Screening of Factors Influencing Pullulan Production by Aureobasidium melanogenum DSM 2404 Using Fractional
Factorial Design
Luo Zaini Mohd Izwan Low
1,2,b), Daniel Joe Dailin
1,2,a), Selly Wong Chew Key
2, Roslinda Abd Malek
1, Dalia Sukmawati
3, Riyaz Sayyed
4, Hesham Ali El-
Enshasy
1,2,51Institute of Bioproduct Development, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia.
2School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor, Malaysia
3Biology Department, Faculty of Mathematics and Natural Sciences, Universitas Negeri Jakarta, Jakarta, Indonesia
4Department of Microbiology, P.S.G.V.P. Mandal’s Arts, Science, and Commerce College, Shahada 425409 (MS), India
5City for Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt
a)Corresponding author: [email protected]
Abstract. Pullulan is a water-soluble exopolysaccharide which is synthesized by the black yeast-like-fungus, Aureobasidium sp. This polysaccharide is of commercial interest with increased applications in industries such as cosmeceutical, pharmaceutical, energy and food. Despite these applications, pullulan is costly as compared with other type of existing commercial polysaccharides which is a key limiting factor for cost effective applications. Therefore, continues study is required for the purpose of improving the production yield of pullulan. In this study, a total of seven factors were investigated including maltose, yeast extract, (NH4)2SO4, peptone, KH2PO4, MgSO4.7H2O and NaCl for their effects on pullulan production in shake-flasks. Aureobasidium melanogenum DSM 2404 was used in this study and all the seven factors were screened by using a two-level fractional factorial design (Plackett–Burman Design). The result of variance analysis (ANOVA) proposed that there were four statistically significant (P < 0.05) factors in the production of pullulan by using this strain. Maltose, yeast extract, peptone and KH2PO4 were found to have significant effects on pullulan production using the Plackett–Burman Design. The statistical analysis shows that the linear mathematical model is significant with the coefficient of determination (R2) value of 0.8945.
INTRODUCTION
Polysaccharides are considered as main ingredients in chemical, food, and pharmaceutical industries (1). Of different biofactories used for the production of polysaccharides, microbial cells gain more attention based on their high capacity for biopolymer production on a large scale economically. Nowadays, the most widely used polysaccharides are xanthan, dextran, pullulan, kefiran, and alginates beside many new functional biopolymers of mushroom origin (2-12).
Pullulan is a linear extracellular water-soluble and neutral polysaccharide obtained from the fermentation broth of black yeast-like-fungus Aureobasidium pullulans. Bauer was the first researcher discovered the presence of pullulan during the fermentation of this fungus (13) and Bernier was first reported to isolate and characterized pullulan from culture broths of A. pullulans (14). Through the studies done by Bender and co-workers, the name of “pullulan” was given based on its positive optical rotation and infrared spectrum that it was composed completely of α-D-glucans with mostly α-(1→4) linkages (15).
The basic structure of pullulan was resolved at the beginning of the 1960s (16-18). The finding of enzyme pullulanase which can hydrolyze α-(1→6) linkages in pullulan and convert to α-(1→6) linked polymer of maltotriose subunits. It had been confirmed that pullulan contained α-(1→4) and α-(1→6) in the ratio of 2:1 (19). Therefore, pullulan is illustrated as a linear and unbranched polysaccharide having maltotriose repeating unit which connected by α-(1→6) glycosidic linkage [20]. Pullulan has molecular formula (C6H10O5)n (21-22). The molecular weight range of pullulan ranges from 1.5×104 to 1×107 Da which is dependent on the physiological conditions and culture strains used (23-24,39).
Pullulan can be described as a white odorless, tasteless, and edible powder. This biopolymer and has a high solubility in water and dilute in alkaline condition but insoluble in alcohol and other organic solvents except dimethylformamide and dimethylsulfoxide (20). Besides that, pullulan has strong mechanical strength and along with other functional properties such as adhesive attributes, enzymatically mediated degradability, the ability to form fibers, compression moldings, and oxygen-impermeable films (19).
There were many pullulan-producing strains reported (22). Yet, many researches directed towards the higher pullulan producing strain, A. pullulans (19). Even though there are much extensive researches in the literature on biochemical mechanisms of pullulan making, but it still not yet well-understood. The pullulan is synthesized intracellularly at the cell wall membrane and the secreted biopolymer is connected to the cell surface to form a slimy and loose layer (25). Research done by Duan et al. (26) suggested the metabolic pathway for pullulan synthesis, where three main enzymes, α-phosphoglucose mutase, uridine diphosphoglucose pyrophosphorylase (UGDP- pyrophosphorylase) and glucosyltransferase are essential in the biosynthesis of pullulan. Nowadays, this biopolymer is widely used in food and pharmaceutical industries in large quantities (46, 54).
Many efforts have been developed to optimize the fermentation conditions to improve pullulan production using the one factor at a time approach. However, this method is not cost-effective, especially there might be interactions between some factors and time-consuming (27). Therefore, in recent years, statistical optimization methods have been considered as efficient alternative tools for bioprocess optimization for both upstream and downstream (30-34).
