Improving mycelium-bound lipase production by aggregating Rhizopus chinensis on a draft tube in a modified stirred tank fermentor

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Improving mycelium-bound lipase production by aggregating Rhizopus chinensis on a draft tube in a modified stirred tank fermentor Dong Wang a , Zengliang Zhu a , Xiaoqing Wang b , Mauricio Bustamante b , Yan Xu a,∗ , Yan Liu b , Wei Liao b a Key Laboratory of Industrial Biotechnology, Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China b Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824, USA

a r t i c l e

i n f o

Article history: Received 4 May 2015 Received in revised form 30 September 2015 Accepted 4 October 2015 Available online xxx Keywords: Rhizopus chinensis Fungal morphology Lipase Synthetic activity Fermentor Computational fluidic dynamics (CFD)

a b s t r a c t Rhizopus chinensis (CCTCC 2010021) is a filamentous fungus that demonstrates a good capability to produce mycelium-bound lipase (mb-RCL) for ester synthesis in non-aqueous solutions. It has been reported that the mycelial aggregation of the fungal growth promotes mb-RCL production compared to free cell growth. This study modified a stirred tank reactor (STR) to accommodate a perforated draft tube so that aggregated growth of the fungal mycelia in the reactor was achieved. The aggregated growth on the draft tube not only improves mb-RCL accumulation but also changes the broth’s rheological properties. Computational fluid dynamic (CFD) modeling and flow pattern verification conclude that the preferred fermentation condition was agitation at 600 rpm and aeration at 1 vvm (4 L/min). Under this condition, the aggregated R. chinensis in the modified STR can fully utilize olive oil to accumulate 19 g/L fungal biomass. The mb-RCL activity reached 325 U/g, which corresponds to a total enzyme activity of 6185 U/L fermentation broth. It was 6.5 times greater than the total enzyme activity in free cell fermentation. These results indicate that controlling the flow pattern of the broth by changing the agitation and aeration in the modified fermentor are able to improve mixing and mass transfer and lead to a better fermentation performance. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Due to their unique properties of protein expression and secretion, filamentous fungi have attracted increasing attention as “cell factories” for various fermentation processes [1–3] and are widely used by the food, pharmaceutical, agricultural, energy and chemical industries to produce a variety of value-added products [2,4]. A filamentous fungus, Rhizopus chinensis, demonstrated a very good capability to accumulate mycelium-bound lipase (mb-RCL) [5]. The mb-RCL exhibits high synthetic activity in non-aqueous solutions, which could facilitate the formation of fatty esters in organic solvents. Unlike normal lipases, mycelium-bound lipases, as naturally immobilized enzymes, are more stable in organic solvents due to being protected from whole fungal cells [6,7]. Therefore, mb-RCL has great potential in catalyzing transesterification used

∗ Corresponding author at: Key Laboratory of Industrial Biotechnology, Ministry of Education, Center for Brewing Science and Enzyme Technology, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, PR China. E-mail address: [email protected] (Y. Xu).

in biodiesel production [8] and enantioselective esterification of racemic mixture [9]. Filamentous fungal fermentation is greatly influenced by fungal morphology [10–13], which varies from dispersed mycelia to compacted pellets of mycelia aggregation [14–17]. Mycelial aggregation might be associated with fungal micro-morphology and influences enzyme biosynthesis and secretion [18]. Papagianni suggested that mycelial aggregates should be viewed as complex differentiated tissues and not as mechanical conglomerates [19]. Mycelia adhesion may be a critical step in triggering certain signaling and gene expression mechanisms that are absent in conventional submerged fermentation and may lead to different fermentation performance [20]. It has been reported that aggregated mycelia is the preferred morphology for R. chinensis to achieve greater mb-RCL production compared with conventional free cell cultures [10]. However, it is difficult to enable aggregated growth of R. chinensis in conventional stirred tank reactors (STRs) [10]. To control the fungal morphology, porous materials, as supportive media, such as cotton towels, polyester fabrics, polyurethane foams and alginate beads, have been used to promote mycelial aggregation for filamentous

