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

Chemical Engineering Science 170 (2017) 670–676

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A new bioreactor design for culturing basidiomycetes: Mycelial biomass production in submerged cultures of Ceriporiopsis subvermispora Marcelo Domingos, Priscila Brasil de Souza-Cruz, André Ferraz, Arnaldo Márcio Ramalho Prata ⇑ Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, 12602-810 Lorena, SP, Brazil

h i g h l i g h t s  A single device was developed for agitation and air injection into a bioreactor for aerobic fermentations.  Rounded surfaces of agitation device avoid adherence during submerged filamentous fungi cultivation and increases homogeneity.  High homogeneity of culture broth promotes decrease in chlamydospores formation during submerged cultivation of a basiomycete.  A specially designed bioreactor provides higher fungal biomass concentration compared to STR bioreactor.

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Article history: Received 31 August 2016 Received in revised form 10 February 2017 Accepted 4 April 2017 Available online 6 April 2017 Keywords: Bioreactor Ceriporiopsis subvermispora Basidiomycete Oxygen transfer Biomass

a b s t r a c t 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 14.1 g
L–1), and avoided pH increase and pellet agglomeration throughout the fungal cultivation for 7 days, resulting in a biomass productivity of 1.72 g
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. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Submerged cultures of basidiomycetes have been used to produce mycelial biomass for inoculation of solid-state cultures used in large-scale processes such as biopulping and fungal pretreatment of lignocellulosic materials (Ferraz et al., 2008; Singh and Singh, 2014; Rouches et al., 2016). Submerged cultures of basidiomycetes also find application in production of extracellular enzymes, exopolysaccharides and bioactive metabolites (Fang and Zhong, 2002; Tinoco-Valencia et al., 2014; Braga et al., 2015). Ceriporiopsis subsvermispora, used as a model basidiomycete in the current work, is a white-rot fungus recognized by its great ⇑ Corresponding author. E-mail address: [email protected] (A.M.R. Prata). http://dx.doi.org/10.1016/j.ces.2017.04.004 0009-2509/Ó 2017 Elsevier Ltd. All rights reserved.

selectivity in lignin degradation, which has been broadly explored in biopulping and lignocellulose pretreatment processes (Akhtar et al., 1998; Ferraz et al., 2008; Machado and Ferraz, 2017). This species produces several extracellular enzymes such as manganese peroxidases, laccases, xylanases, mananases, endoglucanases, b-glucosidases and cellobiose dehidrogenases (RuttimannJohnson et al., 1993; Ferraz et al., 2008; Harreither et al., 2009; Chmelova and Ondrejovic, 2016). Ceriporiopsis Dom. is a poroid genus belonging to the Polyporaceae family (Burdsall, 1998). C. subvermispora is a rare species, found in temperate regions and distributed in southern Canada, northern North America and Central Europe. The development of appropriated technologies for producing large amounts of inoculum from this and other basidiomycete species may be a key step in biopulping and lignocellulose pretreatment processes.

M. Domingos et al. / Chemical Engineering Science 170 (2017) 670–676

Oxygen transfer in submerged cultures is a key factor for efficient fungal growth (Garcia-Ochoa and Gomez, 2009). However, some of the efficient reactors currently used for submerged cultures, such as the stirred tank reactor (STR) and the airlift reactor, are not utterly suitable for basidiomycetes culturing. These fungal class usually produces extracellular exopolysaccharides (Tang and Zhong, 2002; Kim et al., 2005) that performs as a glue, promoting adhesion of the growing mycelium to the prominent parts of the reactor and sensors, resulting in mycelial agglomeration, which limits oxygen transfer to the core of the large formed pellets (Krull et al., 2013; Silvério et al., 2013). Submerged culturing of basidiomycetes could be advantageous to overcome some of the problems related to the control of largescale solid-state fermentation such as low homogeneity and formation of temperature, nutrient and product gradients associated with the low water availability in the cultures (Krishna, 2005). However, few efforts have been performed to develop new bioreactors design that could avoid high shear stress (Garcia-Ochoa and Gomez, 2009; Fazenda et al., 2010; Silvério et al. 2013; TinocoValencia et al., 2014) and agglomeration of mycelial pellets (Krull et al., 2013; Silvério et al., 2013) inside the cultures. Some improvements in the STR and airlift reactors have been described in patents claiming to diminish shear stress to the microorganisms or cell cultures, maintaining high efficiency in oxygen transfer to the culture broth (Prave and Sittig, 1985; Familletti, 1987; Schilling et al., 1998). However, the problems related to the high adhesion capacity of the basidiomycete’s mycelia in bioreactor prominent parts and sensors have not been addressed up to date. In the present work, we described the growing performance of a model basidiomycete, Ceriporiopsis subvermispora, in submerged cultures. Media improvement and the design of a new bioreactor are presented. An original reactor design was developed to overcome limitations imposed by STR and airlift bioreactors to basidiomycetes growing in submerged cultures.

