Tween 20.

Simultaneous removal of carbon and nitrogen by mycelial pellets of a heterotrophic nitrifying fungus-Penicillium sp. L1

A novel heterotrophic nitrifying fungus, defined as Penicillium sp. L1, can form mycelial pellets in liquid medium in this study. The effects of inoculation method, C/N ratio, initial pH, and temperature were gradually evaluated to improve the simultaneous removal of total nitrogen (TN) and chemical oxygen demand (COD) in wastewater by Penicillium sp. L1. Results showed that compared with spore inoculation, 48h pellet inoculum could significantly increase the pellet size (from about 1.5mm to 3.2mm) and improve the removal capability, particularly for COD removal (from less than 50-86.20%). The removal efficiencies of TN and COD reached 98.38% (from 136.01mg/L to 2.20mg/L) and 92.40% (from 10,720mg/L to 815mg/L) under the following conditions: C/N 36, pH 3, 30°C, and inoculation with 48h pellets. The pellet diameter reached 4.8mm after 4-day cultivation. In this case, Penicillium sp. L1 removed TN from 415.93mg/L to 43.39mg/L, as well as COD from 29,533mg/L to 8850mg/L. Overall, the results indicated that the pellet size was closely related to the pollutant-removal ability of Penicillium sp. L1. Furthermore, mycelial pellets (4.8mm, dead) only adsorbed 38.08% TN (from 125.45mg/L to 77.78mg/L), which indicated that adsorption did not play a major role in the nitrogen-removal process.

Efficient decolorization of dye-containing wastewater using mycelial pellets formed of marine-derived Aspergillus niger

In order to improve the efficient decolorization of dye-containing water by biosorbent and understand the biosorption mechanism, the self-immobilization mycelial pellets were prepared using a marine-derived fungus Aspergillus niger ZJUBE-1, and an azo dye, Congo red was chosen as a model dye to investigate batch decolorization efficiency by pellets. The pellets as biosorbent showed strong salt and acid tolerance in biosorption process. The results for dye adsorption showed that the biosorption process fitted well with models of pseudo-second-order kinetic and Langmuir isotherm, with a maximum adsorption capacity of 263.2mg·g−1 mycelium. During 6 batches of continuous decolorization operation, the mycelial pellets could possess efficient decolorization abilities (>98.5%). The appearance of new peak in the UV–Vis spectral result indicated that the decolorization process may also contain biodegradation. The mechanism studies showed that efficient biosorption ability of pellets only relies on the active zone on the surface of the pellet, which can be enhanced by nutrition supplement or be shifted outward by a re-culture process.

Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

ABSTRACTAphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.

Yeast as a Bioremediation Nanoparticle Agent in Piggery-Digested Wastewater Treatment

Abstract The opportunity to produce algal biofuel cost-effectively from waste materials, such as piggery-digested waste, drives research to optimize piggery waste effluent as an algal growth medium. Therefore, the aim of this study was to investigate the potential of newly isolated wastewater-born yeast to optimize growth parameters and maximize pollutant degradation in digested wastewater bioremediation. One isolate was identified as Galactomyces geotrichum through morphological observation and DNA sequencing. Growth parameters regarding pH, temperature, and inoculum amount were optimized by orthogonal design to maximize pollutant removal. Results showed that optimal growth conditions were pH 6, 25°C, and 2.5 g mycelium pellet moisture weight. Chemical oxygen demand (COD) reached 60% and total phosphorus (TP) reduction reached 45% under these conditions. Our observations demonstrate that pH had an overall significant effect on COD and TP reduction. More importantly, results suggested that pretreatment wi...

Mycelial pellet formation by edible ascomycete filamentous fungi, Neurospora intermedia.

Pellet formation of filamentous fungi in submerged culture is an imperative topic of fermentation research. In this study, we report for the first time the growth of filamentous ascomycete fungus, Neurospora intermedia in its mycelial pellet form. In submerged culture, the growth morphology of the fungus was successfully manipulated into growing as pellets by modifying various cultivation conditions. Factors such as pH (2.0–10.0), agitation rate (100–150 rpm), carbon source (glucose, arabinose, sucrose, and galactose), the presence of additive agents (glycerol and calcium chloride) and trace metals were investigated for their effect on the pellet formation. Of the various factors screened, uniform pellets were formed only at pH range 3.0–4.0, signifying it as the most influential factor for N. intermedia pellet formation. The average pellet size ranged from 2.38 ± 0.12 to 2.86 ± 0.38 mm. The pellet formation remained unaffected by the inoculum type used and its size showed an inverse correlation with the agitation rate of the culture. Efficient glucose utilization was observed with fungal pellets, as opposed to the freely suspended mycelium, proving its viability for fast-fermentation processes. Scale up of the pelletization process was also carried out in bench-scale airlift and bubble column reactors (4.5 L).