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Fig. 1. The perforated sleeve-draft tube for the fermentor. (Diameter: 88 mm; high: 80 mm; thickness: 1 mm; mesh: 18).

tation. In addition, because olive oil is lighter than water, mixing is important to enable the fungi to access the substrate. A 45◦ pitched turbine with 6 blades (15.5 mm × 18 mm) was installed on the agitation shaft at a position 5 mm above the top of the draft tube. The pitched turbine created axial and circular flow and enhanced the mixing. The control STR was a standard fermentor without a draft tube or a pitched turbine. After completion of the fermentation, the fungal biomass was harvested from the reactors and was washed with tap water and 25 mM phosphate buffer (pH 7.0) [10]. The biomass was then lyophilized for 36 h using a freeze drying system (Labconco, USA). The lyophilized biomass was used to analyze the mb-RCL activity. The fermentation broth supernatant was also collected after removing the fungal biomass and was centrifuged at 6000 rpm for 15 min to separate the remaining olive oil that floated on the top layer of the supernatant. The utilization of the olive oil was calculated by the consumed olive oil during the fermentation divided by the total olive oil in the original medium. The productivity of fermentation was defined as the total activity of the biomass from 1 L of broth as mb-RCL. Fungal morphologies from both reactors were compared as well. 2.3. CFD modeling

fungi fermentation [13,21–28], although fungal growth on such supportive materials is difficult to control and the separation of fungal mycelia from the supportive materials is a major issue in terms of harvesting the fungal biomass. Therefore, to achieve aggregated growth of R. chinensis mycelia for efficient mb-RCL production, this study developed a new STR configuration with a perforated draft tube to achieve fungal morphology control and to overcome the aforementioned issues related with the supportive media. Computational fluid dynamics (CFD) was used to analyze and optimize the fermentation conditions in the modified STR and, consequently, to improve the performance of R. chinensis for mb-RCL accumulation.

ANSYS 14.5 (ANSYS, Inc. Canonsburg, PA) was used to carry out the CFD modeling. The 3D geometry and grids of the modified SRT with the draft tube were generated using ANSYS ICEM CFD 14.5 (Fig. 2A and B). Considering the complexity of the flow, unstructured tetrahedral meshes were used as the grid type to capture the flow details in the entire reactor. The meshes were refined around the draft tube and the impellers where the flow pattern was significantly changed. The total number of meshes was 113,000, with 200,000 nodes, and the mesh size ranged from 0.3 mm to 10 mm. The governing equations for the CFD analysis included both mass and momentum conservation for each fluid phase. The continuity equation for the mass conservation is the following:

2. Materials and methods

∂ ˛p p



∂t 2.1. Seed and fermentation broth R. chinensis CCTCC (China Center for Type Culture Collection) M201021 was used in this study and maintained on potatodextrose agar (PDA) slants [5]. After 72 h cultivation at 30 ◦ C, the spores were collected from the agar surface and suspended in deionized water. The spore suspension was used as the seed for inoculation. The initial spore concentration in the fermentation broth was maintained at 107 spores/L for all fermentors in this study. The fermentation broth contained olive oil (20 g/L), MgSO4 7H2 O (0.5 g/L), K2 HPO4 (2 g/L), maltose (10 g/L), peptone (40 g/L). The medium pH was adjusted to 5.5 by 0.2 mol/L H3 PO4 solution. 2.2. Comparison of fermentation between the STR with draft tube and control STR The comparison was carried out in stirred-tank fermentors (7L New Brunswick BioFlo110® , Eppendorf, Inc. Enfiled, CT) following the procedure described previously [10], with a slight modification. Briefly, after inoculation, fermentation was carried out in STRs at 30 ◦ C with an effective volume of 4 L, aeration at 1 vvm (4 L/min) and agitation at 200 rpm for 72 h. One of the fermentors was modified to include a perforated stainless draft tube (Fig. 1) for the aggregation of the fungal mycelium. The draft tube had six stainless rods on the side to position the tube in the center of the fermentor so that the tube was not moved by agitation and aeration during fermen-