2. Material and methods 2.1. Fungus, culture media and analytical procedures C. subvermispora (Pilat) Gilbn. & Ryv. (L14807 SS-3 strain) was maintained at 4 °C in 2% (w/v) agar slants containing 2.4% (w/v) potato-dextrose broth (DIFCO, Maryland), 0.7% (w/v) yeast extract (Vetec, Brazil) (PD/YE medium). A culture medium composed of 2% (w/v) sucrose and 3.2% (w/v) corn steep liquor was used for fungal growth in submerged cultures. Corn steep liquor is a commercial product containing 47% (w/w) solids composed mainly of corn protein hydrolysate, minor amounts of lactic acid and reducing sugars and micronutrients as reported elsewhere (Akhtar et al., 1998; Agarwal et al., 2006; Masarin and Ferraz, 2008). Analytical procedures used for monitoring fungal growth in the cultures included pH and mycelial dry weight determinations. Residual sugars in the cultures was determined by the dinitrosalicylic acid procedure (Dubois et al., 1956). When sucrose was used as the carbon source, the liquid medium was acidified with 1 mol
L 1 HCl to a final concentration of 0.5 mol
L 1 followed by heating at 100 °C for 10 min. After heating, the mixture was neutralized with 1 mol
L 1 NaOH and analyzed by the dinitrosalicylic acid method (Dubois et al., 1956). Light microscopic evaluation of the grown mycelium was used for estimating chlamydospores accumulation in the fungal hyphae. Freshly collected mycelium was dispersed in a 5% (w/v) sodium hydroxide solution and stained with 1% phloxine B (Alic et al., 1987). Chlamydospores counting was semi-quantitatively ranked from (none) to ++++ (copious). Volumetric oxygen transfer coefficient (kLa) was determined using the procedure described by Schmidell (2001): after

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filling the bioreactor with the sucrose/corn steep liquor medium nitrogen was sparged abundantly, under stirring, until the dissolved oxygen reached zero, verified by an O2 polarographic electrode previously calibrated to oxygen saturation. After that, the desired agitation and aeration were started. The variation of dissolved oxygen concentration with time was registered and kLa was calculated according to the equation ln (1 C/CS) = kLa. t, where C and CS are the oxygen concentration in the liquid and oxygen saturation concentration, respectively. By plotting ln (1 C/CS) against time (t) kLa was obtained by linear regression. The influence of the culture pH on the C. subvermispora growth rates was estimated in Petri dishes with PD/YE, 2% (w/v) agar medium at pH 3.5, 4.0, 4.5, and 5.5. Culture broths were adjusted to the defined pH by adding 1 mol
L 1 HCl or 1 mol
L 1 NaOH before autoclaving at 121 °C for 30 min. The colony diameter was measured every day up to reach the Petri dishes border. Triplicate cultures for each pH were performed. Growth rates were determined as the slope in the curves relating the colony diameter versus growing period. Average values followed by standard deviations are reported in the text. 2.2. Submerged-unshaken cultures Unshaken submerged cultures of C. subvermispora were conducted in 250 mL Erlenmeyer flasks maintained at 27 °C using PD/YE or the sucrose/corn-steep liquor media. Each culture flask contained 20 mL of medium that was inoculated with 2 discs (8 mm in diameter) of C. subvermispora precultured in PD/YE solid medium. For each culturing period, fungal biomass, residual sugars and pH were determined from three independent culture flasks. 2.3. Cultures in STR bioreactor An 1.25-L (working volume) STR bioreactor (Bioflo III, New Brunswick) was inoculated with C. subvermispora mycelium previously grown in sucrose/corn-steep liquor for 15 days. For inoculum preparation, the mycelium mass from several cultures grown on 250 mL Erlenmeyer flasks were blended in autoclaved water by 10 cycles lasting 15 s each cycle. A 45 s resting time between each blending cycle was used to avoid excessive heating of the mycelium suspension. The bioreactor contained 1.25 L of sucrose/cornsteep liquor medium and was inoculated with blended mycelium at the ratio of 250 mg
L–1 (mass of mycelium at the dry weight basis per L of medium). Cultures were agitated at 166 rpm and aerated at 1.16 vvm, based on the work from Lee et al. (2004), who used submerged culture to produce mycelial biomass and exopolysaccharides by basidiomycetes. Adecanol was used as an anti-foaming agent at 0.77 mL
L 1. Periodic sampling of the culture was performed using the bioreactor facilities. Samples were assayed for mycelial and residual sugar contents and for pH. 2.4. New bioreactor design and culture conditions The bioreactor consisted of a simple 14-L Marriote’s bottle with a single-bottom outlet for sampling. The main top entrance of the Marriote’s bottle was fitted with an innovative cap as briefly described in previous patent (Prata et al., 2011), performing several features: (a) to close the main bottle entrance providing one gasexhaust tube and one liquid inlet tube; (b) to hold one air inlet tube connected to the central air inlet chamber; (c) to hold an L-shaped stainless steel tube, fixed with two ball bearings, that cross the air inlet chamber (Fig. 1). The L-shaped tube had the top aperture closed and was fixed into an external electric motor providing its rotation. Inside the air inlet chamber, the L-shaped tube has a ring of small holes permitting air entrance to the inside of the

