- Email: [email protected]
Efficient production of ulvan lyase from Ulva prolifera by Catenovulum sp. LP based on stage-controlled fermentation strategy
Algal Research 46 (2020) 101812
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Efficient production of ulvan lyase from Ulva prolifera by Catenovulum sp. LP based on stage-controlled fermentation strategy
T
Leke Qiaoa,b, Xiaoke Yangc, Ruize Xiea, Chunying Dua, Yongzhou Chia, Jingliang Zhangb, ⁎ Peng Wanga, a
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, PR China Marine Biomedical Research Institute of Qingdao, Qingdao 266100, PR China c Tsingtao Brewery Company Limited, Qingdao 266023, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Ulvan lyase U. prolifera Fermentation Catenovulum sp. LP
Ulvan lyase, specifically depolymerizing ulvan to unsaturated sulfated oligosaccharides with diverse biological activities, is becoming more and more popular. To provide an economical fermentative strategy, Catenovulum sp. LP cultured in low-cost medium that directly used Ulva prolifera as the main carbon source was used to produce ulvan lyase. The fermentation conditions were established in a shake flask containing 5% (w/v) U. prolifera powder, 0.2% K2HPO4, 0.4% NH4Cl, 0.5% NaCl, and 0.05% MgSO4. In a 5-L fermentor, series of strategies were used to systematically discuss the fermentation process. The enzyme with the best activity was obtained by the condition of a two-stage temperature (28–32 °C), initial pH 7.0, 1.5 vvm and two-stage agitation (200–400 rpm). The fermentation kinetic model of substrate consumption indicated that a shorter culture time may improve the ulvan lyase activity. Moreover, 10.09% improvement in enzyme activity was observed in a 30-L fermentor by shortening the fermentation time to 20 h. This fermentation method offers a promising prospect to enlarge the production of ulvan lyase and also provides a potential possibility to mildly prepare bioactive unsaturated oligosaccharides.
1. Introduction Green macroalgae can proliferate rapidly in eutrophic water. The green-tides and algal blooms that can result are a significant environmental concern for cities along the Yellow Sea [1,2]. Despite their abundant biomass, the utilization of green macroalgae is currently limited [3]. The green algal cell wall, especially the two main genera Ulva and Enteromorpha, is composed of a long chain anionic heteropolysaccharide polymer termed “ulvan”. And there are two major polysaccharide families in Ulva sp.: the water-soluble ulvan and insoluble cellulose. The ulvan is a kind of intercellular polysaccharide which mainly exists between cuticle and cellulose layer [4,7]. The ulvan content can reach 29% of the total dry weight. Its degradation products, which include D-glucuronic acid (GLcA), D-xylose (Xyl), and 3-sulfated rhamnose (Rha3S), have potential applications in the food, agriculture, pharmaceutical, and chemistry sectors [5–7]. However, these bioactive oligosaccharides are normally present in low quantities, especially the disaccharide portion of Rha3S linked to Xyl [8]. Ulvan lyase (EC 4.2.2) is a depolymerase that precisely cleaves the β (1–4) glycosidic bond between Rha3S and uronic acid through the β⁎
elimination mechanism [3,8]. This mild enzymatic method can yield all the constituent oligosaccharides without isomerization [7]. Developing a fermentation process applied to the industrial production of ulvan lyase would be beneficial to prepare these oligosaccharides. This potential is limited by the lack of knowledge on ulvan lyase. Studies on ulvan lyase have mainly been directed toward the screening microorganisms exhibiting enzyme activity and the characteristic analysis of catalysis [3,7,9,10]. The scale of fermentation has been limited to shake flask, while the substrate used as the main carbon source has been limited to the pure soluble polysaccharide extracted using the hot water method [7]. Poor production, limited fermentative substrates, and high output cost of the enzyme have limited the generation of ulvan oligosaccharides. Consequently, an improved ulvan lyase production method is required. The production of polysaccharide degradases including agarase and alginate lyase have been studied more intensively in brown and red algae than in the green algae species Ulva prolifera. Hassairi et al. [11] presented results of the production of α-agarase in a 2-L fermentor with batch, fed-batch, and continuous fermentation strategies. Zhou et al. [12] reported the successful scale-up of fermentation in 5-L and 30-L
Corresponding author at: College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (P. Wang).
https://doi.org/10.1016/j.algal.2020.101812 Received 18 July 2019; Received in revised form 19 January 2020; Accepted 19 January 2020 2211-9264/ © 2020 Elsevier B.V. All rights reserved.
Algal Research 46 (2020) 101812
L. Qiao, et al.
bioreactors. More importantly and different from the use of a common carbon source, a novel strategy using mixed carbon sources (starch and alginate) was reported by the authors. This improved culture medium was less expensive and could expand alginate lyase production. These findings highlight the importance of the culture medium used in the fermentation process of ulvan lyase. U. prolifera is a marine green algae that is distributed globally in ocean coastal regions [13,14]. The purified polysaccharide of U. prolifera (PU) is usually used as the main material for shake flask production of ulvan lyase. In this study, we tried to directly use U. prolifera as the main carbon source. To the best of our knowledge, only limited information regarding the direct fermentation of algal bodies by a microorganism is available. The objectives of the study were (1) to test the possibility of replacing PU with solid algae powders in the culture medium, (2) to optimize the fermentation conditions in a 5-L laboratory-scale fermentor, and (3) to establish the fermentation kinetics models and scale-up the fermentation to a 30-L fermentor. As the first step in the scale-up process of ulvan lyase fermentation, we attempted to reduce the costs of raw materials and increase the economic feasibility of the large-scale production.
the growth curve and ulvan lyase production were plotted using the software Origin 8.0. 2.4. Fermentor experiments 2.4.1. 5-L laboratory fermentation The optimization of ulvan lyase production was carried out in a 5-L glass fermentor (Bailun Bioscience Company, Shanghai, China) with 3 L working volume. Before fermentation, the fermentor was pre-sterilized at 121 °C for 15 min. Then, temperature-shift procedure (24–32 °C), agitation speed shift procedure (200–600 rpm), aeration rate control (0.5–2.0 vvm), and pH control (6.0–9.0) were all taken into consideration to determine the optimal fermentation parameters. The pH control was achieved by adding either 0.1 M HCl or NaOH. 2.4.2. Fermentation kinetics models Three preliminary rate equations for cell biomass (X), enzyme activity (P) and residual sugar (S) were employed to describe the whole fermentation process. The cell growth rate of strain LP was described by Logistic's equation. This equation reflected the cell growth rate as a function of biomass concentration [16,18,42]. In this study, the biomass concentration (X, Xm) was presented by the values of OD 600 measured by spectrophotometrically.
2. Materials and methods 2.1. Feedstock and microorganism
dX = µmax 1 dt
U. prolifera was collected from the coast of Qingdao, China in 2018. It was cleaned carefully and dried in an oven (50 °C, 48 h). Subsequently, it was milled in a mechanical grinder to obtain a homogeneous powder with 60 meshes, and used in the following experiments. All the other reagents were analytical grade and commercially available. The strain Catenovulum sp. LP which could produce ulvan lyase used in the study was supplied by the Microbiology Laboratory of Ocean University of China. It was maintained on a Luria-Bertani (LB) agar slant at 4 °C.
X X Xm
(1)
where X was biomass concentration, t was fermentation time, μmax was max specific growth rate, and Xm was max biomass concentration. The Luedeking-Piret equation was employed to indicate the formation rate of the main product [17]. This equation illustrated that the product formation was associated with cell growth.
dP = dt
dx + X dt
(2)
where P was product concentration, α was growth associated constant, β was non- growth associated constant. The consumption rate of substrate is mainly driven by cell growth, product synthesis and cell metabolic activities. It could be described by the following equation [18]:
2.2. The extraction of PU and culture medium Two different methods of PU extraction were investigated. The first one was the improved hot water method as described by Li et al. [13]. Briefly, the milled alga (100 g) was dipped into 2 L of tap water, homogenized and refluxed at 95 °C for 2 h. The soluble polysaccharide was collected by centrifugation (4800 ×g, 15 min). The second one was the autoclave method. With the same solid-liquid ratio, appropriate amounts of U. prolifera powder and distilled water were added into an Erlenmeyer flask, mixed evenly and autoclaved at 115 °C for 40 min. After cooling, the autoclaved polysaccharide was also collected by centrifugation (4800 ×g, 15 min). The culture medium consisted of (g/L):12 PU, 2.0 K2HPO4, 4.0 NH4Cl, 5.0 NaCl, and 0.5 MgSO4. The initial pH of the medium was neutral.
dS = dt
dX 1 dt Yx / s
dP 1 dt Yp / s
mx X
(3)
where S was substrate concentration, Yx/s was the cell yield coefficient, Yp/s was the product yield coefficient, and mx was the coefficient for maintenance functions. During the fermentation of strain LP, substrate consumption was mainly focused on cell yield coefficient and product yield coefficient. Therefore, we could further simplify the equation as follow:
k1 =
2.3. Shake flask experiments
kk2 =
2.3.1. Effect of autoclaved conditions on PU concentration U. prolifera powder medium with 5% (w/v) solid algae powder was autoclaved at different temperatures (115 °C and 121 °C) and for different durations (20–60 min). The concentration of polysaccharides was determined by the phenol‑sulfuric acid method at 490 nm using glucose as a standard [15].