+ ∇ × ˛p p Up = Sp

(1)

where p is the phase index, Up is the average velocity, p is the density, Sp is a source term, and ˛p is the phase volume fraction (liquid or gas volume vs. the overall broth volume). The momentum equation is the following: ∂ (˛p p Up ) + ∇ × (˛p p Up Up ) = −˛p ∇ P + ˛p p g + ∇ × (˛p ˜p ) + Fp ∂t

(2)

where P is the pressure, g is the acceleration due to gravity, ˜p is the phase p stress tensor, and Fp is the interaction force between the liquid and gas phases. A two-fluid Euler model was adopted to describe the interfacial momentum transfer between the liquid and gas phases [29]. In the two-fluid Euler model, the fermentation broth is in the continuous phase, and the air is in the discrete phase. The air bubble size was set to a diameter of 2 mm. Interfacial momentum transfer is influenced by the drag force, the weight and the buoyant force. Among these forces, the drag force is the major force in this particular case. Therefore, the drag force was the only force considered in the interfacial momentum transfer, and the Grace model was used to simulate the drag force. The – turbulence model was applied to the continuous phase of the fermentation broth, and a zero-equation turbulence model was used for the discrete phase of the air bubbles. The boundaries of the reactor, impeller, and aeration domains are demonstrated in Fig. 2C. The air balance was included in the simulation using the following equation: air Balance =

volumeInt (Air velocity) @location (1, 2, 3) volume () @location (1, 2, 3)

(3)

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Fig. 2. 3D sketches of the fermentor with the draft tube. (A) Geometric model; (B) mesh model; (C) domains for the CFD modeling.

Where volumeInt()@location and volume()@location are the CFX expressions; locations 1, 2, and 3 are for the any given point in the reactor, Impeller 1 domain, and Impeller 2 domain, respectively; Air velocity was the air velocity in the aeration domain. The geometry and the operational and boundary parameters for the CFD modeling are summarized in Table 1. 2.4. CFD model verification The flow patterns under different mixing conditions (aeration and agitation) from the CFD modeling were verified using a tracer experiment. A transparent plastic draft tube that is identical to the stainless draft tube was used to observe the flow pattern inside the draft tube. Granular activated carbon (at a concentration of 4 g/L) was the tracer in the water. Three agitation speeds of 200, 400, and 600 rpm and six aeration rates of 0–5 L/min were tested to conclude the relationship between agitation, aeration and flow type.

the fermentation performance between different agitation and aeration conditions. 2.6. Lipase activity determination Lipase activity was measured by an ester synthesis method [7]. One milliliter of the substrate solution (1.2 mol L−1 octoic acid and ethanol in heptane, acid-to-alcohol molar ratio of 1:1) was added in a 5 mL capped tube. The reaction was started by adding 20 mg of the lyophilized biomass followed by incubation at 40 ◦ C for 30 min with a shaking speed of 200 rpm. After the reaction, the liquid/solid separation was achieved by centrifugation at 10,000 × g for 5 min at 4 ◦ C. A liquid sample of 200 ␮L was mixed with an internal standard (50 ␮L of 2-hexanol) and was then analyzed using a gas chromatograph (Agilent 6820, flame-ionization detector, 30 m × 0.22 mm PEG 20 M (AC20) capillary column). One unit of lipase activity was defined as the amount of the lyophilized biomass required to produce 1 ␮mol of ester per minute.

2.5. mb-RCL production under different flow conditions 2.7. Rheology measurement of fermentation broth According to the results from CFD model, selected agitation and aeration conditions were applied to carry out the fermentation in the SRT with draft tube. Dissolved oxygen and pH were monitored during the fermentation. The olive oil utilization, biomass production, and lipase activity were also measured in order to compare

The rheological properties of the fermentation broth were determined using the DV-II + Pro Programmable viscometer (Brookfield Engineering Laboratories, MA). Fermentation broth (3–5 ml) was added to the sample cup and was incubated in a water bath at a

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Table 1 The geometry and the operating and boundary parameters of the fermentor for the CFD modeling. Geometry parameter of the fermentor

Value

Operating parameter

Value

Boundary condition (aeration and agitation)