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A

B Motor

Liquid inlet

Exhaust Gas outlet Air inlet

Bioreactor’s cap

“L” shapped tube

14-L Marriote's bottle

C Air inlet Air chamber

Ball bearings

Fig. 1. Schematic diagram of the low-shear aerated-agitated bioreactor (LSAAB). (A) Front view (14-L Marriote’s bottle has 23 cm diameter and the horizontal part of the ‘‘L” shapped tube is 8.5 cm long); (B) side view; (C) bioreactor’s cap: Once inside the chamber, the air is forced through four holes in the ‘‘L” tube and falls down to its lower end.

bioreactor. The rotational movement of the L-shaped tube serves for aeration and agitation of the culture simultaneously. S1-supplementary material presents a short video showing the airflow patterns inside this bioreactor (https://www.dropbox. com/s/sic3mlz1tw51pbu/S1%20aeration%20pattern%20in%20new% 20designed%20bioreactor.mp4?dl=0). This new designed 14-L bioreactor was named low-shear aerated-agitated bioreactor (LSAAB). Cultures of C. subvermsipora in the LSAAB was performed as described for the 1.25-L STR bioreactor. The L-shaped aerating tube rotated at 166 rpm and the air inlet flow corresponded to 1.0 vvm. Aeration was slightly lower than that used in STR experiments owing to some compressor limitation. The bioreactor was kept at a temperature controlled room (27 ± 2 °C). Periodic sampling of the culture was performed using the single bioreactor outlet located at the bottom of the Marriote’s flask. Samples were assayed for mycelial and residual sugar contents, pH and dissolved oxygen. For dissolved oxygen measurements, sampled liquid filled up a 10-mL flask up to the top and the flask was promptly screw-caped to avoid further oxygen dissolution or release. Liquid was assayed for dissolved oxygen using a Hansatech oxymeter (Cunha et al., 2010).

3. Results and discussion 3.1. Biomass production by C. subvermispora in conventional submerged culturing systems Inoculum production is considered a bottleneck for costeffective industrial application of basidiomycetes in solid-state fermentation processes such as biopulping, biological pretreatment of lignocellulosic residues and edible mushroom production (Akhtar et al., 1998; Ferraz et al., 2008; Singh and Singh, 2014; Rouches et al., 2016). In the present work, searching for less expensive culture broths, an appropriate culture system for the selective whiterot basidiomycete C. subvermispora was evaluated. Culturing C. subvermispora in potato-dextrose/yeast extract (PD/ YE) agar Petri dishes indicated that high growth rates were obtained at low pH values from 3.5 to 5.0 (growth rates were 20.2 ± 0.3, 20.3 ± 0.8, 19.5 ± 0.8 and 14.9 ± 0.1 mm
day–1 for initial pH values of 3.5, 4.0, 5.0 and 5.5, respectively). Fungal biomass accumulation and sugars consumption using the same medium buffered at pH 4.0 in unshaken cultures are shown in Fig. 2a. Biomass dry weight reached 5.7 ± 0.2 g L–1 after 17 days of culturing. This culturing system, broadly used for inoculum production at