1 Yx / s
(4)
1 Yp / s
(5)
Rearrangement Eq. (3), Eq. (4) and Eq. (5):
dS = dt
dX k1 dt
dP k2 dt
(6)
2.4.3. 30-L bench fermentation In a 30-L fermentor (Bailun Bioscience company, Shanghai, China), the optimal strategy with two-stage agitation control and temperatureshift procedure was further studied. Similar to the pretreatment of the 5-L fermentor, it was also pre-sterilized at 121 °C for 20 min before fermentation. Agitation control and temperature-shift were both
2.3.2. Growth curve and ulvan lyase activity The Catenovulum sp. LP (strain LP) was cultured on a rotary shaker (ZWF-200, Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China) at 170 rpm and 28 °C for 50 h. Samples were withdrawn to determine the ulvan lyase activity and cell biomass. Fitting of 2
Algal Research 46 (2020) 101812
L. Qiao, et al.
initiated after 6 h. The initial pH was around 7.0 and aeration rate was 1.5 vvm.
method. This phenomenon may be attributed to the differences in stirring condition and extraction time between the two methods. Stirring continuously and a longer extraction time were both beneficial for water soluble polysaccharide dissolution [23]. Compared with the hot water method, autoclaving was a closed system. Some operations such as stirring and refluxing were unavailable. In order to achieve better polysaccharide dissolution, extending the autoclave time and enhancing the autoclave temperature could be taken into consideration.
2.5. Analytical methods To remove the U. prolifera powder residue, the withdrawn samples were filtered through gauze (200 meshes) and centrifuged (2000 ×g, 5 min). The supernatant collected was applied to determine the enzyme activity and cell biomass. The ulvan lyase activity was determined based on two kinds of methods. The first one was measuring the formation of double bonds in the assay at 235 nm according to the method of Collen et al. [19]. And the second was measuring the release of the reducing sugar [20,21]. During the fermentation process, the ulvan lyase activity experimental data demonstrate formation of unsaturated bond (Linker's method) was consistent with the level of sugars that measured by DNS method. We chose the DNS method under the assay conditions (hydrolysis at pH 5.5 and 35 °C) to evaluate the ulvan lyase activity. One unit (U) of degradase activity was defined as the amount of enzyme required to produce 1 μmoL of reducing sugar (glucose equivalent) in 1 min under the aforementioned conditions. The cell biomass was measured spectrophotometrically by monitoring the optical density at 600 nm. The PU with the same concentration was employed as control. The iterative regression of kinetic models was obtained using mathematical software Matlab.
3.1.2. Effect of autoclave conditions on polysaccharide of U. prolifera (PU) extraction Extraction time and temperature are factors that would influence the extraction efficiency as well as the polysaccharides diffusion coefficient in the extracting solution [24]. To study the effect of different autoclave conditions on the dissolution of PU, various experiments were carried out with different autoclaved temperatures between 115 °C and 121 °C as well as different autoclave durations from 20 min to 60 min. As shown in Table. 2, the PU extracted at 115 °C for 40 min was higher than that for 30 min. Prolonged durations of autoclaving did not significantly affect the dissolution of polysaccharides, but led to the increase of reducing sugar concentration. When the sterilization temperature was increased to 121 °C, the reducing sugar content was enhanced further at all tested autoclave durations. Longer extraction time and higher temperature may lead to decomposition or degradation during the extraction process [25]. In this study, for the PU extracted by autoclaving, prolonged extraction time and higher temperature may damage the structure of the polysaccharide chains, producing large numbers of reducing sugars. Similar to agarase, κ-carrageenase and alginate lyase, the characteristic polysaccharides in the culture medium is necessary for Catenovulum sp. LP to induce the high production of ulvan lyase [26–28]. When the reducing sugars accumulate in the culture, microorganisms would prior to use it for cell growth, reducing PU utilization and the induction of enzyme secretion. Thus, the polysaccharide extracted at 115 °C for 40 min was more suitable to be supplemented in the culture medium.
2.6. Statistical analysis All experiments were conducted in triplicate. The values of different parameters were all expressed as the mean (n = 3). The corresponding standard deviations were determined by using one-way analysis of variance (ANOVA). 3. Results and discussion
3.1.3. Growth curve and ulvan lyase production in two kinds of media In order to assess the possibility of replacing soluble polysaccharides with algae powder, the cell growth and ulvan lyase production from Catenovulum sp. LP under two different conditions were investigated. As shown in Fig. 1, the two curves exhibited similar trends. During the first 6 h, strain LP was in the lag phase. The exponential phase occurred between 6 h and 24 h, and was the phase associated with the production of ulvan lyase. When the culture time extended to 36 h, the bacterial culture reached the stationary phase and the production of ulvan lyase also came to a plateau. Any further increase in incubation time showed no effect on the enzyme production. The curve of bacterial growth and ulvan lyase production were both fitted between soluble polysaccharide medium and solid algae powder medium, which confirmed that algae powder may be used as the main carbon source. This result indicated that ulvan lyase can be obtained without a complicated polysaccharide extraction procedure, which is of great value from a cost effectiveness standpoint. Moreover, this kind of improved culture medium could benefit to realize the large scale production of ulvan lyase. Thus, subsequent studies on a 5-L laboratory scale fermentor were carried out to optimize ulvan lyase production.
3.1. Fermentation in shake flasks 3.1.1. The possibility of U. prolifera as the main carbon source Strain LP domesticated over a long period of time has been reported to be a typical inducible enzyme production strain, with the polysaccharide concentration being the main factor that directly influences cell growth and ulvan lyase production [22]. Ulvan lyase was reported to be the major enzyme product during the fermentation process. In the previous study, the polysaccharide used as the main carbon source in the culture medium was extracted using the hot water method, followed by a series of processing steps that included concentration, dialysis, ethanol precipitation, and drying. This process was tedious and complex. If we could obtain the carbon resource directly by autoclaving the algae powder, the fermentation production of ulvan lyase would be markedly improved, especially in large-scale production. The comparison of polysaccharide dissolution between the hot water method and the autoclave method was conducted. Results are shown in Table 1. The concentration of polysaccharides and reducing sugars obtained by autoclaving was lower than that by hot water Table 1 The polysaccharide of U. prolifera (PU) dissolution with different method. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. Extraction condition
Polysaccharide concentration (g/L)
Reducing sugar concentration (g/L)
Hot water method Autoclaved method
11.72 ± 0.71 8.95 ± 0.42
0.33 ± 0.02 0.21 ± 0.01
3.2. Fermentation in 5-L laboratory-scale batch fermentor Transferring a fermentation process from shake flasks to fermentors is considered to be a great challenge due to substantial differences in the mode of controlling the temperature, pH, aeration, agitation, as well as the geometric variety in both conditions [29]. In order to scale up the ulvan lyase production, four strategies including temperatureshift procedure, pH control, agitation-shift procedure and aeration 3
Algal Research 46 (2020) 101812
L. Qiao, et al.
Table 2 Effect of autoclaved conditions on polysaccharide of U. prolifera (PU) extraction. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
The hot water method The autoclaved method
Extraction condition
The polysaccharide concentration (g/L)
The reducing sugar concentration (g/L)
Ulvan lyase activity (U/mL)
Hot water reflux 115 °C 30 min 115 °C 40 min 115 °C 50 min 115 °C 60 min 121 °C 20 min 121 °C 30 min 121 °C 40 min
11.72 10.67 12.02 12.17 12.66 12.63 12.60 12.85
0.33 0.32 0.35 0.40 0.43 0.45 0.48 0.49
0.715 0.736 0.748 0.690 0.705 0.720 0.702 0.708
± ± ± ± ± ± ± ±
0.71 0.39 0.76 0.35 0.19 0.79 0.60 0.84
control were all conducted to seek the optimal condition for the culture of strain LP in a 5-L laboratory-scale fermentor.
± ± ± ± ± ± ± ±
0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.03
± ± ± ± ± ± ± ±
0.018 0.038 0.031 0.073 0.019 0.019 0.042 0.041
at 28 °C probably positively impacted the stability of strain LP and was consequently used for the latter stage of fermentation. With the temperature-shift process, the growth rate of microorganisms was enhanced, and the fermentation time was shortened to 36 h.