Air normal velocity (m/s)

Inside diameter of the fermentor Height of broth level Diameter of turbine Height of lower turbine Height of upper turbine Inside diameter of draft tube Length of draft tube Height of the lower edge of draft tube

178 mm 173 mm 62 mm 48 mm 158 mm 88 mm 80 mm 63 mm

Broth volume Temperature Viscosity of broth Aeration Agitation

4L 30 ◦ C 2.75 cp 2–5 L/min 200–600 rpm

2 L/min and 400 rpm 2 L/min and 600 rpm 3 L/min and 600 rpm 4 L/min and 200 rpm 4 L/min and 400 rpm 4 L/min and 600 rpm 5 L/min and 600 rpm

6.06 6.06 9.1 12.13 12.13 12.13 15.16

constant temperature of 30 ◦ C. The spindle and sequential speed were selected based on the liquid viscosity, and the program was then run to measure the rheological parameters. Viscosities of the broth at culture time of 0, 24, 48, 72 h with variable shear strain rates were determined, respectively. 3. Results and discussion 3.1. Comparison of fermentation between the STR with the draft tube and control STR The STRs with and without the draft tube were used to carry out R. chinensis fermentation. Fermentation in the STR without the draft tube (the control) had a majority of the biomass dispersed in the suspended culture with a small amount of the biomass attached to the baffles and impellers (Fig. 3A), whereas in the STR with the draft tube, R. chinensis grew directly on the draft tube and formed an aggregated biomass with much less dispersed biomass in the broth (Fig. 3B). It is apparent that the draft tube can function as a supportive structure and facilitate aggregated growth on the tube during fermentation. After completion of the fermentation, the separation of the aggregated biomass from the tube can easily be achieved using water blasting in situ, which is superior to other immobilization approaches using supportive materials made of cotton towels, polyester fabrics, polyurethane foams and alginate beads. The fermentation data demonstrated that the aggregated culture in the modified STR had a much better performance than the suspended culture. The biomass production and lipase activity from the aggregated culture with an agitation of 200 rpm and aeration of 4 L/min were 14 g/L and 283 U/g, respectively, and these values were greater than the corresponding values (11 g/L and 88 U/g) from the control (Table 2). The aggregated culture had an olive oil utilization efficiency of 84%, which was also greater than in the control (59%). The olive oil was used as a major carbon source and an inducer for mb-RCL production [30]. The better utilization of olive oil resulted in a much greater yield of lipase production (236 U/g oil) in the aggregated culture compared with the control (81 U/g oil), although the yield of biomass to oil (0.83 g/g oil) was slightly lower than in the control (0.93 g/g oil). The experimental results also showed that the aggregated biomass had better mb-RCL production than the dispersed biomass. Enzyme activities from the aggregated biomass on the draft tube and baffles in the suspended culture reached 283 U/g and 180 U/g, respectively, which are approximately 6–10 times greater than the enzyme activities from the dispersed biomass (28 U/g). In addition, the culture in the modified STR had approximately 30% more fungal biomass than the control; therefore, the enzyme productivity (3958 U/L) of the modified STR was 4 times greater than the enzyme productivity (950 U/L) from the control. These results clearly demonstrate that adopting the draft tube in the STR facilitated R. chinensis growth in an aggregated form, which significantly improved mb-RCL production.