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18

4

12

2

6

0

0 5

b

10

10

15

20

Biomass pH Sugars

8

30 24

6

18

4

12

2

6

0

0 0

5

10

15

20

Culturing time (days) Fig. 2. Biomass accumulation and sugar consumption during submerged culturing of C. subvermispora in unshaken conditions and two different culture media: (a) potato-dextrose/yeast extract and (b) sucrose/corn steep liquor.

laboratory scale (Leatham et al., 1990; Mendonça et al., 2004; Hunt et al., 2004; Masarin et al., 2016), is not optimized and is unsuitable for large-scale inoculum production. Less expensive culture broths such as sucrose/corn steep liquor (Akhtar et al., 1998; Agarwal et al., 2006) used in similar unshaken cultures provided higher biomass yield, 9.2 ± 0.5 g
L–1 after 17 days (Fig. 2b). Corn steep liquor provided an efficient buffering of the medium once the pH was maintained over the evaluated culturing period in this unshaken cultures. However, available sugars were not depleted until the end of the monitored fermentation time (17 days) in both culture broths. The incomplete sugar consumption showed in Fig. 2 suggests some limitations in the culture broth due to nitrogen, phosphorous or micronutrients depletion or inefficient oxygen transfer in the unshaken cultures. Microscopic evaluation of the mycelium grown in these cultures showed the occurrence of chlamydospores (as also verified by Saxena et al., 2001) from the 6th culturing day on, suggesting the occurrence of stressing conditions in this period (Fig. 3). With cultures aging, the chlamydospore amounts increased rapidly. The low mycelial biomass productivity in the C. subvermispora cultures means a limited capacity for inoculum production aiming

3.2. Original bioreactor design and test for biomass production by C. subvermispora in submerged cultures The developed bioreactor was tested for a basidiomycete growth in submerged cultures. The new reactor drawing aimed minimal shear stress to avoid mycelia damage. The internal area of the reactor was maintained as unobstructed as possible to avoid anchoring places, which serve for initiation points of pellets agglomeration. High kLa was also a target, but the slow growth rate of basidiomycetes in submerged cultures indicates that oxygen transfer in the reactor is not a critical feature. Traditional airlift reactors (Garcia-Ochoa and Gomez, 2009) served as a model; however, previous experience growing basidiomycetes in this reactor type indicate that mycelia sheaves often obstruct the central tube, minimizing aeration and agitation capabilities (data not shown).

Biomass

10

30

pH 8

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4

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2

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0

Total sugars (g.L-1)

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large scale processes such as biopulping or fungal pretreatment of lignocellulosic materials (Ferraz et al., 2008; Singh and Singh, 2014; Rouches et al., 2016). Stirred tank reactors (STR) are often used for large scale culturing of bacteria and yeasts (GarciaOchoa and Gomez, 2009; Braga et al., 2015). However, filamentous fungi, especially basidiomycetes, suffer from high shear stress occurring inside these reactors (Fazenda et al., 2010; Silvério et al. 2013; Tinoco-Valencia et al., 2014). In general, basidiomycetes tend to form large pellets with poor oxygen transfer capacity in these reactors. Another limited effectiveness of STR for basidiomycetes culturing is that mycelia damage derived from high shear stress induces nutrient consumption for mycelium repair instead of growth (Fazenda et al., 2010; Krull et al., 2013). A reference C. subvermispora submerged culturing in a 1.25-L STR was performed to check the performance of this fungal species in this bioreactor model (Fig. 4). Agitation and aeration levels were set at previously optimized conditions described for growth of the basidiomycete Grifola frondosa (Lee et al., 2004). Biomass dry weight reached 7.0 g
L–1 after 12 days of culturing in the STR and sugars were consumed up to 77% of the initial value in the same period. The biomass accumulation plateau occurred already from the 7th culturing day on, corresponding to a productivity of 0.93 g
L–1
day 1. However, large pellets were observed inside the STR from the 6th culturing day. The pellets agglomerate from the 7th culturing day on, forming large mycelia sheaves that adhered to the reactor sensors as well as on the tank wall (Fig. 5). Low nutrient and oxygen diffusion inside these large mycelia sheaves decrease the growth efficiency (Krull et al., 2013; Silvério et al., 2013).