Statistical methods have been proved to resolve similar problems in a large number of scientific disciplines efficiently (28-34). In this investigation, a two-level fractional factorial design (Plackett–Burman Design) was used to reveal the effects of seven bioprocessing parameters and their interactions on pullulan production.
RESEARCH METHOD
Microorganism and Maintenance
Aureobasidium melanogenum DSM 2404 (formerly known as Aureobasidium pullulans var. melanogenum) was originally obtained from Leibniz Institute DSMZ - German Collection of Microorganism and Cell Cultures GmbH in
lyophilized form. The strain was cultured on Potato Dextrose Agar (PDA) and sub-cultured bi-weekly to maintain its viability. For long-term storage, stock cultures were maintained at -80ºC in 50% (v/v) glycerol solution.
Inoculum Preparation in Seed Medium
The inoculum was prepared in the 250 mL Erlenmeyer flask containing 50 mL of sterile seed medium (SM) (35) by transferring the culture from PDA into the SM. The flask was then incubated at 200 rpm, 28ºC for 48 h in a rotary shaker incubator.
Medium for Pullulan Production
Pullulan production medium by A. melanogenum DSM 2404 was prepared as described by Chen et al. (36). The medium composition was composed of (g L-1) maltose, 50.0; yeast extract, 10; (NH4)2SO4, 0.6; peptone, 20.0; NaCl, 1.0; K2HPO4, 5.0; MgSO4.7H2O, 0.6. The medium was first adjusted to pH 6.0 before sterilized at 121ºC for 20 minutes.
Pullulan Production in Shake-Flask
Each experiment was carried out in 250 mL Erlenmeyer flasks containing 50 mL of production medium. 5% of the inoculum from SM was used to inoculation production medium. The inoculated flasks were incubated in a rotary shaker incubator at 200 rpm and 28ºC for 4 days. The experiments were performed in duplicate.
Experimental Design
The factors that influenced the pullulan production were screened using two-level factorial design (Plackett–Burman Design) by Minitab 16 software. Total seven bioprocessing parameters were chosen, which were maltose (A), yeast extract (B), (NH4)2SO4 (C), peptone (D), KH2PO4 (E), MgSO4.7H2O (F) and NaCl (G) with each variable denoted at two levels, a high (+) level and low (-) level to evaluate their effects on pullulan production (Table 1). A design of 24 experiments was used to investigate the most significant variables that affected pullulan production as shown in Table 3. All experiments were conducted in duplicates and carried out in 250 ml of Erlenmeyer flask containing 50 ml of various media formulations and cultivated at 2ºC, 200 rpm for 4 days.
TABLE 1. Experimental range and levels of factors using Plackett-Burman design.
Variables Units Symbol code
Low level (-)
High level (+)
Maltose g L-1 A 10 50
Yeast Extract g L-1 B 4 10
(NH4)2SO4 g L-1 C 0.2 0.8
Peptone g L-1 D 5 20
KH2PO4 g L-1 E 1 5
MgSO4.7H2O g L-1 F 0.1 0.5
NaCl g L-1 G 0.5 1.0
Cell Dry Weight Determination
50 mL of culture broth in flasks were harvested by transferring 40 mL of the culture broth into 50 mL of the conical tubes. The samples were then centrifuged (Zentrifugen, Hettich, D-78532, Tuttlingen Germany) at 8000 rpm at 4ºC for 20 minutes to precipitate the cells. The supernatant was used for pullulan determination. After that, the cell-pellets were dried at 80ºC until it achieved the constant weight.
Crude Pullulan Determination
Pullulan was recovered from the supernatant and determined according to Özcan et al. (35). Briefly, 10 mL of supernatant was transferred into 50 ml of the conical tube, then the pullulan was precipitated with two volumes (20 mL) of pre-chilled 95% of ethanol at 4ºC overnight. The next day, the samples were centrifuged at 4ºC, 8000 rpm for 20 minutes. The supernatant was discarded, and the final precipitate was dried at 80ºC for overnight before weighing.
pH Determination
The final pH of the medium was measured by using a pH meter (Mettler-Toledo Delta 320, Greifensee, Switzerland).
RESULTS AND DISCUSSION
Significant Factors Affecting Production of Pullulan
A total of seven variables were analyzed concerning their effects on pullulan production using Plackett–Burman design (Table 1). The design consisted of 24 experiments is shown in Table 2. Statistical analysis is represented in Table 3. From Table 2, a Pareto chart (Fig. 1) can be obtained by analyzing the results by using Minitab 16 software to identify which factor is significantly affecting the production of pullulan.
The most significant effect for the variables was presented on the upper part and progressed down to the minimal effect. Variables that passed the reference line of 2.12 were selected for further optimization. From the Pareto chart, it shows that only maltose, peptone, K2HPO4, and yeast extract had a significant effect on pullulan production.