3.2.1. Effect of temperature on fermentation Temperature is an important factor affecting protein production and the synthesis of metabolites of many strains [30]. In this study, a time course experiment for cell biomass and ulvan lyase activity was conducted at different temperatures (24 °C, 28 °C, and 32 °C). The 32 °C temperature resulted in the greatest increase in cell biomass and ulvan lyase activity within 24 h (Fig. 2A and B). After 24 h of fermentation, the yield at 28 °C reached 0.851 U/mL, which was almost equal to the yield achieved at 32 °C. However, when fermentation was extended to 36 h, the degradase activity at 32 °C decreased, while the activity was the highest at 28 °C. The experimental setup at 24 °C resulted in the least increase in cell biomass and ulvan lyase production during the entire fermentation process. These findings indicate that 32 °C was suitable for the earlier stage of fermentation by strain LP, while 28 °C was more appropriate for the later stage. The temperature-shift process was beneficial to cell growth (Fig. 2C). According to the report of Fang et al. [31], high temperatures for initial fermentation can improve cell biomass accumulation and effectively increase the yield of the target product. For the fermentation of strain LP, a high temperature (32 °C) stimulated the accumulation of cell biomass in the early stage (data not show). However, sustained fermentation at high temperature may decresae the synthesis of proteins essential for growth and other vital physiological processes of microorganisms [13]. Some disadvantageous soluble metabolic products such as organic bases (amine substances) could weaken the accumulation of cell biomass [32,33]. Moreover, enzyme activity is not always enhanced at high temperatures. According to the report of Unsworth et al. [34], high temperature is a harsh condition for enzymes to retain their activity and stability; it might affect the structural rearrangement of enzymes and result in decreased activity. Fermentation
3.2.2. Effect of agitation speed on fermentation It is well known that mixing by agitation is a crucial aspect of microbial fermentation to achieve the maximum productivity [29]. As shown in Fig. 3A and B, the effect of agitation speed ranging from 200 rpm to 600 rpm on cell biomass and ulvan lyase activity was investigated with 1.5 vvm aeration rate. During the 12 h of fermentation, the optimal agitation speed was found to be 200 rpm. When the fermentation time reached 24 h, the ulvan lyase activity at agitation speeds of 400 rpm and 600 rpm increased faster than that at 200 rpm. The improved fermentation yield at 400 rpm was maintained until the end of the fermentation process. In contrast, the enzyme activity at 600 rpm decreased when the fermentation time extended to 36 h. A well-directed control strategy would be beneficial to metabolite biosynthesis [35]. In this study, when the sterilized U. prolifera powder culture medium was cooled to the fermentation temperature (24–32 °C), the viscosity caused by PU increased significantly. This resulted in a lower oxygen requirement, which was mainly used to maintain the seed liquid growth. With the strain LP culture, the viscosity of the broth decreased with time due to PU degradation and increased cell concentration. Accompanied by agitation, microorganisms could mix with the culture medium homogenously, requiring more dissolved oxygen for its growth. Thus, fermentation conducted at 400 rpm provided the appropriate levels of oxygen between 12 h and 36 h of fermentation. The dissolved oxygen was maintained at a level of 32%. However, 600 rpm with 45% dissolved oxygen was not favorable for ulvan lyase activity. The inappropriate agitation speed would be a disadvantage for cell growth and metabolite biosynthesis [29,35]. Therefore, a two-stage agitation strategy was employed during the
Fig. 1. Growth profile and production of ulvan lyase by Catenovulum sp. LP, (A) the growth condition of polysaccharide of U. prolifera (PU) medium, (B) the growth condition of algae medium. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. 4
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 2. The effect of temperature on the ulvan lyase fermentation, (A) the OD 600 at different temperatures, (B) the ulvan lyase activity at different temperatures, (C) the fermentation condition of two-stage temperature strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
Fig. 3. The effect of agitation speed on the ulvan lyase fermentation, (A) the OD 600 at different agitation speeds, (B) the ulvan lyase activity at different agitation speeds, (C) the fermentation condition of two-stage agitation strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
aeration rate (2.0 vvm) may negatively affect the formation and release of ulvan lyase, leading to poor enzyme activity. Moreover, 1.5 vvm was the appropriate aeration rate for the ulvan lyase production.
whole fermentation process. Fermentation time was further shortened to 24 h. 3.2.3. Effect of initial pH on fermentation Controlling the pH of the culture medium during fermentation has also been reported to enhance growth and enzyme production [36]. As shown in Fig. 4A–B, cell growth and ulvan lyase production were found to be dependent on pH, and the optimal pH condition was 7.0. When the pH conditions of the culture medium were 6.0, 8.0, and 9.0, a loss of 32.9%, 19.6% and 71.7% of ulvan lyase production was observed, respectively. This phenomenon may be attributed to the cytoplasmic transmission of microbial cell. The pH of the medium is a critical environmental parameter that strongly affects bacterial metabolism and the transport of various components across the cell membrane [13,37]. Changes in pH result in different metabolic product distribution and ulvan lyase performance. Similar phenomena have been reported in many researches, such as in the production of carboxymethylcellulase, chitinase, xylanase and β-galactosidase [36–39].
3.3. Establishing fermentation kinetics models For ulvan lyase fermentation, a preliminary kinetic model including cell growth, product synthesis and substrate consumption was proposed to describe the fermentation performance. The relative regressed parameters were determined and kinetic model equations under the optimal conditions were as follows:
dX = 0.27 1 dt
3.2.4. Effect of aeration rate on fermentation The effects of aeration rate on cell growth and ulvan lyase production are depicted in Fig. 4C–D. As the results show, a higher aeration rate enhanced the cell growth of strain LP and the maximal cell biomass accumulation occurred at 2.0 vvm. However, the optimal aeration rate for ulvan lyase production was 1.5 vvm. According to the study of Roukas & Kyriakides [40], aeration may be beneficial to the growth and performance of microorganisms by improving the mass transfer characteristics with respect to the substrate, products, and oxygen. In contrast, a large quantity of oxygen transferred into microbial cells intensively could affect product formation by impacting metabolic pathways and altering metabolic fluxes [41]. In this study, excessive
X X 1.14
(7)
dP dx = 1.35 + 0.024X dt dkt
(8)
dS = dt
(9)
7.68
dX dt
2.37
dP dt
The kinetic equations agreed with the experimental data and fitting curves are shown in Fig. 5. The value of Xm was close to the actual value. The coefficient values of α and β were 1.347 and 0.024 respectively, showing that the whole fermentation process was coupled with cell growth [42]. Furthermore, the cell growth curve and the substrate consumption curve both matched the experimental data well. However, the fitting curve of product formation had some bias and showed a lower production trend in the final stage of ulvan lyase fermentation. The kinetic models reflected that a shorter fermentation time may be beneficial to obtain maximal enzyme activity. 5
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 4. The effect of pH and aeration rate on the ulvan lyase fermentation, (A) the ulvan lyase activity and cell biomass at different pH conditions on the fermentation end-point, (B) the fermentation condition of optimal pH strategy, (C) the ulvan lyase activity and cell biomass at different aeration conditions on the fermentation end-point, (D) the fermentation condition of optimal aeration strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
3.4. Developing fermentation in a 30-L fermentor
activity achieved within 20 h was 1.20 U/mL, which was nearly a 10.09% improvement compared with that obtained over the same period data in a 5-L fermentor. The accumulation of cell biomass was similar; the lowest level of residual sugar was obtained at 12 h, which is earlier than that in the 5-L fermentor. Generally speaking, microbial fermentation is usually developed in laboratory scale bioreactors and then scaled-up to larger bioreactors, first in pilot-scale and then in industrial scale fermentors for
The objective of the kinetic study is not only to develop a model for describing the fermentation process but also to be further applied to predict the data under various fermentation conditions [18]. Based on the predictions using the kinetic models, ulvan lyase fermentation was carried out in a 30-L fermentor to explore the possibility of industrial scale production. As depicted in Fig. 6, the maximum ulvan lyase
Fig. 5. The fitting curve of fermentation kinetic models, (A) the cell growth, (B) the ulvan lyase activity, (C) the substrate consumption. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. 6
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 6. The comparison of fermentation between 5-L fermentor and 30-L fermentor, (A) the cell growth, (B) the ulvan lyase activity (C) the substrate consumption. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
commercial production [43]. The aims of a process scale-up are to enlarge the production quantities with similar or higher productivity and product quality [44], and we intended to achieve similar objectives by developing ulvan lyase fermentation in a 30-L fermentor. As shown in Fig. 6, the positive result indicates the potential to scale-up ulvan lyase production. Fang et al. [45] reported that a 30-L fermentor was close to pilot-scale level and its fermentation conditions could be easily enhanced for scale-up fermentation. Collectively, the findings indicate that it might be possible to achieve the industrial-scale production of ulvan lyase directly using U. prolifera powder medium.