Mycoremediation of manganese and phenanthrene by Pleurotus eryngii mycelium enhanced by Tween 80 and saponin.

Bioremediation of areas co-contaminated with metals and polycyclic aromatic hydrocarbons (PAHs) by mushrooms has attracted considerable attention in recent years. In this study, Pleurotus eryngii was introduced for the removal of Mn and phenanthrene (Phe) from potato liquid medium (PDL) simultaneously. Effects of Tween 80 and saponin on P. eryngii growth together with Mn uptake as well as Phe removal were investigated. Although pollutants had a negative effect on mycelial morphology and growth, P. eryngii could still tolerate and remove Mn and Phe. Tween 80 increased removal of Mn and Phe through increase of P. eryngii growth, Phe solubility, pollutants bioavailability, and specific surface area of mycelium pellets, moreover, the activities of manganese peroxidase (MnP) and laccase, which played an important role on PAHs biodegradation. The maximal removal of Mn and Phe was achieved (92.17 and 93.85% after 15days incubation, respectively) with 0.6gL(-1) Tween 80. Treatments with saponin markedly inhibited P. eryngii growth (50.17-66.32% lower relative to control) due to its fungistatic activity. Nevertheless, saponin could slightly enhance Phe removal through increasing solubility of Phe, and Phe removal rate varied from 80.53 to 87.06% in saponin treatments. Joint stress of Mn and Phe induced a strong antioxidative response, and superoxide dismutase (SOD) activity decreased in surfactants-treated mycelium compared with control. Generally, Tween 80 was more suitable for strengthening mycoremediation by P. eryngii than saponin, and could be a promising alternative for the remediation of heavy metals and PAHs co-contaminated sites by mushrooms.

Carbonate Mineral Formation under the Influence of Limestone-Colonizing Actinobacteria: Morphology and Polymorphism.

Microorganisms and their biomineralization processes are widespread in almost every environment on earth. In this work, Streptomyces luteogriseus DHS C014, a dominant lithophilous actinobacteria isolated from microbial mats on limestone rocks, was used to investigate its potential biomineralization to allow a better understanding of bacterial contributions to carbonate mineralization in nature. The ammonium carbonate free-drift method was used with mycelium pellets, culture supernatant, and spent culture of the strain. Mineralogical analyses showed that hexagonal prism calcite was only observed in the sub-surfaces of the mycelium pellets, which is a novel morphology mediated by microbes. Hemispheroidal vaterite appeared in the presence of spent culture, mainly because of the effects of soluble microbial products (SMP) during mineralization. When using the culture supernatant, doughnut-like vaterite was favored by actinobacterial mycelia, which has not yet been captured in previous studies. Our analyses suggested that the effects of mycelium pellets as a molecular template almost gained an advantage over SMP both in crystal nucleation and growth, having nothing to do with biological activity. It is thereby convinced that lithophilous actinobacteria, S. luteogriseus DHS C014, owing to its advantageous genetic metabolism and filamentous structure, showed good biomineralization abilities, maybe it would have geoactive potential for biogenic carbonate in local microenvironments.

Removal of phenol in phenolic resin wastewater by a novel biomaterial: the Phanerochaete chrysosporium pellet containing chlamydospore-like cells.

A novel biomaterial, the Phanerochaete chrysosporium pellet (CP) composed of chlamydospore-like cells (CLCs), was prepared and its potential in treating phenolic resin wastewater was evaluated. CP possesses higher phenol removal ability in contrast with mycelial pellets of P. chrysosporium, and CLC can be seen as the naturally immobilized enzymes. At shake-flask level, the ideal pH value, temperature, and inoculation quantity of CP for treatment of 1430mg/l phenol wastewater were pH4-6, 30°C, and 5.0g/l, respectively, and the maximum specific removal rate, 41.1mg phenol/g CP/h, was obtained in fixed bed reactor (FBR) when the flow rate of wastewater was 3.4l/h. During the treatment, FBR harbored amounts of bacteria (135 genera) and eukaryotes, as analyzed by metagenomic sequencing. Bacterial pollution not only decreased reactor performance but also had a negative impact on reusability of CP. Hot water treatment (80-85°C) is effective to inhibit bacterial pollution, and heat resistance of CLC makes the repeated regrowing of CP be feasible. This work presents an innovative and low-cost biomaterial for phenol removal and will be helpful for the practical application of P. chrysosporium in wastewater treatment.

Biodegradation, Biosorption of Phenanthrene and Its Trans-Membrane Transport by Massilia sp. WF1 and Phanerochaete chrysosporium.

