Basidiomycota strains

The fungal strain L. edodes (M3102) was purchased from Mycelia®, Deinze, Belgium. The stock culture was maintained on agar slants and sub-cultured once a month. The agar slant media consisted of the following reagents (bought from Sigma-Aldrich, Dublin, Ireland): 5 g/L of yeast extract (YE), 3 g/L of malt extract (ME), 2 g/L of soy peptone (SP), 2 g/L of glucose, and 15 g/L of agar. The pH of the media was 6.9.

Conical flasks growth and inoculum preparation

To prepare the inoculum for the submerged cultivation in conical flasks, the following procedure was used: the selected fungal strain was grown by adding vegetatively growing and cryopreserved malt agar (MA) plates and incubated at 28 °C for 5 days. Five agar cubes (about 4 mm in diameter) were cut from the agar at the leading edge of the mycelium and placed into 250-mL conical flasks containing 100 mL of media that consisted of 15 mL/L of corn-steep liquor, CSL (Santa Cruz Biotechnology, Heidelberg, Germany) 10 g/L of glucose (Sigma-Aldrich, Dublin, Ireland), and 3.3 g/L of KH2PO4 (Fisher Scientific, Dublin, Ireland); the flasks were kept at a pH of 5.6. These cubes were then homogenised using an IKA T18 digital ULTRA TURRAX® homogeniser (IKA, Staufen, Germany), the tip of which was sterilised by autoclave. The flasks were placed in a New Brunswick Scientific Innova® (Eppendorf AG, Hamburg, Germany) 44 rotary shaker and shaken at 150 RPM at 25 °C for 7 days, which resulted in the growth of fungal pellets. The fungal biomass was maintained in liquid media stocks on a rotary shaker table shaking at 150 RPM, with the temperature maintained at 25° C. The culture was refreshed every 7 days. The stocks consisted of 100 mL of media in 250-mL flasks. The stocks were refreshed by homogenising the contents of one flask after 7 days of growth and pipetting 5 mL of the homogenised suspension to inoculate another 250-mL flask, which was subsequently shaken in a rotary shaker at 150 RPM for 7 days at 25 °C. This process was repeated every 7 days to maintain stocks. An 800-mL inoculation flask (to be used in the stirred tank reactor (STR) was prepared in the following manner. Two 7-day-old 250-mL flasks with a fungal strain containing 100 mL growth media (15 mL/L CSL, 10 g/L glucose, 3.3 g/L KH2PO4) were homogenised with a homogeniser and were each transferred to two 2-L flasks containing 700 mL of 15 mL/L CSL, 10 g/L glucose, and 3.3 g/L KH2PO4, making the total volume for each flask 800 mL. After 10 days, the biomass of one flask was drained through a sieve. The contents of the other flask and the drained biomass were transferred to a 1-L Duran bottle. Due to the fact that some evaporation had occurred during flask growth, the total resulting volume of medium and pellets in the Duran bottle was roughly 800 mL. The biomass of this bottle was homogenised for 30 s. Following the homogenisation, the contents of the Duran bottle were transferred to a fermenter (bioreactor) using a peristaltic pump connected to the Biostat C (Sartorius™ AG, Göttingen, Germany) stirred tank reactor (STR) using a Watson Marlow (Spirax Group PLC, Cheltenham, UK) peristaltic pump.

Cryopreservation of the fungal strain

L. edodes was grown in cultivation flasks in the above-mentioned media for 5 days, until a homogenous population of pellets developed an approximate size of 3 mm. Pelletised aggregates cultivated in flasks were homogenised in sterile conditions using an IKA® T18 UltraTurrax® (IKA, Staufen, Germany) submergible homogeniser, equipped with a stainless steel S18 N 10G accessory that facilitates dispersion of the mycelia aggregates. After homogenisation, 100 μL of the culture was transferred into sterile cryovials already containing 450 μL of sterile glycerol (60% v/v solution) and 450 μL of sterile sucrose (30% w/v solution) for a final volume of 1 mL. The cryovials were kept in an ice bath at 0 °C for 5 min before being transferred into a − 70 °C Revco® (Thermo Scientific, Dublin, Ireland) Elite plus® freezer.

In order to mix the mycelia with the cryoprotectants, the following steps were taken: firstly, fungal pellets that had been grown in 100 mL of 15 mL/L CSL, 10 g/L glucose, and 3.3 g/L KH2PO4 media in 250-mL flasks at 250 RPM were homogenised. Fifty microliters of the homogenised pellets and spent media was transferred to a cryovial. For cryovials (i) and (ii), 950 µL of the cryoprotectants was added. For the other cryovials in which each cryoprotectant was added along with skimmed milk, 475 µL of the cryoprotectant was added along with 475 µL skimmed milk. The cryovials had a total volume of 1 mL. The vials were put into an ice bath for 5 min and then transferred to a − 80 °C freezer.

In order to test for the viability of the strains after cryopreservation, the vials were thawed and the contents transferred onto an agar plate (30 g/L ME, 3 g/L SP, 15 g/L agar) and their growth analysed.