The mechanism between mycelial aggregation and enzyme productivity is still not clear [31], even though it has been reported that cell aggregation is considered to be a trigger for some Rhizopus species to enhance intracellular lipase production [32] and the fungal morphology could influence the lipase localization within the cell and could influence its secretion [22]. Because the mycelial aggregation method studied in this work is different from other morphology control approaches, such as pelletization, cotton towel or alginate bead immobilization, the rheology and mass transfer of the fermentation need to be investigated to understand the broth flow behavior and to delineate the relationships between the fluid dynamics, fungal biomass growth, and enzyme production efficiency. 3.2. Simulation of the modified STR with the draft tube using CFD model The aggregated fermentation in the STR with the draft tube implied that flow pattern and rheological property of fermentation broth had been changed (Fig. 3). Therefore, flow pattern in the modified STR was simulated using CFD model in order to understand the relationship between the flow pattern and operational parameters, and their influence on the enzyme production. The fluid type of the fermentation broth was first determined before applying CFD analysis to the fermentation. The viscosities of the broth at different culture times in the modified STR were measured (data are not shown). The results show that the trends in the viscosity change under different shear stresses and time were almost the same and fairly stable. In addition, the viscosity of the broth in the modified STR was relatively low (approximately 2.75 cp at an average shear strain rate of 15 s−1 ) during the fermentation. These results mean that the fermentation broth of the aggregated culture behaved like a Newtonian fluid under the fermentation conditions, which is different from the suspended control culture that behaved like a pseudo-plastic non-Newtonian fluid [10] (Fig. 3B). Therefore, Newtonian fluid was adopted as the fluid type to run the CFD simulation on the modified STR. The modeling results are presented in Fig. 4. The verification experiment using deionized water with granular activated carbon as the tracer (for the Newtonian fluid) was conducted under the conditions listed in Table 3. The flow patterns from the verification are summarized in Table 3 and Fig. 5. The flow patterns were consistent with the corresponding simulated velocity profiles (Fig. 4-left), which indicates that the CFD models successfully described the fluid motion under different aeration and agitation conditions in the modified STR with the draft tube. According to the CFD model and tracer experiment, three typical flow patterns, which are referred to as Types A, B, and C, can be observed (Fig. 5) in the modified STR and are very different from those of typical submerged fermentation in the conventional STR. Under lower agitation (200 rpm) with moderate aeration (4 L/min), Type C was the main flow pattern in the reactor (Figs. 4 A-left and

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Fig. 3. R. chinensis morphology in STRs. (A) The control STR; (B) the modified STR with draft tube (a) dispersed biomass in the broth; (b) aggregated biomass attached on the baffles; (c) aggregated biomass accumulated on the draft tube.

Table 2 Performance of the suspended and aggregated fermentation in the STR by R. chinensis at 200 rpm agitation and 4 L/min aeration. Fermentation

Mycelia

Biomass (g/L broth)

Lipase activity (U/g dry cell)

Productivity (U/L broth)

Utilization of olive oil (%)

Yield

Biomass (g/g oil)

Lipase production (U/g oil)

6.57 ± 0.61 4.26 ± 0.27 10.83 ± 0.88

28.06 ± 3.39 180.33 ± 28.60 88.11 ± 10.28

185.41 ± 39.33 764.36 ± 73.36 949.77 ± 34.03

58.86 ± 6.70

0.93 ± 0.18

81.04 ± 6.34

Aggregated culture Aggregated biomass on the draft tube 14.01 ± 0.57

283.16 ± 31.75

3958.09 ± 284.63 84.25 ± 4.92

0.83 ± 0.02

235.80 ± 30.67

Suspended culture

Dispersed biomass in the broth Aggregated biomass on the baffles Total biomass

5). Based on Nienow’s calculation on flooding [33], an aeration of 4 L/min is at the flooding point for an agitation of 200 rpm, which reduced the air retention time in the broth inside the draft tube and negatively influenced oxygen transfer (Fig. 4A-right) and olive oil dispersion even if a pitched turbine was used to improve the mixing. Increasing agitation to 400 rpm at an aeration of 4 L/min changed the flow pattern to Type B, which has a similar flow pattern as Type C in the outside of the draft tube. However, a complicated flow pattern inside the draft tube appeared in that both the up and down flows co-existed at the same time due to the limitation of

the tube to the broth flow (Figs. 5 and 4 C-left). Type B did not significantly improve the air dissolution in the broth, and a portion of the air was not dissolved into the broth (Fig. 4C-right). When the agitation was further increased to 600 rpm at the same aeration of 4 L/min, the flow pattern changed to Type A (Figs. 5 and 4 F-left), which had better mixing compared to Types B and C. Type A was mainly caused by the full broth cycling inside and outside the draft tube. The down flow inside the tube enhanced the mixing of the olive oil and formed a cross flow with the air moving up, which resulted in a better air volume fraction and improved