Acknowledgment This work was supported by the National Key Research and Development Program (2018YFC0311203), and the Key Project of New and Old Energy Transformation in Shandong Province. References [1] Y. Yang, J. Boncoeur, S.G. Liu, P. Nyvall-Collen, Economic assessment and environmental management of green tides in the Chinese Yellow Sea, Ocean Coast. Manage. 161 (2018) 20–30. [2] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice, Int. J. Biol. Macromol. 64 (2014) 1–5. [3] V.R. Konasani, C.S. Jin, N.G. Karlsson, E. Albers, Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species, Algal Res. 36 (2018) 106–114. [4] O. Coste, E. Malta, J.C. López, C. Fernandez-Diaz, Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new ulvan-degrading enzymatic bacterial crude extract, Algal Res. 10 (2015) 224–231. [5] S.J. Charnock, I.E. Brown, J.P. Turkenburg, G.W. Black, G.J. Davies, Convergent evolution sheds light on the anti-β-elimination mechanism common to family 1 and 10 polysaccharide lyases, Proc. Natl. Acad. Sci. 99 (2002) 12067–12072. [6] A. Robic, C. Gaillard, J.F. Sassi, Y. Lerat, M. Lahaye, Ultrastructure of ulvan: a polysaccharide from green seaweeds, Biopolymers 91 (2009) 652–664. [7] M. Lahaye, A. Robic, Structure and functional properties of ulvan, a polysaccharide from green seaweeds, Biomacromolecules 8 (2007) 1765–1774. [8] H.M. Qin, P. Xu, Q. Guo, X.T. Cheng, D.K. Gao, D.Y. Sun, Z.J. Zhu, F.P. Lu, Biochemical characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV, RSC Adv. 8 (2018) 2610–2615. [9] A. Salinas, C.E. French, The enzymatic ulvan depolymerisation system from the alga-associated marine flavobacterium Formosa agariphila, Algal Res. 27 (2017) 335–344. [10] C. He, H. Muramatsu, S. Kato, K. Ohnishi, Characterization of an Alteromonas longtype ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi, Biosci. Biotechnol. Biochem. 81 (2017) 2145–2151. [11] I. Hassairi, R.B. Amar, M. Nonus, B.B. Gupta, Production and separation of αagarase from Altermonas agarlyticus strain GJ1B, Bioresour. Technol. 79 (2001) 47–51. [12] J.S. Zhou, M.H. Cai, T. Jiang, W.Q. Zhou, W. Shen, X.S. Zhou, Y.X. Zhang, Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102, Bioprocess Biosyst. Eng. 37 (2014) 575–584. [13] Y.P. Li, J. Wang, Y. Yu, X. Li, X.L. Jiang, H. Hwang, P. Wang, Production of enzymes by Alteromonas sp. A321 to degrade polysaccharides from Enteromorpha prolifera, Carbohydr. Polym. 98 (2013) 988–994. [14] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice, Int. J. Biol. Macromol. 64 (2014) 1–5. [15] M. Dubios, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [16] R.E. Ricklefs, A graphical method of fitting equations to growth curves, Ecology 48 (1967) 978–983. [17] R. Luedeking, E.L. Piret, A kinetic study of the lactic acid fermentation. Batch process at controlled pH, J. Biochem. Microbiol. Technol. Eng. 1 (1959) 393–412. [18] L. He, X.B. Zhao, K.K. Cheng, Y. Sun, D.H. Liu, Kinetic modeling of fermentative production of 1, 3-propanediol by Klebsiella pneumoniae HR526 with consideration of multiple product inhibitions, Appl. Biochem. Biotechnolo. 169 (2013) 312–326. [19] P.N. Collén, J.F. Sassi, H. Rogniaux, H. Marfaing, W. Helbert, Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family, J. Biol. Chem. 286 (2011) 42063–42071.
4. Conclusion Present study confirms the successful production of ulvan lyase directly by consuming U. prolifera powder culture medium. Fermentation of the improved culture medium was established in a shake flask. Based on the two-stage developed strategy, the ulvan lyase activity reached 1.09 U/mL after shortening the fermentation time to 24 h in a 5-L fermentor. After establishing the kinetic models, investigation of scaling up the fermentation in a 30-L fermentor was further conducted. Higher enzyme activity and shorter fermentation time were obtained with 1.20 U/mL and 20 h, which revealed that ulvan lyase production may be done at the industrial scale. Authors contributions to the work All the authors carried out the studies together. Peng Wang and Jing-liang zhang mainly offered the conception of the study. Le-ke Qiao and Xiao-ke Yang analyzed the experiment data and drafted the article. Rui-ze Xie, Chun-ying Du and Yong-zhou Chi revised the article critically. All authors read the final manuscript and agreed to authorship and its submission to the Algal Research for the peer review and publication. Statement of informed consent Neither humans nor animals was employed during the experiments. And no conflicts, informed consent, or human or animal rights are applicable to this study. CRediT authorship contribution statement Leke Qiao:Writing original draft.Xiaoke Yang:Data curation.Ruize Xie:Investigation.Chunying Du:Visualization. Yongzhou Chi:Conceptualization, Supervision.Jingliang Zhang: Conceptualization, Supervision.Peng Wang:Project administration, Writing - review & editing. 7
Algal Research 46 (2020) 101812
L. Qiao, et al. [20] G.L. Miller, Use of dinitrosalicylic acid reagants for the determination of reducing sugar, Anal. Chem. 31 (1959) 6–8. [21] M. Fenice, L. Selbmann, L. Zucconi, S. Onofri, Production of extracellular enzymes by Antarctic fungal strain, Polar Biol. 17 (1997) 275–280. [22] Y.P. Li, Z.J. Huang, L.K. Qiao, Y. Gao, H.S. Guan, H. Aker, W. Hwang, P. Wang, Purification and characterization of a novel enzyme produced by Catenovulum sp. LP and its application in the pre-treatment to Ulva prolifera for bio-ethanol production, Process Biochem. 50 (2015) 799–806. [23] J.P. Maran, S. Manikandan, K. Thiruganasanbandham, C.V. Nivetha, R. Dinesh, Box-Behnken design based statistical modeling for ultrasound-assisted extraction of corn silk polysaccharide, Carbohydr. Polym. 92 (2013) 604–611. [24] C.L. Ye, C.J. Jiang, Optimization of extraction process of crude polysaccharides from Plantago asiatica L. by response surface methodology, Carbohydr. Polym. 84 (2011) 495–502. [25] R. Thangam, V. Suresh, S. Kannan, Optimized extraction of polysaccharides from Cymbopogon citrates and its biological activities, Int. J. Biol. Macromol. 65 (2014) 415–423. [26] I. Hassairi, R.B. Amar, M. Nonus, B.B. Gupta, Production and separation of αagarase from Altermonas agarlyticus strain GJ1B, Bioresour. Technol. 79 (2001) 47–51. [27] Y. Khambhaty, K. Mody, B. Jha, V. Gohel, Statistical optimization of medium components for κ-carrageenase production by Pseudomonas elongate, Enzym. Microb. Technol. 40 (2007) 813–822. [28] Q.D. An, G.L. Zhang, H.T. Wu, Z.C. Zhang, W. Gong, Y. Liu, X.Z. Li, Y. Murata, Production and partial properties of alginase from newly isolated Flavobacterium sp. LXA, Process Biochem. 43 (2008) 842–847. [29] T.I. Zanghloul, A.M. Embaby, A.R. Elmahdy, Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor, Bioresour. Technol. 102 (2011) 2387–2393. [30] M.S. Tanyildizi, D. Özer, M. Elibol, Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation, Biochem. Eng. J. 37 (2007) 294–297. [31] Z. Fang, J. Zhang, B.H. Liu, G.C. Du, J. Chen, Biochemical characterization of three keratinolytic enzymes from Stenotrophomonas maltophilia BBE11-1 for biodegrading keratin wastes, Int. Biodeterior. Biodegrad. 82 (2013) 166–172. [32] N. Wieczorek, M.A. Kucuker, K. Kuchta, Microalgae-bacteria flocs (MaB-Flocs) as a substrate for fermentative biogas production, Bioresour. Technol. 194 (2015)
130–136. [33] X. Shi, D.H. Kim, H.S. Shin, K.W. Jung, Effect of temperature on continuous fermentative hydrogen production from Laminaria japonica by anaerobic mixed cultures, Bioresour. Technol. 144 (2013) 225–231. [34] L.D. Unsworth, D. Van, J. Oost, S. Koutsopoulos, Hyperthermophilic enzymes-stability, activity and implementation strategies for high temperature applications, FEBS J. 274 (2007) 4044–4056. [35] J. Yang, Q. Chen, W.P. Wang, J.J. Hu, C. Hu, Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation, J. Biosci. Bioeng. (5) (2015) 564–569. [36] C.A. Hsu, R.C. Yu, S.L. Lee, C.C. Chou, Cultural condition affecting the growth and production of β-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermentor, Int. J. Food Microbiol. 116 (2007) 186–189. [37] M. Kapoor, L.M. Nair, R.C. Kuhad, Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora, Biochem. Eng. J. 38 (2008) 88–97. [38] B.L. Liu, P.M. Kao, Y.M. Tzeng, K.C. Feng, Production of chitinase from Verticillium lecanii F091 using submerged fermentation, Enzym. Microb. Technol. 33 (2003) 410–415. [39] B.H. Lee, B.K. Kim, Y.J. Lee, C.H. Chung, J.W. Lee, Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. Subtilis A-53, Enzym. Microb. Technol. 46 (2010) 38–42. [40] T. Roukas, M.L. Kyriakides, Production of pullulan from beet molasses by Aureobasidium pullulan in a stirred tank fermentor, J. Food Eng. 40 (1999) 89–94. [41] L. Qu, L.J. Ren, H. Huang, Scale-up of docosahexaenoic acid production in fed-batch fermentation by Schizochytrium sp. based on volumetric oxygen-transfer coefficient, Biochem. Eng. J. 77 (2013) 82–87. [42] L.Q. Zhu, X. Yang, C.Y. Xue, Y. Chen, L. Qu, W.Y. Lu, Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fed-batch fermentation process, Bioresour. Technol. 117 (2012) 208–213. [43] C. Heath, R. Kiss, Cell culture process development: advances in process engineering, Biotechnol. Prog. 23 (2007) 46–51. [44] F.R. Schmidt, Optimization and scale up of industrial fermentation processes, Appl. Microbiol. Biotechnol. 68 (2005) 425–435. [45] Z. Fang, J. Zhang, B.H. Liu, G.C. Du, J. Chen, Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1, Bioresour. Technol. 140 (2013) 286–291.