Batch cultivation in stirred tank reactor (STR)

A Biostat-C (Sartorius™ AG, Göttingen, Germany) 5-L total working volume vessel was chosen for batch fermentation. Dissolved oxygen (DO) and pH were constantly monitored for the duration of the growth period using online probes. In particular, DO was maintained using a constant flow of air (provided with an air compressor) of 1 vvm (4 slpm); a limit of 40% of oxygen saturation in the media was maintained as a reference point; the background agitation rate was set at 400 RPM, which equates to an impeller tip speed of 1.3 m/s. The agitation rate was modified by sets of burst speed increases for specific time frames. If ever the DO limit of 40% was reached, an increase in agitation rate (RPM) was also set up to be activated as a cascade control. The pH was controlled automatically at 5.6 ± 0.1 by the addition of 20% v/v NH4OH solution (Sigma-Aldrich, Dublin Ireland) or 15% v/v H2SO4 (Sigma-Aldrich, Dublin Ireland); foaming was controlled by the automatic addition of antifoam (polypropylene glycol P2000, Sigma-Aldrich, Dublin Ireland) when required. A laptop computer was used to remotely record data every 5 s. The accumulated data were recorded into BioPAT® MFCS SCADA fermentation software (Sartorius™ AG, Göttingen, Germany). A homogenised inoculum of 800 mL was used for a 4-L working volume. The temperature of the fermenter was set to 25 °C.

Semicontinuous cultivations in stirred tank reactor (STR)

A Biostat-C (Sartorius™ AG, Göttingen, Germany) 5-L total working volume vessel was also chosen for semicontinuous fermentation studies. Dissolved oxygen (DO) and pH were constantly monitored for the duration of the growth period using online probes. In particular, DO was maintained using a constant flow of air (provided with an air compressor) of 1 vvm (4 slpm); a limit of 40% of oxygen saturation into the media was maintained as a reference point; the background agitation rate was set at 400 RPM, which equates to an impeller tip speed of 1.3 m/s. The agitation rate was modified by sets of burst speed increases for specific time frames. If ever the DO limit of 40% was reached, an increase in agitation rate (RPM) was also set up to be activated as a cascade control. The pH was controlled automatically at 5.6 ± 0.1 by the addition of 20% v/v NH4OH solution (Sigma-Aldrich, Dublin, Ireland) or 15% v/v H2SO4 (Sigma-Aldrich, Dublin, Ireland); foaming was controlled by the automatic addition of antifoam (polypropylene glycol P2000, Sigma-Aldrich, Dublin, Ireland) when required. A laptop computer was used to remotely record data every 5 s. The accumulated data were recorded into BioPAT® MFCS SCADA fermentation software (Sartorius™ AG, Göttingen, Germany). A homogenised inoculum of 800 mL was used for a 4-L working volume. The temperature of the fermenter was set to 25 °C. The semicontinuous regime was obtained by the use of a continuous process modulus (CPM) manufactured by Bionet™ (Alamo de Murcia, Spain) externally controlled by Rosita™, proprietary software of the same company. The CPM is provided with two peristaltic pumps with different heads, which can either work in continuous or semicontinuous mode for the addition of fresh medium or the withdrawal of spent medium. The head of the pump for the media addition works in a range of 17 to 300 mL/min, while the head of the pump for the media withdrawal works in a range of 368 to 1760 mL/min.

Analytical procedures

After harvesting, the pelletised fungal biomass was filtered through a 90-µm membrane filter. The filter was made of stainless steel, and its diameter was 200 mm. The filtration was carried out by gravity and at room temperature. The mycelium was then resuspended in water for 2 min to clear away any excess media and was filtered again using the same method. The amount of water that the mycelia were resuspended in was equal to the quantity of media from which the mycelia were obtained. The harvested pelletised fungal biomass was frozen into a − 70 °C Revco® (Thermo Scientific, Dublin, Ireland) Elite plus® freezer, on bulk trays designed for a Labconco™, (Kansas City, MO, USA) freeze dryer. After freezing, the mycelia were transferred to the Labconco™ freeze-dryer, and the freeze-dryer created a near-vacuum of 0.1 mbar, which caused the water present in fungal samples to evaporate by sublimation. Freeze drying lasted for 72 h. After freeze-drying, the biomass was then weighed using an Ohaus™ Explorer weighing scale (Ohaus™, Nänikon, Switzerland), and the value was used to determine the concentration (g/L) of mycelium harvested from shake flasks and bioreactors. Glucose and analyses of other monosaccharides were detected by using a Shimadzu®, (Duisburg, Germany) HPLC equipment provided with a RID detector. The used mobile phase was a 0.014N H2SO4 solution, prepared in Milli-Q water, subsequently vacuum filtered with a 0.2 μm pore size filter; this mobile phase was used in an isocratic fashion. The column used was an Aminex HPX-87H (BioRad®, Watford, England) with a solid polymeric matrix composed of polystyrene divinylbenzene. The flow rate of the mobile phase was 0.55 mL/min. α- and β-glucans were detected by an enzymatic-based colorimetric method using a specific yeast/fungi glucan detection kit (Megazyme®, Wicklow, Ireland) following the company provided protocol.

Statistical analysis of results

The averaged values of CDW obtained varying the amount of bursts speed per day, or varying the impeller constant speed or varying the impeller burst speed are tabulated in Tables 1, 2 and 3, respectively, and were obtained by triplicate experiments. The achieved values of fungal CDW in batch and semicontinuous fermentations were also obtained by triplicate independent experiments. Analyses of variance (specifically, one-way ANOVA) of the mean values of CDW obtained with different impeller conditions (batch fermentations) and with different dilution rates (semicontinuous fermentations) were performed to determine a statistical difference across results, and they were performed using the corresponding function in Excel™; T-test and ad hoc Tukey test were performed on the ANOVA results to allow both pairwise and total comparisons of them; T-test and Tukey’s test were also performed using the corresponding function in Excel™.

Table 1 Impact of the number of impeller tip speed bursts on biomass and glucan content of L. edodes cells grown in batch cultureTable 2 The CDW and glucan content of L. edodes grown in a stirred tank bioreactor with various “constant impeller” tip speedsTable 3 The effect of impeller tip speed variation in a stir tank reactor (STR) on the growth and glucan content of L. edodes when grown in batch mode