Mycelial biomass (g.L-1) and pH values

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0

Mycelial biomass (g.L-1) and pH values

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Biomass pH Sugars

Total sugars (g/L)

a

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Mycelial biomass (g.L-1) and pH values

M. Domingos et al. / Chemical Engineering Science 170 (2017) 670–676

0 0

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4

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12

Culturing time (days) Fig. 3. Example of C. subvermispora mycelium containing chlamydospores. Mycelium recovered from a submerged culture was dispersed in 5% sodium hydroxide and stained with 1%phloxine B.

Fig. 4. Biomass accumulation and sugar consumption during submerged culturing of C. subvermispora in 1.25-L stirred tank reactor (STR) on sucrose/corn steep liquor medium.

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Fig. 5. Large mycelia sheaves of C. subversmispora formed from the 7th culturing day inside of a 1.25-L stirred tank reactor (STR) containing sucrose/corn steep liquor broth.

The kLa obtained at 1.0 vvm with 10 L of sucrose/corn steep liquor medium at pH 4.5 was 9.4 h–1. This was a low kLa value as compared to airlift and STR bioreactors previously used for filamentous fungi cultivation. For example, Michelin et al. (2013) used air lift and STR bioreactors in Aspergillus niger fermentations. In this case, kLa of 12 h–1 and 30 h–1 were reported for the airlift and STR (400 min 1 agitation) at 0.5 vvm, respectively. Lin et al. (2010) reported 26 h–1 for the kLa of a 18.4–L STR bioreactor when used 1.0 vvm and 173 min–1 agitation. In the present work, using the 1.25–L STR bioreactor at 166 rpm and 1.16 vvm, a kLa of 9.5 h–1 was obtained, similar to the value from LSAAB. This fact suggests that at least at low agitation rates the proposed agitation/aeration strategy could be compared to STR in terms of oxygen transfer. Further studies must be performed in order to verify the efficiency of LSAAB using higher agitation rates. The kLa of 9.5 h–1 is quite lower than that obtained by Michelin et al. (2013) and Lin et al. (2010), which used synthetic medium for fungal cultivations. The lower kLa value can be attributed to the composition of sucrose/corn steep liquor medium (item 2.1), used for C. subvermispora cultivation. Corn steep liquor is a quite complex material resulting from the corn wet-milling process. According to Nienow (2010) all material in broths, whether in solution or in suspension, affect both kL and a, compared to water. Nevertheless, cultures requiring high oxygen transfer would require improvements of the LSAAB, particularly in its air sparger, as reported by Garcia-Ochoa and Gomez (2009). For example, a ‘‘T” shaped tube could be used instead of the ‘‘L” shaped tube, providing an improvement in air distribution inside the bioreactor. Indeed, this is important to enable using higher agitation speed, which also favors the oxygen transfer. Three serial culturing schedules were performed in LSAAB to demonstrate the suitability of the bioreactor for culturing C. subvermispora as a model basidiomycete. The first trial used the same batch culturing schedule already evaluated for the conventional 1.25-L STR reported in Fig. 4. Data for sugar consumption and biomass accumulation are shown in Fig. 6a. Under these conditions, intense fungal growth was observed, reaching 6.2 g
L–1 at the 4th culturing day and 9.1 g
L–1 at day 13. Fungal growth occurred mainly through formation of numerous small pellets, as shown in

Fig. 6. Biomass accumulation and sugar consumption during submerged culturing of C. subvermispora in 14-L bioreactor (LSAAB) on sucrose/corn steep liquor broth: (a) normal batch culturing, (b) normal batch culturing with pH control and (c) batch culturing with a pulse of sucrose at the 5th culturing day.