Based on the p values (Table 3), it was identified that maltose (A), yeast extract (B), peptone (D) and KH2PO4 (E) has given significant effect on pullulan production. The interpretation of the study was analyzed statistically by using the variance analysis (ANOVA). A p-value less than 0.05 indicates that the factor is significant to the responses studied. This is based on a confidence level that is set at 95%.
FIGURE 1. Pareto chart for the Plackett-Burman parameter
Coefficient of determination R2 was used to check the fit of model. The R2 for pullulan was 0.8945 and about 10.55% of variation could not be explained by this model. The predicted R2 was 0.7625 and is in reasonable agreement with the adjusted R2 obtained that was 0.8483.
Carbon source had the most significant influence on polysaccharide production. This is followed by nitrogen sources, salt, as well as other factors (22, 37-38). Many previous studies reported that carbon source plays significant role in pullulan production (39-42). In this study, maltose was screened for its probability to enhance pullulan production. However, less study reported the efficiency of maltose to be used as potential carbon source in pullulan production (36). Besides maltose, other type of commercially available carbon sources that were tested were glucose, sucrose, fructose (32,43-44,50). In addition to that, agro-industrial waste was also reported to have significant effect and potential substitute in pullulan production (45,46). However, all studies were conducted using different strains of pullulan producer. The carbon source that significantly affect pullulan production is normally strain dependent.
TABLE 2. Factorial design for pullulan production
TABLE 3. Analysis of variance (ANOVA) on pullulan production.
Run
Maltose (A)
Yeast extract (B)
(NH4)2SO4
(C)
Peptone (D)
KH2PO4
(E)
MgSO4
(F)
NaCl (G)
Pullulan (g L-1)
1 50 4 0.8 5 1 0.1 1.0 0.4
2 50 10 0.2 20 1 0.1 0.5 2.0
3 10 10 0.8 5 5 0.1 0.5 0.7
4 50 4 0.8 20 1 0.5 0.5 2.3
5 50 10 0.2 20 5 0.1 1.0 3.8
6 50 10 0.8 5 5 0.5 0.5 3.0
7 10 10 0.8 20 1 0.5 1.0 1.6
8 10 4 0.8 20 5 0.1 1.0 1.6
9 10 4 0.2 20 5 0.5 0.5 1.6
10 50 4 0.2 5 5 0.5 1.0 1.9
11 10 10 0.2 5 1 0.5 1.0 0.6
12 10 4 0.2 5 1 0.1 0.5 0.2
13 50 4 0.8 5 1 0.1 1.0 0.3
14 50 10 0.2 20 1 0.1 0.5 2.4
15 10 10 0.8 5 5 0.1 0.5 0.9
16 50 4 0.8 20 1 0.5 0.5 2.0
17 50 10 0.2 20 5 0.1 1.0 4.0
18 50 10 0.8 5 5 0.5 0.5 3.2
19 10 10 0.8 20 1 0.5 1.0 1.5
20 10 4 0.8 20 5 0.1 1.0 1.8
21 10 4 0.2 20 5 0.5 0.5 1.7
22 50 4 0.2 5 5 0.5 1.0 1.8
23 10 10 0.2 5 1 0.5 1.0 0.7
24 10 4 0.2 5 1 0.1 0.5 0.3
Sources Degree of
freedom
Sequential
sum of
squares
Adjusted sum of squares
Adjusted mean sum of squares
F value p value
Maltose (A) 1 8.0504 8.0504 8.05042 46.06 0.000
Yeast Extract (B) 1 3.0104 3.0104 3.01042 17.22 0.001
(NH4)2SO4 (C) 1 0.1204 0.1204 0.12042 0.69 0.419
Peptone (D) 1 6.3037 6.3037 6.30375 36.06 0.000
KH2PO4 (E) 1 5.7038 5.7038 5.70375 32.63 0.000
MgSO4 (F) 1 0.5104 0.5104 0.51042 2.92 0.107
NaCl (G) 1 0.0037 0.0037 0.00375 0.02 0.885
Nitrogen source was found to have a significant effect on the production of pullulan, biomass as well as the activity of UDPG-pyrophosphorylase enzyme that plays a major role in pullulan biosynthesis (47). There are two categories of nitrogen sources that are organic and inorganic. This research found out that organic nitrogen sources such as yeast extract and peptone had a greater influence on the pullulan production, which similar to some of the previous findings (36,49). While several researches found out that some strains favor inorganic nitrogen sources especially from ammonium or nitrate salts (47,50,51). The effect of minerals in this study was also found to enhance the EPS production which in line with some of the previous findings, which showed the significance of minerals in pullulan production (51,52-53).
CONCLUSION
Two-level fractional factorial design (Plackett–Burman Design) has been used to screen for the significant factors that affect the pullulan production by A. melanogenum DSM 2404 in the shake flask level. Pullulan production was significantly affected by maltose, yeast extract, peptone and KH2PO4 (p value<0.05). The determination coefficient of R2 was 0.8945 and about 10.55% of the variation could not be explained by this model.
ACKNOWLEDGMENTS
The authors would like to extend their sincere appreciation to MOHE and Universiti Teknologi Malaysia for HICOE grant no R.J130000.7851.4J386..
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