8
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Efficient production of ulvan lyase from Ulva prolifera by Catenovulum sp. LP based on stage-controlled fermentation strategy
T
Leke Qiaoa,b, Xiaoke Yangc, Ruize Xiea, Chunying Dua, Yongzhou Chia, Jingliang Zhangb, ⁎ Peng Wanga, a
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, PR China Marine Biomedical Research Institute of Qingdao, Qingdao 266100, PR China c Tsingtao Brewery Company Limited, Qingdao 266023, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Ulvan lyase U. prolifera Fermentation Catenovulum sp. LP
Ulvan lyase, specifically depolymerizing ulvan to unsaturated sulfated oligosaccharides with diverse biological activities, is becoming more and more popular. To provide an economical fermentative strategy, Catenovulum sp. LP cultured in low-cost medium that directly used Ulva prolifera as the main carbon source was used to produce ulvan lyase. The fermentation conditions were established in a shake flask containing 5% (w/v) U. prolifera powder, 0.2% K2HPO4, 0.4% NH4Cl, 0.5% NaCl, and 0.05% MgSO4. In a 5-L fermentor, series of strategies were used to systematically discuss the fermentation process. The enzyme with the best activity was obtained by the condition of a two-stage temperature (28–32 °C), initial pH 7.0, 1.5 vvm and two-stage agitation (200–400 rpm). The fermentation kinetic model of substrate consumption indicated that a shorter culture time may improve the ulvan lyase activity. Moreover, 10.09% improvement in enzyme activity was observed in a 30-L fermentor by shortening the fermentation time to 20 h. This fermentation method offers a promising prospect to enlarge the production of ulvan lyase and also provides a potential possibility to mildly prepare bioactive unsaturated oligosaccharides.
1. Introduction Green macroalgae can proliferate rapidly in eutrophic water. The green-tides and algal blooms that can result are a significant environmental concern for cities along the Yellow Sea [1,2]. Despite their abundant biomass, the utilization of green macroalgae is currently limited [3]. The green algal cell wall, especially the two main genera Ulva and Enteromorpha, is composed of a long chain anionic heteropolysaccharide polymer termed “ulvan”. And there are two major polysaccharide families in Ulva sp.: the water-soluble ulvan and insoluble cellulose. The ulvan is a kind of intercellular polysaccharide which mainly exists between cuticle and cellulose layer [4,7]. The ulvan content can reach 29% of the total dry weight. Its degradation products, which include D-glucuronic acid (GLcA), D-xylose (Xyl), and 3-sulfated rhamnose (Rha3S), have potential applications in the food, agriculture, pharmaceutical, and chemistry sectors [5–7]. However, these bioactive oligosaccharides are normally present in low quantities, especially the disaccharide portion of Rha3S linked to Xyl [8]. Ulvan lyase (EC 4.2.2) is a depolymerase that precisely cleaves the β (1–4) glycosidic bond between Rha3S and uronic acid through the β⁎
elimination mechanism [3,8]. This mild enzymatic method can yield all the constituent oligosaccharides without isomerization [7]. Developing a fermentation process applied to the industrial production of ulvan lyase would be beneficial to prepare these oligosaccharides. This potential is limited by the lack of knowledge on ulvan lyase. Studies on ulvan lyase have mainly been directed toward the screening microorganisms exhibiting enzyme activity and the characteristic analysis of catalysis [3,7,9,10]. The scale of fermentation has been limited to shake flask, while the substrate used as the main carbon source has been limited to the pure soluble polysaccharide extracted using the hot water method [7]. Poor production, limited fermentative substrates, and high output cost of the enzyme have limited the generation of ulvan oligosaccharides. Consequently, an improved ulvan lyase production method is required. The production of polysaccharide degradases including agarase and alginate lyase have been studied more intensively in brown and red algae than in the green algae species Ulva prolifera. Hassairi et al. [11] presented results of the production of α-agarase in a 2-L fermentor with batch, fed-batch, and continuous fermentation strategies. Zhou et al. [12] reported the successful scale-up of fermentation in 5-L and 30-L
Corresponding author at: College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (P. Wang).
https://doi.org/10.1016/j.algal.2020.101812 Received 18 July 2019; Received in revised form 19 January 2020; Accepted 19 January 2020 2211-9264/ © 2020 Elsevier B.V. All rights reserved.
Algal Research 46 (2020) 101812
L. Qiao, et al.
bioreactors. More importantly and different from the use of a common carbon source, a novel strategy using mixed carbon sources (starch and alginate) was reported by the authors. This improved culture medium was less expensive and could expand alginate lyase production. These findings highlight the importance of the culture medium used in the fermentation process of ulvan lyase. U. prolifera is a marine green algae that is distributed globally in ocean coastal regions [13,14]. The purified polysaccharide of U. prolifera (PU) is usually used as the main material for shake flask production of ulvan lyase. In this study, we tried to directly use U. prolifera as the main carbon source. To the best of our knowledge, only limited information regarding the direct fermentation of algal bodies by a microorganism is available. The objectives of the study were (1) to test the possibility of replacing PU with solid algae powders in the culture medium, (2) to optimize the fermentation conditions in a 5-L laboratory-scale fermentor, and (3) to establish the fermentation kinetics models and scale-up the fermentation to a 30-L fermentor. As the first step in the scale-up process of ulvan lyase fermentation, we attempted to reduce the costs of raw materials and increase the economic feasibility of the large-scale production.
the growth curve and ulvan lyase production were plotted using the software Origin 8.0. 2.4. Fermentor experiments 2.4.1. 5-L laboratory fermentation The optimization of ulvan lyase production was carried out in a 5-L glass fermentor (Bailun Bioscience Company, Shanghai, China) with 3 L working volume. Before fermentation, the fermentor was pre-sterilized at 121 °C for 15 min. Then, temperature-shift procedure (24–32 °C), agitation speed shift procedure (200–600 rpm), aeration rate control (0.5–2.0 vvm), and pH control (6.0–9.0) were all taken into consideration to determine the optimal fermentation parameters. The pH control was achieved by adding either 0.1 M HCl or NaOH. 2.4.2. Fermentation kinetics models Three preliminary rate equations for cell biomass (X), enzyme activity (P) and residual sugar (S) were employed to describe the whole fermentation process. The cell growth rate of strain LP was described by Logistic's equation. This equation reflected the cell growth rate as a function of biomass concentration [16,18,42]. In this study, the biomass concentration (X, Xm) was presented by the values of OD 600 measured by spectrophotometrically.
2. Materials and methods 2.1. Feedstock and microorganism
dX = µmax 1 dt
U. prolifera was collected from the coast of Qingdao, China in 2018. It was cleaned carefully and dried in an oven (50 °C, 48 h). Subsequently, it was milled in a mechanical grinder to obtain a homogeneous powder with 60 meshes, and used in the following experiments. All the other reagents were analytical grade and commercially available. The strain Catenovulum sp. LP which could produce ulvan lyase used in the study was supplied by the Microbiology Laboratory of Ocean University of China. It was maintained on a Luria-Bertani (LB) agar slant at 4 °C.
X X Xm
(1)
where X was biomass concentration, t was fermentation time, μmax was max specific growth rate, and Xm was max biomass concentration. The Luedeking-Piret equation was employed to indicate the formation rate of the main product [17]. This equation illustrated that the product formation was associated with cell growth.
dP = dt
dx + X dt
(2)
where P was product concentration, α was growth associated constant, β was non- growth associated constant. The consumption rate of substrate is mainly driven by cell growth, product synthesis and cell metabolic activities. It could be described by the following equation [18]:
2.2. The extraction of PU and culture medium Two different methods of PU extraction were investigated. The first one was the improved hot water method as described by Li et al. [13]. Briefly, the milled alga (100 g) was dipped into 2 L of tap water, homogenized and refluxed at 95 °C for 2 h. The soluble polysaccharide was collected by centrifugation (4800 ×g, 15 min). The second one was the autoclave method. With the same solid-liquid ratio, appropriate amounts of U. prolifera powder and distilled water were added into an Erlenmeyer flask, mixed evenly and autoclaved at 115 °C for 40 min. After cooling, the autoclaved polysaccharide was also collected by centrifugation (4800 ×g, 15 min). The culture medium consisted of (g/L):12 PU, 2.0 K2HPO4, 4.0 NH4Cl, 5.0 NaCl, and 0.5 MgSO4. The initial pH of the medium was neutral.
dS = dt
dX 1 dt Yx / s
dP 1 dt Yp / s
mx X
(3)
where S was substrate concentration, Yx/s was the cell yield coefficient, Yp/s was the product yield coefficient, and mx was the coefficient for maintenance functions. During the fermentation of strain LP, substrate consumption was mainly focused on cell yield coefficient and product yield coefficient. Therefore, we could further simplify the equation as follow:
k1 =
2.3. Shake flask experiments
kk2 =
2.3.1. Effect of autoclaved conditions on PU concentration U. prolifera powder medium with 5% (w/v) solid algae powder was autoclaved at different temperatures (115 °C and 121 °C) and for different durations (20–60 min). The concentration of polysaccharides was determined by the phenol‑sulfuric acid method at 490 nm using glucose as a standard [15].