Fig. 7a and b. A closer view of the pellets (Fig. 7c) indicated noncompacted mycelium bundles, which permitted efficient diffusion of nutrients and oxygen. However, the intense fungal growth was followed by rapid oxygen consumption, reaching a dissolved oxygen concentration of 5.9 lmol dL–1 at day 7. The oxygen depletion in the culture seemed to have induced cell death and lysis, since pH increased from day 4, reaching 7.1 at the day 13. Oxygen depletion in the medium could be a consequence of the rapid fungal growth associated with the low kLa value in the bioreactor. To overcome pH increase in the cultures, two additional experiments were performed in the LSAAB. One included periodic correction of pH to maintain the range of 4–5, as shown in Fig. 6b. The other used a slightly lower sucrose concentration at the culture beginning, and a sucrose pulse of 5 g
L–1 at the 5th culturing day, as shown in Fig. 6c. Control of pH during the culturing extended the period of intense fungal growth up to 7 days, providing a fungal biomass

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Biomass productivity (g.L-1.day-1)

Fig. 7. LSAAB bioreactor (a) with C. subvermispora growing on sucrose/corn steep liquor at the 5th culturing day. Small, homogeneous (b) and non-compacted (c) pellets were formed. Pellet micrograph was obtained after phloxine B staining and magnification of 40 times.

2.5

LSAAB(c) LSAAB(b) LSAAB(a) STR

2 1.5 1 0.5 0 2

4

6

8

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14

Culturing time (days) Fig. 8. Biomass productivity of C. subermispora cultured in sucrose/corn steep liquor broth inside STR and LSAAB. Letters a, b and c according to Fig. 6.

concentration of 10.6 g
L–1. The oxygen depletion was less severe since 12–13 lmol
dL–1 was maintained from the 7th to the 13th culturing day. However, cell lysis occurred from day 7, since fungal biomass decreased from this period (Fig. 6b). The use of sucrose pulse was performed with basis on previous studies reported by Fang and Zhong (2002) evaluating biomass and exopolysaccharides production by the basidiomycete Ganoderma lucidum. This approach was very efficient to avoid pH increase in the cultures of C. subvermispora in the LSAAB, as shown in Fig. 6c. Consequently, the period of intense fungal growth continued up to day 11, which caused almost complete consumption of the available sucrose in the medium and a biomass concentration of 14.1 g
L–1, meaning a productivity of 1.72 g
L–1
day–1 at the 7th culturing day. Dissolved oxygen was over 10 lmol
d L–1 up to day 9 and decreased to 7.5 lmol
d L–1 at the end of the culturing period, which matched with the biomass decrease owing to cell lysis observed on the 14th culturing day (Fig. 6c).

New bioreactors design for cultivation of filamentous fungi has been poorly exploited (Garcia-Ochoa and Gomez, 2009). Most of available work deals with medium and culture conditions optimization using conventional STR (Fang and Zhong, 2002; Wei et al., 2014). Basidiomycetes growth rate and biomass accumulation depends on several factors. Fungal species or even strains of the same species vary in their abilities to convert nutrients into fungal biomass (Wei et al., 2014). Several researches have focused on enzyme secretion or exopolysaccharide production by basidiomycetes in STR reactors. So far, the highest biomass accumulation levels in fed-batch cultures of Ganoderma species have been reported to be in the range of 20–26 g
L–1 under optimal nutritional, agitation and aeration conditions (Tang and Zhong, 2002; Wei et al., 2014). Cultivation of other basidiomycetes species usually reach lower biomass accumulation: 8 g
L–1 for Pleurotus ostreatus (Tinoco-Valencia et al., 2014), 8–10 g
L–1 for Tremella fuciformis (Cho et al., 2006), 13 g
L–1 for Antrodia camphorat and 10 g
L–1 for Agrocybe cylindracea (Kim et al., 2005). The current reported data for C. subvermispora in submerged cultures performed in the LSAAB with substrate pulse attained 14.1 g
L–1, which is in the same range of several previously studied basidiomycetes. Biomass productivities obtained in the cultures of C. subvermispora along culturing time in the STR and LSAAB are shown in Fig. 8. The substrate pulsed cultures performed in LSAAB provided around twice the productivity of the STR along the first 9 culturing days. Data compiled from the C. subvermispora growth in the STR and LSAAB suggest that LSAAB operated with substrate pulse provided the most intense fungal growth with minimal stress induced to the cultures. A semi-quantitative microscopic evaluation of the grown mycelium in each culture confirmed that minimal chlamydospores accumulation occurred in the substrate pulsed cultures performed in LSAAB (Table 1). Chlamydospores counting was semiquantitatively ranked from (none) to ++++ (copious). Data in Table 1 show that chlamydospores accumulated significantly from the third culturing day in STR, reaching a maximum on day 8. In contrast, a similar batch culture in LSAAB presented scarce chlamydospores only on the 6th culturing day, with a maximum

Table 1 Chlamydospores accumulation (*) in the C. subvermispora mycelium grown on sucrose/corn steep liquor broth in STR and LSAAB. Culturing period (days)

STR batch culture

LSAAB batch culture

LSAAB batch culture with pH control

LSAAB substrate pulsed culture

3 6 7 8 9 12

+ +++ +++ ++++ ++++ ++++

+ ++ +++ ++++ ++++

+ ++ ++ +++ +++

+ + + ++

(*) Semi-quantitative counts based on microscopic evaluation of the mycelium varying from none ( ) to copious (++++) chlamydospores.