Reducing phenanthrene (PHE) in the environment is critical to ecosystem and human health. Biodegradation, biosorption, and the trans-membrane transport mechanism of PHE by a novel strain, Massilia sp. WF1, and an extensively researched model fungus, Phanerochaete chrysosporium were investigated in aqueous solutions. Results showed that the PHE residual concentration decreased with incubation time and the data fitted well to a first-order kinetic equation, and the t1/2 of PHE degradation by WF1, spores, and mycelial pellets of P. chrysosporium were about 2 h, 87 days, and 87 days, respectively. The biosorbed PHE was higher in P. Chrysosporium than that in WF1, and it increased after microorganisms were inactivated and inhibited, especially in mycelial pellets. The detected intracellular auto-fluorescence of PHE by two-photon excitation microscopy also proved that PHE indeed entered into the cells. Based on regression, the intracellular (Kdin) and extracellular (Kdout) dissipation rate constants of PHE by WF1 were higher than those by spores and mycelial pellets. In addition, the transport rate constant of PHE from outside solution into cells (KinS/Vout) for WF1 were higher than the efflux rate constant of PHE from cells to outside solution (KoutS/Vin), while the opposite phenomena were observed for spores and mycelial pellets. The amount of PHE that transported from outside solution into cells was attributed to the rapid degradation and active PHE efflux in the cells of WF1 and P. Chrysosporium, respectively. Besides, the results under the inhibition treatments of 4°C, and the presence of sodium azide, colchicine, and cytochalasin B demonstrated that a passive trans-membrane transport mechanism was involved in PHE entering into the cells of WF1 and P. Chrysosporium.

Self-immobilization of a magnetic biosorbent and magnetic induction heated dye adsorption processes

Biosorbents for wastewater treatments is one of the most widely studied techniques in environmental and bioresource research. A marine-derived Penicillium janthinellum strain fungus was used in this study to prepare self-immobilized mycelial pellets. Fe3O4 nanoparticles were added in the culturing media, which were automatically incorporated in the mycelial pellets during culturing process to endow the pellets with magnetic properties. These magnetic mycelial pellets were used as biosorbents for water treatments, and their morphology was characterized by microscopic observation, Energy dispersive X-ray analysis, Fourier transform infrared spectroscopy and thermal gravimetric analysis. The effects of nanoparticles in culture media on pellet formation were discussed and the content of nanoparticle in culturing media was optimized. Study on Congo red adsorption from aqueous solutions showed that the pellets with 71.6wt% Fe3O4 nanoparticles had a maximum adsorption capacity of 102.4mg/g pellets (360.6mg/g mycelia), and the solution heated by magnetic induction showed faster adsorption rates than that of bulk liquid heating processes under the same adsorption temperature. These pellets were easily separated by magnetic forces and the magnetic induction heating is beneficial in energy saving when comparing to traditional heating processes.

Removal of Chromium (VI) from Aqueous Solution Using Mycelial Pellets of Penicillium simplicissimum Impregnated with Powdered Biochar

ABSTRACTThe mycelia pellets of Penicillium simplicissimum impregnated with powdered biochar (MPPSIPB) were synthesized and applied to remove chromium (VI) from aqueous solution. The effects of pH, MPPSIPB dosage, initial Cr(VI) concentration, and contact time were investigated via batch experiments. Results indicated that the percentage removal of Cr(VI) was significantly dependent on the pH of the solution. Ten grams mycelial pellets and 0.2 g powdered biochar could form the most stable pellets. The maximum value of biosorption of Cr(VI) was 28.0 mg/g. Scanning electron microscopy (SEM) analysis showed that the mycelia pellets of Penicillium simplicissimum had abundant filamentous network, which entrapped powdered biochar firmly. Fourier transform infrared (FTIR) analysis suggested that O‒H, N‒H, C‒H, C˭O, and C‒OH groups from MPPSIPB were involved in chromium binding and the subsequent reduction. Kinetic studies indicated that the pseudo-second-order equation fit best for Cr(VI) removal from aqueous solution. Freundlich isotherm was found to apply better for the adsorption equilibrium data with respect to the Langmuir isotherm. Furthermore, MPPSIPB can be separated from aqueous solution completely by filtration. Both experimental study and modeling results indicated that MPPSIPB exhibited remarkable affinity for chromate and had a potential application in Cr(VI) removal from water.

Research Progress and Development Trend of Mycelial Pellets

Research Progress and Development Trend of Mycelial Pellets

Mycelial Pellet Formation in Fungal Lipid Production by Cunninghamella bainieri 2A1 Using Repeated Batch Culture

Repeated batch culture of Cunninghamella bainieri 2A1 was performed to investigate mycelial pellet formation in fungal lipid production process. Experimental studies were performed based on the size and structural morphology of pellets formed with consideration of harvesting times and harvested volumes of repeated batch culture. Electron microscope images showed that fungal cells formed mycelial pellets with varied size from 575 µm × 586 µm in 60 % harvesting volume with a hairy loose morphology to 1.01 mm × 1.18 mm in 90 % harvesting volume with a tightly packed dense morphology.