1 Yx / s
(4)
1 Yp / s
(5)
Rearrangement Eq. (3), Eq. (4) and Eq. (5):
dS = dt
dX k1 dt
dP k2 dt
(6)
2.4.3. 30-L bench fermentation In a 30-L fermentor (Bailun Bioscience company, Shanghai, China), the optimal strategy with two-stage agitation control and temperatureshift procedure was further studied. Similar to the pretreatment of the 5-L fermentor, it was also pre-sterilized at 121 °C for 20 min before fermentation. Agitation control and temperature-shift were both
2.3.2. Growth curve and ulvan lyase activity The Catenovulum sp. LP (strain LP) was cultured on a rotary shaker (ZWF-200, Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China) at 170 rpm and 28 °C for 50 h. Samples were withdrawn to determine the ulvan lyase activity and cell biomass. Fitting of 2
Algal Research 46 (2020) 101812
L. Qiao, et al.
initiated after 6 h. The initial pH was around 7.0 and aeration rate was 1.5 vvm.
method. This phenomenon may be attributed to the differences in stirring condition and extraction time between the two methods. Stirring continuously and a longer extraction time were both beneficial for water soluble polysaccharide dissolution [23]. Compared with the hot water method, autoclaving was a closed system. Some operations such as stirring and refluxing were unavailable. In order to achieve better polysaccharide dissolution, extending the autoclave time and enhancing the autoclave temperature could be taken into consideration.
2.5. Analytical methods To remove the U. prolifera powder residue, the withdrawn samples were filtered through gauze (200 meshes) and centrifuged (2000 ×g, 5 min). The supernatant collected was applied to determine the enzyme activity and cell biomass. The ulvan lyase activity was determined based on two kinds of methods. The first one was measuring the formation of double bonds in the assay at 235 nm according to the method of Collen et al. [19]. And the second was measuring the release of the reducing sugar [20,21]. During the fermentation process, the ulvan lyase activity experimental data demonstrate formation of unsaturated bond (Linker's method) was consistent with the level of sugars that measured by DNS method. We chose the DNS method under the assay conditions (hydrolysis at pH 5.5 and 35 °C) to evaluate the ulvan lyase activity. One unit (U) of degradase activity was defined as the amount of enzyme required to produce 1 μmoL of reducing sugar (glucose equivalent) in 1 min under the aforementioned conditions. The cell biomass was measured spectrophotometrically by monitoring the optical density at 600 nm. The PU with the same concentration was employed as control. The iterative regression of kinetic models was obtained using mathematical software Matlab.
3.1.2. Effect of autoclave conditions on polysaccharide of U. prolifera (PU) extraction Extraction time and temperature are factors that would influence the extraction efficiency as well as the polysaccharides diffusion coefficient in the extracting solution [24]. To study the effect of different autoclave conditions on the dissolution of PU, various experiments were carried out with different autoclaved temperatures between 115 °C and 121 °C as well as different autoclave durations from 20 min to 60 min. As shown in Table. 2, the PU extracted at 115 °C for 40 min was higher than that for 30 min. Prolonged durations of autoclaving did not significantly affect the dissolution of polysaccharides, but led to the increase of reducing sugar concentration. When the sterilization temperature was increased to 121 °C, the reducing sugar content was enhanced further at all tested autoclave durations. Longer extraction time and higher temperature may lead to decomposition or degradation during the extraction process [25]. In this study, for the PU extracted by autoclaving, prolonged extraction time and higher temperature may damage the structure of the polysaccharide chains, producing large numbers of reducing sugars. Similar to agarase, κ-carrageenase and alginate lyase, the characteristic polysaccharides in the culture medium is necessary for Catenovulum sp. LP to induce the high production of ulvan lyase [26–28]. When the reducing sugars accumulate in the culture, microorganisms would prior to use it for cell growth, reducing PU utilization and the induction of enzyme secretion. Thus, the polysaccharide extracted at 115 °C for 40 min was more suitable to be supplemented in the culture medium.
2.6. Statistical analysis All experiments were conducted in triplicate. The values of different parameters were all expressed as the mean (n = 3). The corresponding standard deviations were determined by using one-way analysis of variance (ANOVA). 3. Results and discussion
3.1.3. Growth curve and ulvan lyase production in two kinds of media In order to assess the possibility of replacing soluble polysaccharides with algae powder, the cell growth and ulvan lyase production from Catenovulum sp. LP under two different conditions were investigated. As shown in Fig. 1, the two curves exhibited similar trends. During the first 6 h, strain LP was in the lag phase. The exponential phase occurred between 6 h and 24 h, and was the phase associated with the production of ulvan lyase. When the culture time extended to 36 h, the bacterial culture reached the stationary phase and the production of ulvan lyase also came to a plateau. Any further increase in incubation time showed no effect on the enzyme production. The curve of bacterial growth and ulvan lyase production were both fitted between soluble polysaccharide medium and solid algae powder medium, which confirmed that algae powder may be used as the main carbon source. This result indicated that ulvan lyase can be obtained without a complicated polysaccharide extraction procedure, which is of great value from a cost effectiveness standpoint. Moreover, this kind of improved culture medium could benefit to realize the large scale production of ulvan lyase. Thus, subsequent studies on a 5-L laboratory scale fermentor were carried out to optimize ulvan lyase production.
3.1. Fermentation in shake flasks 3.1.1. The possibility of U. prolifera as the main carbon source Strain LP domesticated over a long period of time has been reported to be a typical inducible enzyme production strain, with the polysaccharide concentration being the main factor that directly influences cell growth and ulvan lyase production [22]. Ulvan lyase was reported to be the major enzyme product during the fermentation process. In the previous study, the polysaccharide used as the main carbon source in the culture medium was extracted using the hot water method, followed by a series of processing steps that included concentration, dialysis, ethanol precipitation, and drying. This process was tedious and complex. If we could obtain the carbon resource directly by autoclaving the algae powder, the fermentation production of ulvan lyase would be markedly improved, especially in large-scale production. The comparison of polysaccharide dissolution between the hot water method and the autoclave method was conducted. Results are shown in Table 1. The concentration of polysaccharides and reducing sugars obtained by autoclaving was lower than that by hot water Table 1 The polysaccharide of U. prolifera (PU) dissolution with different method. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. Extraction condition
Polysaccharide concentration (g/L)
Reducing sugar concentration (g/L)
Hot water method Autoclaved method
11.72 ± 0.71 8.95 ± 0.42
0.33 ± 0.02 0.21 ± 0.01
3.2. Fermentation in 5-L laboratory-scale batch fermentor Transferring a fermentation process from shake flasks to fermentors is considered to be a great challenge due to substantial differences in the mode of controlling the temperature, pH, aeration, agitation, as well as the geometric variety in both conditions [29]. In order to scale up the ulvan lyase production, four strategies including temperatureshift procedure, pH control, agitation-shift procedure and aeration 3
Algal Research 46 (2020) 101812
L. Qiao, et al.
Table 2 Effect of autoclaved conditions on polysaccharide of U. prolifera (PU) extraction. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
The hot water method The autoclaved method
Extraction condition
The polysaccharide concentration (g/L)
The reducing sugar concentration (g/L)
Ulvan lyase activity (U/mL)
Hot water reflux 115 °C 30 min 115 °C 40 min 115 °C 50 min 115 °C 60 min 121 °C 20 min 121 °C 30 min 121 °C 40 min
11.72 10.67 12.02 12.17 12.66 12.63 12.60 12.85
0.33 0.32 0.35 0.40 0.43 0.45 0.48 0.49
0.715 0.736 0.748 0.690 0.705 0.720 0.702 0.708
± ± ± ± ± ± ± ±
0.71 0.39 0.76 0.35 0.19 0.79 0.60 0.84
control were all conducted to seek the optimal condition for the culture of strain LP in a 5-L laboratory-scale fermentor.
± ± ± ± ± ± ± ±
0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.03
± ± ± ± ± ± ± ±
0.018 0.038 0.031 0.073 0.019 0.019 0.042 0.041
at 28 °C probably positively impacted the stability of strain LP and was consequently used for the latter stage of fermentation. With the temperature-shift process, the growth rate of microorganisms was enhanced, and the fermentation time was shortened to 36 h.