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on the day 9. The use of substrate pulse during the culture in LSAAB provided mycelium almost free of chlamydospores up to day 6, accumulating only a minor amount at the end of the 12 culturing days. The observed low chlamydospore accumulation can be due to the low shear stress provided by the proposed bioreactor configuration. 4. Conclusions Most of available work on fungi cultivations deals with medium and culture conditions optimization using conventional reactors. High biomass productivities were obtained in the cultures of C. subvermispora using a new designed bioreactor that causes low shear stress to the mycelial hyphae, showing the importance of bioreactor configuration for improving the cultivation process. An important characteristic of the proposed bioreactor is the high homogeneity of the culture broth during basidiomycete cultivation. It can be concluded that this characteristic provided efficient mass transfer phenomena (both gas-liquid and solid-liquid) in LSAAB comparing to conventional STR reactors, making possible an efficient pH control, a representative sampling and a more efficient substrate uptake during the process, important to reach high mycelial biomass concentration of the basidiomycete used. The use of sucrose/corn steep liquor medium in the new designed bioreactor provided appropriated levels of dissolved oxygen during 7 days of culturing and at the same time avoided pellets agglomeration, showing to be a promising bioreactor for basidiomycetes cultivation when fungal biomass growth is the target. Acknowledgements Financial support from FAPESP, CNPq and CAPES. PSC thanks FAPESP for student fellowship under contract number 02/07747-0. References Agarwal, L., Isar, J., Meghwanshi, G.K., Saxena, R.K., 2006. A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli. J. Appl. Microbiol. 100, 1348–1354. Akhtar, M., Blanchette, R.A., Myers, G., Kirk, K., 1998. An overview of biomechanical pulping research. In: Young, R., Akhtar, M. (Eds.), Environmentally Friendly Technologies for the Pulp and Paper Industry. John Wiley and Sons, New York, pp. 309–383. Alic, M., Letzring, C., Gold, M.H., 1987. Mating system and basidiospore formation in the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53, 1464–1469. Braga, A., Mesquita, D.P., Amaral, A.L., Ferreira, E.C., Belo, I., 2015. Aroma production by Yarrowia lipolytica in airlift and stirred tank bioreactors: differences in yeast metabolism and morphology. Biochem. Eng. J. 93, 55–62. Burdsall, S., 1998. Taxonomy of industrially important white-rot fungi. In: Young, R., Akhtar, M. (Eds.), Environmentally Friendly Technologies for the Pulp and Paper Industry. John Wiley and Sons, New York, pp. 259–272. Chmelova, D., Ondrejovic, M., 2016. Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes. J. Basic Microbiol. 56, 1173–1182. Cho, E.J., Oh, J.Y., Chang, H.Y., Yun, J.W., 2006. Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis. J. Biotechnol. 127, 129–140. Cunha, G.S., Masarin, F., Norambuena, M., Freer, J., Ferraz, A., 2010. Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures. Enzyme Microbial Technol. 46, 262–267. Dubois, M., McDonald, C., Robert, C.R., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Fang, Q.H., Zhong, J.J., 2002. Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolites-ganoderic acid and polysaccharide. Biochem. Eng. J. 10, 61–65. Familletti, P.C., 1987. Airlift Bioreactor. US Patent 4,649,117. Fazenda, M.L., Harvey, L.M., McNeil, B., 2010. Effects of dissolved oxygen on fungal morphology and process rheology during fed-batch processing of Ganoderma lucidum. J. Microbiol. Biotechnol. 20, 844–851. Ferraz, A., Guerra, A., Mendonca, R., Masarin, F., Vicentim, M.P., Aguiar, A., Pavan, P. C., 2008. Technological advances and mechanistic basis for fungal biopulping. Enzyme Microbial Technol. 43, 178–185.

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