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Fig. 4. Velocity (left) and air volume fraction (right) of the flow fields under different fermentation conditions in the modified STR with the draft tube (A) agitation 200 rpm. Aeration 4 L/min; (B) agitation 400 rpm. Aeration 2 L/min; (C) agitation 400 rpm. Aeration 4 L/min; (D) agitation 600 rpm. Aeration 2 L/min; (E) agitation 600 rpm. Aeration 3 L/min; (F) agitation 600 rpm. Aeration 4 L/min; (G) agitation 600 rpm. Aeration 5 L/min.

oxygen transfer (Fig. 4F-right). These simulation results indicate that low agitation and high aeration are not favorable for the STR with the draft tube to enhance the fungal fermentation. Furthermore, increasing agitation to 800 rpm resulted in the appearance of a whirlpool (data are not shown) and a stronger shear force around the impellers, which is also unfavorable for the culture of R. chinensis. Decreasing the aeration can also achieve a Type A flow pattern in the reactor. Maintaining the agitation at 400 rpm and reducing the aeration to 2 L/min changed the flow pattern to Type A. This change improved the air distribution so that more air was dissolved in the broth even with the lower aeration (Figs. 5 and 4B). By decreasing

the aeration to 2 and 3 L/min with an agitation of 600 rpm, Type A fluid was maintained and the air distribution did not show a significant difference, although there was a small amount of down flow in the outside of the draft tube (Figs. 4 D, 4E and 5). When the aeration was increased to 5 L/min, the flow pattern remained Type A, but more uneven air was distributed into the broth (Fig. 4G), which could have negatively influenced the corresponding fermentation performance. These results suggest that the flow pattern is determined by the agitation and aeration and is critical to the mass transfer for the aggregated fungus fermentation in the modified STR. Therefore, by considering the benefits of the flow pattern and air distributed in the broth, an agitation of 600 rpm and an aeration

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Fig. 4. (Continued)

of 4 L/min were selected as the preferred conditions for the fungal fermentation in the modified STR. 3.3. Aggregated cultures for mb-RCL production under different operation parameters Three different conditions, including the preferred one, were chosen to carry out R. chinensis fermentation in the modified STR. The preferred condition, Condition A (600 rpm and 4 L/min, Type A flow pattern), was compared with Condition B (400 rpm and 2 L/min, Type A flow pattern) and Condition C (200 rpm and 4 L/min, Type C flow pattern as a control) for the mb-RCL production. The

fungal fermentation in the modified STR under the preferred condition exhibited better fermentation performance than Conditions B and C, although the aggregated growth of the mycelia on the draft tube was achieved under all three conditions. A biomass concentration of 19 g/L was achieved from Condition A, which was greater than from Condition B (18 g/L) and Condition C (14 g/L). A lipase activity of 325 U/g from Condition A was also better than Condition B (307 U/g) and Condition C (283 U/g). Olive oil utilization for Condition A reached 100%. As a result, the mb-RCL productivity in Condition A was increased to 6185 U/L, which was 1.6 and 6.5 times greater than the mb-RCL productivity from Condition C (control) and the free cell fermentation, respectively (Tables 2 and 4).

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Fig. 4. (Continued)

Table 3 Flow pattern of fermentation broth under different aeration and agitation conditions in the tracer experiment. Aeration (L/min)

Agitation velocity at flooding pointa (rpm)

Agitation (rpm)

Observed flow pattern typeb

2

158

3

180

4

200

5

215

200 400–600 200 400 600 200 400 600 200 400 600

B A C B A C B A C B A

a b

Based on Nienow’s calculation on flooding [33]. Type A–C refers to the flow patterns in Fig. 5.

Fig. 5. Major flow patterns observed in the aggregated mycelia fermentation in the modified STR.