3.2.1. Effect of temperature on fermentation Temperature is an important factor affecting protein production and the synthesis of metabolites of many strains [30]. In this study, a time course experiment for cell biomass and ulvan lyase activity was conducted at different temperatures (24 °C, 28 °C, and 32 °C). The 32 °C temperature resulted in the greatest increase in cell biomass and ulvan lyase activity within 24 h (Fig. 2A and B). After 24 h of fermentation, the yield at 28 °C reached 0.851 U/mL, which was almost equal to the yield achieved at 32 °C. However, when fermentation was extended to 36 h, the degradase activity at 32 °C decreased, while the activity was the highest at 28 °C. The experimental setup at 24 °C resulted in the least increase in cell biomass and ulvan lyase production during the entire fermentation process. These findings indicate that 32 °C was suitable for the earlier stage of fermentation by strain LP, while 28 °C was more appropriate for the later stage. The temperature-shift process was beneficial to cell growth (Fig. 2C). According to the report of Fang et al. [31], high temperatures for initial fermentation can improve cell biomass accumulation and effectively increase the yield of the target product. For the fermentation of strain LP, a high temperature (32 °C) stimulated the accumulation of cell biomass in the early stage (data not show). However, sustained fermentation at high temperature may decresae the synthesis of proteins essential for growth and other vital physiological processes of microorganisms [13]. Some disadvantageous soluble metabolic products such as organic bases (amine substances) could weaken the accumulation of cell biomass [32,33]. Moreover, enzyme activity is not always enhanced at high temperatures. According to the report of Unsworth et al. [34], high temperature is a harsh condition for enzymes to retain their activity and stability; it might affect the structural rearrangement of enzymes and result in decreased activity. Fermentation
3.2.2. Effect of agitation speed on fermentation It is well known that mixing by agitation is a crucial aspect of microbial fermentation to achieve the maximum productivity [29]. As shown in Fig. 3A and B, the effect of agitation speed ranging from 200 rpm to 600 rpm on cell biomass and ulvan lyase activity was investigated with 1.5 vvm aeration rate. During the 12 h of fermentation, the optimal agitation speed was found to be 200 rpm. When the fermentation time reached 24 h, the ulvan lyase activity at agitation speeds of 400 rpm and 600 rpm increased faster than that at 200 rpm. The improved fermentation yield at 400 rpm was maintained until the end of the fermentation process. In contrast, the enzyme activity at 600 rpm decreased when the fermentation time extended to 36 h. A well-directed control strategy would be beneficial to metabolite biosynthesis [35]. In this study, when the sterilized U. prolifera powder culture medium was cooled to the fermentation temperature (24–32 °C), the viscosity caused by PU increased significantly. This resulted in a lower oxygen requirement, which was mainly used to maintain the seed liquid growth. With the strain LP culture, the viscosity of the broth decreased with time due to PU degradation and increased cell concentration. Accompanied by agitation, microorganisms could mix with the culture medium homogenously, requiring more dissolved oxygen for its growth. Thus, fermentation conducted at 400 rpm provided the appropriate levels of oxygen between 12 h and 36 h of fermentation. The dissolved oxygen was maintained at a level of 32%. However, 600 rpm with 45% dissolved oxygen was not favorable for ulvan lyase activity. The inappropriate agitation speed would be a disadvantage for cell growth and metabolite biosynthesis [29,35]. Therefore, a two-stage agitation strategy was employed during the
Fig. 1. Growth profile and production of ulvan lyase by Catenovulum sp. LP, (A) the growth condition of polysaccharide of U. prolifera (PU) medium, (B) the growth condition of algae medium. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. 4
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 2. The effect of temperature on the ulvan lyase fermentation, (A) the OD 600 at different temperatures, (B) the ulvan lyase activity at different temperatures, (C) the fermentation condition of two-stage temperature strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
Fig. 3. The effect of agitation speed on the ulvan lyase fermentation, (A) the OD 600 at different agitation speeds, (B) the ulvan lyase activity at different agitation speeds, (C) the fermentation condition of two-stage agitation strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
aeration rate (2.0 vvm) may negatively affect the formation and release of ulvan lyase, leading to poor enzyme activity. Moreover, 1.5 vvm was the appropriate aeration rate for the ulvan lyase production.
whole fermentation process. Fermentation time was further shortened to 24 h. 3.2.3. Effect of initial pH on fermentation Controlling the pH of the culture medium during fermentation has also been reported to enhance growth and enzyme production [36]. As shown in Fig. 4A–B, cell growth and ulvan lyase production were found to be dependent on pH, and the optimal pH condition was 7.0. When the pH conditions of the culture medium were 6.0, 8.0, and 9.0, a loss of 32.9%, 19.6% and 71.7% of ulvan lyase production was observed, respectively. This phenomenon may be attributed to the cytoplasmic transmission of microbial cell. The pH of the medium is a critical environmental parameter that strongly affects bacterial metabolism and the transport of various components across the cell membrane [13,37]. Changes in pH result in different metabolic product distribution and ulvan lyase performance. Similar phenomena have been reported in many researches, such as in the production of carboxymethylcellulase, chitinase, xylanase and β-galactosidase [36–39].
3.3. Establishing fermentation kinetics models For ulvan lyase fermentation, a preliminary kinetic model including cell growth, product synthesis and substrate consumption was proposed to describe the fermentation performance. The relative regressed parameters were determined and kinetic model equations under the optimal conditions were as follows:
dX = 0.27 1 dt
3.2.4. Effect of aeration rate on fermentation The effects of aeration rate on cell growth and ulvan lyase production are depicted in Fig. 4C–D. As the results show, a higher aeration rate enhanced the cell growth of strain LP and the maximal cell biomass accumulation occurred at 2.0 vvm. However, the optimal aeration rate for ulvan lyase production was 1.5 vvm. According to the study of Roukas & Kyriakides [40], aeration may be beneficial to the growth and performance of microorganisms by improving the mass transfer characteristics with respect to the substrate, products, and oxygen. In contrast, a large quantity of oxygen transferred into microbial cells intensively could affect product formation by impacting metabolic pathways and altering metabolic fluxes [41]. In this study, excessive
X X 1.14
(7)
dP dx = 1.35 + 0.024X dt dkt
(8)
dS = dt
(9)
7.68
dX dt
2.37
dP dt
The kinetic equations agreed with the experimental data and fitting curves are shown in Fig. 5. The value of Xm was close to the actual value. The coefficient values of α and β were 1.347 and 0.024 respectively, showing that the whole fermentation process was coupled with cell growth [42]. Furthermore, the cell growth curve and the substrate consumption curve both matched the experimental data well. However, the fitting curve of product formation had some bias and showed a lower production trend in the final stage of ulvan lyase fermentation. The kinetic models reflected that a shorter fermentation time may be beneficial to obtain maximal enzyme activity. 5
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 4. The effect of pH and aeration rate on the ulvan lyase fermentation, (A) the ulvan lyase activity and cell biomass at different pH conditions on the fermentation end-point, (B) the fermentation condition of optimal pH strategy, (C) the ulvan lyase activity and cell biomass at different aeration conditions on the fermentation end-point, (D) the fermentation condition of optimal aeration strategy. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
3.4. Developing fermentation in a 30-L fermentor
activity achieved within 20 h was 1.20 U/mL, which was nearly a 10.09% improvement compared with that obtained over the same period data in a 5-L fermentor. The accumulation of cell biomass was similar; the lowest level of residual sugar was obtained at 12 h, which is earlier than that in the 5-L fermentor. Generally speaking, microbial fermentation is usually developed in laboratory scale bioreactors and then scaled-up to larger bioreactors, first in pilot-scale and then in industrial scale fermentors for
The objective of the kinetic study is not only to develop a model for describing the fermentation process but also to be further applied to predict the data under various fermentation conditions [18]. Based on the predictions using the kinetic models, ulvan lyase fermentation was carried out in a 30-L fermentor to explore the possibility of industrial scale production. As depicted in Fig. 6, the maximum ulvan lyase
Fig. 5. The fitting curve of fermentation kinetic models, (A) the cell growth, (B) the ulvan lyase activity, (C) the substrate consumption. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations. 6
Algal Research 46 (2020) 101812
L. Qiao, et al.
Fig. 6. The comparison of fermentation between 5-L fermentor and 30-L fermentor, (A) the cell growth, (B) the ulvan lyase activity (C) the substrate consumption. The values of different parameters were all expressed as the mean (n = 3), and the variability was shown as standard deviations.
commercial production [43]. The aims of a process scale-up are to enlarge the production quantities with similar or higher productivity and product quality [44], and we intended to achieve similar objectives by developing ulvan lyase fermentation in a 30-L fermentor. As shown in Fig. 6, the positive result indicates the potential to scale-up ulvan lyase production. Fang et al. [45] reported that a 30-L fermentor was close to pilot-scale level and its fermentation conditions could be easily enhanced for scale-up fermentation. Collectively, the findings indicate that it might be possible to achieve the industrial-scale production of ulvan lyase directly using U. prolifera powder medium.
Acknowledgment This work was supported by the National Key Research and Development Program (2018YFC0311203), and the Key Project of New and Old Energy Transformation in Shandong Province. References [1] Y. Yang, J. Boncoeur, S.G. Liu, P. Nyvall-Collen, Economic assessment and environmental management of green tides in the Chinese Yellow Sea, Ocean Coast. Manage. 161 (2018) 20–30. [2] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice, Int. J. Biol. Macromol. 64 (2014) 1–5. [3] V.R. Konasani, C.S. Jin, N.G. Karlsson, E. Albers, Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species, Algal Res. 36 (2018) 106–114. [4] O. Coste, E. Malta, J.C. López, C. Fernandez-Diaz, Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new ulvan-degrading enzymatic bacterial crude extract, Algal Res. 10 (2015) 224–231. [5] S.J. Charnock, I.E. Brown, J.P. Turkenburg, G.W. Black, G.J. Davies, Convergent evolution sheds light on the anti-β-elimination mechanism common to family 1 and 10 polysaccharide lyases, Proc. Natl. Acad. Sci. 99 (2002) 12067–12072. [6] A. Robic, C. Gaillard, J.F. Sassi, Y. Lerat, M. Lahaye, Ultrastructure of ulvan: a polysaccharide from green seaweeds, Biopolymers 91 (2009) 652–664. [7] M. Lahaye, A. Robic, Structure and functional properties of ulvan, a polysaccharide from green seaweeds, Biomacromolecules 8 (2007) 1765–1774. [8] H.M. Qin, P. Xu, Q. Guo, X.T. Cheng, D.K. Gao, D.Y. Sun, Z.J. Zhu, F.P. Lu, Biochemical characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV, RSC Adv. 8 (2018) 2610–2615. [9] A. Salinas, C.E. French, The enzymatic ulvan depolymerisation system from the alga-associated marine flavobacterium Formosa agariphila, Algal Res. 27 (2017) 335–344. [10] C. He, H. Muramatsu, S. Kato, K. Ohnishi, Characterization of an Alteromonas longtype ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi, Biosci. Biotechnol. Biochem. 81 (2017) 2145–2151. [11] I. Hassairi, R.B. Amar, M. Nonus, B.B. Gupta, Production and separation of αagarase from Altermonas agarlyticus strain GJ1B, Bioresour. Technol. 79 (2001) 47–51. [12] J.S. Zhou, M.H. Cai, T. Jiang, W.Q. Zhou, W. Shen, X.S. Zhou, Y.X. Zhang, Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102, Bioprocess Biosyst. Eng. 37 (2014) 575–584. [13] Y.P. Li, J. Wang, Y. Yu, X. Li, X.L. Jiang, H. Hwang, P. Wang, Production of enzymes by Alteromonas sp. A321 to degrade polysaccharides from Enteromorpha prolifera, Carbohydr. Polym. 98 (2013) 988–994. [14] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice, Int. J. Biol. Macromol. 64 (2014) 1–5. [15] M. Dubios, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [16] R.E. Ricklefs, A graphical method of fitting equations to growth curves, Ecology 48 (1967) 978–983. [17] R. Luedeking, E.L. Piret, A kinetic study of the lactic acid fermentation. Batch process at controlled pH, J. Biochem. Microbiol. Technol. Eng. 1 (1959) 393–412. [18] L. He, X.B. Zhao, K.K. Cheng, Y. Sun, D.H. Liu, Kinetic modeling of fermentative production of 1, 3-propanediol by Klebsiella pneumoniae HR526 with consideration of multiple product inhibitions, Appl. Biochem. Biotechnolo. 169 (2013) 312–326. [19] P.N. Collén, J.F. Sassi, H. Rogniaux, H. Marfaing, W. Helbert, Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family, J. Biol. Chem. 286 (2011) 42063–42071.
4. Conclusion Present study confirms the successful production of ulvan lyase directly by consuming U. prolifera powder culture medium. Fermentation of the improved culture medium was established in a shake flask. Based on the two-stage developed strategy, the ulvan lyase activity reached 1.09 U/mL after shortening the fermentation time to 24 h in a 5-L fermentor. After establishing the kinetic models, investigation of scaling up the fermentation in a 30-L fermentor was further conducted. Higher enzyme activity and shorter fermentation time were obtained with 1.20 U/mL and 20 h, which revealed that ulvan lyase production may be done at the industrial scale. Authors contributions to the work All the authors carried out the studies together. Peng Wang and Jing-liang zhang mainly offered the conception of the study. Le-ke Qiao and Xiao-ke Yang analyzed the experiment data and drafted the article. Rui-ze Xie, Chun-ying Du and Yong-zhou Chi revised the article critically. All authors read the final manuscript and agreed to authorship and its submission to the Algal Research for the peer review and publication. Statement of informed consent Neither humans nor animals was employed during the experiments. And no conflicts, informed consent, or human or animal rights are applicable to this study. CRediT authorship contribution statement Leke Qiao:Writing original draft.Xiaoke Yang:Data curation.Ruize Xie:Investigation.Chunying Du:Visualization. Yongzhou Chi:Conceptualization, Supervision.Jingliang Zhang: Conceptualization, Supervision.Peng Wang:Project administration, Writing - review & editing. 7
Algal Research 46 (2020) 101812
L. Qiao, et al. [20] G.L. Miller, Use of dinitrosalicylic acid reagants for the determination of reducing sugar, Anal. Chem. 31 (1959) 6–8. [21] M. Fenice, L. Selbmann, L. Zucconi, S. Onofri, Production of extracellular enzymes by Antarctic fungal strain, Polar Biol. 17 (1997) 275–280. [22] Y.P. Li, Z.J. Huang, L.K. Qiao, Y. Gao, H.S. Guan, H. Aker, W. Hwang, P. Wang, Purification and characterization of a novel enzyme produced by Catenovulum sp. LP and its application in the pre-treatment to Ulva prolifera for bio-ethanol production, Process Biochem. 50 (2015) 799–806. [23] J.P. Maran, S. Manikandan, K. Thiruganasanbandham, C.V. Nivetha, R. Dinesh, Box-Behnken design based statistical modeling for ultrasound-assisted extraction of corn silk polysaccharide, Carbohydr. Polym. 92 (2013) 604–611. [24] C.L. Ye, C.J. Jiang, Optimization of extraction process of crude polysaccharides from Plantago asiatica L. by response surface methodology, Carbohydr. Polym. 84 (2011) 495–502. [25] R. Thangam, V. Suresh, S. Kannan, Optimized extraction of polysaccharides from Cymbopogon citrates and its biological activities, Int. J. Biol. Macromol. 65 (2014) 415–423. [26] I. Hassairi, R.B. Amar, M. Nonus, B.B. Gupta, Production and separation of αagarase from Altermonas agarlyticus strain GJ1B, Bioresour. Technol. 79 (2001) 47–51. [27] Y. Khambhaty, K. Mody, B. Jha, V. Gohel, Statistical optimization of medium components for κ-carrageenase production by Pseudomonas elongate, Enzym. Microb. Technol. 40 (2007) 813–822. [28] Q.D. An, G.L. Zhang, H.T. Wu, Z.C. Zhang, W. Gong, Y. Liu, X.Z. Li, Y. Murata, Production and partial properties of alginase from newly isolated Flavobacterium sp. LXA, Process Biochem. 43 (2008) 842–847. [29] T.I. Zanghloul, A.M. Embaby, A.R. Elmahdy, Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor, Bioresour. Technol. 102 (2011) 2387–2393. [30] M.S. Tanyildizi, D. Özer, M. Elibol, Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation, Biochem. Eng. J. 37 (2007) 294–297. [31] Z. Fang, J. Zhang, B.H. Liu, G.C. Du, J. Chen, Biochemical characterization of three keratinolytic enzymes from Stenotrophomonas maltophilia BBE11-1 for biodegrading keratin wastes, Int. Biodeterior. Biodegrad. 82 (2013) 166–172. [32] N. Wieczorek, M.A. Kucuker, K. Kuchta, Microalgae-bacteria flocs (MaB-Flocs) as a substrate for fermentative biogas production, Bioresour. Technol. 194 (2015)
130–136. [33] X. Shi, D.H. Kim, H.S. Shin, K.W. Jung, Effect of temperature on continuous fermentative hydrogen production from Laminaria japonica by anaerobic mixed cultures, Bioresour. Technol. 144 (2013) 225–231. [34] L.D. Unsworth, D. Van, J. Oost, S. Koutsopoulos, Hyperthermophilic enzymes-stability, activity and implementation strategies for high temperature applications, FEBS J. 274 (2007) 4044–4056. [35] J. Yang, Q. Chen, W.P. Wang, J.J. Hu, C. Hu, Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation, J. Biosci. Bioeng. (5) (2015) 564–569. [36] C.A. Hsu, R.C. Yu, S.L. Lee, C.C. Chou, Cultural condition affecting the growth and production of β-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermentor, Int. J. Food Microbiol. 116 (2007) 186–189. [37] M. Kapoor, L.M. Nair, R.C. Kuhad, Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora, Biochem. Eng. J. 38 (2008) 88–97. [38] B.L. Liu, P.M. Kao, Y.M. Tzeng, K.C. Feng, Production of chitinase from Verticillium lecanii F091 using submerged fermentation, Enzym. Microb. Technol. 33 (2003) 410–415. [39] B.H. Lee, B.K. Kim, Y.J. Lee, C.H. Chung, J.W. Lee, Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. Subtilis A-53, Enzym. Microb. Technol. 46 (2010) 38–42. [40] T. Roukas, M.L. Kyriakides, Production of pullulan from beet molasses by Aureobasidium pullulan in a stirred tank fermentor, J. Food Eng. 40 (1999) 89–94. [41] L. Qu, L.J. Ren, H. Huang, Scale-up of docosahexaenoic acid production in fed-batch fermentation by Schizochytrium sp. based on volumetric oxygen-transfer coefficient, Biochem. Eng. J. 77 (2013) 82–87. [42] L.Q. Zhu, X. Yang, C.Y. Xue, Y. Chen, L. Qu, W.Y. Lu, Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fed-batch fermentation process, Bioresour. Technol. 117 (2012) 208–213. [43] C. Heath, R. Kiss, Cell culture process development: advances in process engineering, Biotechnol. Prog. 23 (2007) 46–51. [44] F.R. Schmidt, Optimization and scale up of industrial fermentation processes, Appl. Microbiol. Biotechnol. 68 (2005) 425–435. [45] Z. Fang, J. Zhang, B.H. Liu, G.C. Du, J. Chen, Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1, Bioresour. Technol. 140 (2013) 286–291.
8