Table 4 Performance of aggregated mycelia fermentation by R. chinensis in the modified STR under different conditions. Fermentation conditions Biomass(g/L) Lipase activity (U/g) Productivity (U/L) Utilization of olive oil (%) Yield of biomass (g/g oil) Yield of lipase production (U/g oil)

600 rpm, 4 L/min(Condition A) 19.08 ± 0.93 324.78 ± 24.55 6185.34 ± 165.28 100 0.96 ± 0.05 309.27 ± 8.26

400 rpm, 2 L/min(Condition B)

200 rpm, 4 L/min(Condition C)

17.88 ± 1.12 306.62 ± 13.82 5465.85 ± 224.50 88.67 ± 1.88 1.00 ± 0.08 308.82 ± 11.91

14.01 ± 0.57 283.16 ± 31.75 3958.09 ± 284.63 84.25 ± 4.92 0.83 ± 0.02 235.80 ± 30.67

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ing can be concluded to significantly enhance cell growth and olive oil utilization and to increase mb-RCL activity and productivity. 4. Conclusions

Fig. 6. Changes in the relative dissolved oxygen concentration and pH during aggregated mycelia fermentation for different operation parameters.

This study developed a new morphology control approach for fungal fermentation by implementing a modified STR with a perforated draft tube to achieve aggregated growth of R. chinensis mycelia. The aggregated culture significantly improved mb-RCL production and changed the rheology and mass transfer patterns of the broth in the reactor. Flow pattern control by changing the agitation and aeration is critical for aggregated fungal fermentation in modified STR. The CFD modeling and verification concluded a preferred culture condition (600 rpm and 4 L/min) with better mixing and oxygen transfer for mb-RCL production. Under the preferred condition, the STR with the draft tube produced 19 g/L fungal biomass with a mb-RCL activity of 325 U/g and the fermentation productivity reached 6185 U/L, which was 6.5 times greater than the suspended fermentation. Acknowledgements

In addition, even though Condition B, which had a Type A flow pattern, indicated slightly less lipase activity, productivity and olive oil utilization than Condition A, its fermentation performance was better than Condition C. The mb-RCL productivity from Condition B reached 5466 U/L, which was approximately 1.4 and 5.8 times greater than the mb-RCL productivity from Condition C and the free cell fermentation, respectively (Tables 2 and 4). Interestingly, although olive oil utilization for Condition B was less than 90%, the yields of biomass and lipase production were similar to those for Condition A and were greater than those for Condition C (Tables 4). This result suggests that a Type A flow pattern could improve olive oil utilization by the aggregated R. chinensis. These results biologically verify the importance of the flow pattern on R. chinensis fermentation for mb-RCL production in the modified STR. It is apparent that a Type A flow pattern greatly improved the mass transfer and air distribution. The CFD model concluded that Conditions A and B (Type A flow patterns) had better flow patterns and exhibited better air volume fractions compared with Condition C (Type C flow pattern) (Fig. 4A,B,F). Fig. 6 shows the changes in the dissolved oxygen and pH during the aggregated culture under these different agitation and aeration conditions. For Condition C, with an agitation of 200 rpm and an aeration of 4 L/min, the dissolved oxygen decreased to less than 5% after 24 h and the gas distribution was relatively poor as well (Fig. 4A-left). Whereas, for Conditions A and B, the dissolved oxygen was maintained at approximately 50% during the entire fermentation. It has been reported that immobilized fungal fermentation can achieve better oxygen transfer in static or rotating fibrous bed bioreactor [13,28]. Although the oxygen transfer coefficient was not determined in this work, the CFD simulation demonstrates that oxygen dissolution in the broth with a Type A flow pattern increased significantly. In addition, the Type A flow pattern in Condition A allowed all of the broth to cycle around the draft tube, which also led to a more effective dispersion of the oil. The lower agitation of Condition B did not sufficiently mix the oil into the broth, even though its flow pattern was Type A. This difference could be the reason why a better oil utilization in Condition A was achieved compared with Condition B and why low agitation was not favorable for the fungal enzyme production in the modified STR. Due to mycelia aggregation on the tube and the heterogeneous system of the broth, the kinetics of mycelial growth and enzyme production cannot be determined during fermentation. However, based on the results of the final biomass amount, enzyme activity and olive oil consumption, the Type A flow pattern with increased dissolved oxygen and good mix-

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