Influence of immobilization parameters on endopolygalacturonase productivity by hybrid Aspergillus sp. HL entrapped in calcium alginate

Biochemical Engineering Journal 32 (2006) 43–48

Influence of immobilization parameters on endopolygalacturonase productivity by hybrid Aspergillus sp. HL entrapped in calcium alginate N. Reyes a,∗ , I. Rivas-Ruiz a , R. Dom´ınguez-Espinosa b , S. Sol´ıs a a

Divisi´on de Estudios de Posgrado e Investigaci´on, Instituto Tecnol´ogico de M´erida, Av. Tecnol´ogico s/n, Apdo. Postal 9-11 Chuburn´a, C.P. 97118 M´erida, Yuc., Mexico b Universidad Aut´ onoma de Yucat´an, Av. Ju´arez No. 421 Cd. Industrial C.P. 97288, Apdo. Postal 26 Suc. Las Fuentes M´erida, Yuc., Mexico Received 4 March 2006; received in revised form 31 August 2006; accepted 2 September 2006

Abstract Culture cells of the hybrid Aspergillus HL were immobilized in calcium alginate beads. Different spore concentrations and bead sizes produced surfaces with different mechanical resistances. Bead size of 2.2 mm made with 2% alginate and inoculum of 4 × 107 spores/ml gel was determined to be adequate to reduce bead fracture by 32%. The production of endopolygalacturonases (endoPG) was strongly dependent on the temperature alone or in interaction (P < 0.05) with others factors, particularly with the pH of the medium. High temperature (39 ◦ C) increased the production of endoPG 10-fold in comparison with low temperature (27 ◦ C) without modifying the growth of HL (2.2 g/l). Acid pH (2.8) facilitated the production of the enzyme (235 U/(l h−1 )), achieving a reduction in growth of 71% in relation to the maximum obtained (3.2 g/l) at pH 4.5. The best endoPG efficiency was found at 39 ◦ C, pH 2.8 using 5 ml of bead volume. Under these conditions, the immobilized cells doubled the production of endoPG in comparison with free cells, and minimum support cell detachment. © 2006 Elsevier B.V. All rights reserved. Keywords: Biosynthesis; Endopolygalacturonase; Enzyme production; Filamentous fungy; Immobilized cells; Mechanical strength

1. Introduction Endopolygalacturonases (endoPG) are an extracellular enzyme group that randomly break ␣-(1–4) glucosidic bonds, reducing pectin’s viscosity, thus they are a fundamental element to degrade pectin from juice and to clarify wine [1]. Cell immobilization is a way of producing pectinases at large scales and low bioprocessing costs that has received only limited attention. Entrapping calcium alginate can improve yields. The immobilization of Aspergillus niger 26 fungus in calcium alginate beads, provides a microenvironment favorable to microorganism growth that tripled polymethylgalacturonase production [2,3]. This method is done under mild conditions, resulting in low loss of cellular viability, and the components used are nontoxic and stable at acid pH’s [4]. However, during immobilizing by entrapment some parameters such as gel concentration, bead diameter, temperature, pH, etc. can affect the catalytic efficiency ∗

Corresponding author. Tel.: +52 999 9448479; fax: +52 999 9448479. E-mail address: [email protected] (N. Reyes).

1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.09.001

[4,5] and can also change the system’s physical and mechanical properties [6,7]. With the objective of developing an immobilization system for the interspecific hybrid Aspergillus HL [8], the effect of different gel concentrations during bead formation, bead diameter, spore concentration, bead volume in the fermentation, pH, temperature and fermentation time on endoPG yield and bead mechanical resistance were studied. 2. Materials and methods 2.1. Microorganism The interspecific hybrid Aspergillus HL was chosen for its ability to produce extracellular endoPG in acid media [8]. 2.2. Inoculum preparation A fungal inoculum was prepared by growing the strain in standard potato dextrose agar (PDA) for 6 days at 37 ◦ C. Spores

44

N. Reyes et al. / Biochemical Engineering Journal 32 (2006) 43–48

were then harvested by addition of Tween 80 at 0.01% (v/v), and diluted in sterile distilled water to achieve 2 × 107 and 4 × 108 spores/ml.

buffer at pH 5, 30 ◦ C and 200 rpm for 48 h. Free biomass was recovered by filtration and weighed after drying (60 ◦ C for 48 h). 2.6. EndoPG activity

2.3. Immobilization Calcium alginate beads were prepared by needle dropping 2% and 3% (w/v) aqueous solutions of sodium alginate (Sigma) and HL spores (2 × 106 and 4 × 107 spores/ml gel) into 0.05 M CaCl2 (Baker) solution under stirring (15 min). Bead size was controlled by needle diameter (Table 1) and mean bead size (1.78 and 2.2 mm) was characterized by direct measurement using an optic microscope based on the method of Sriamornsak [9]. 2.4. Production of endoPG with immobilized cells Five and 15 ml beads were added to 100 ml of sterile medium with 1% (w/v) commercial citric pectin (57% degree of esterification, Sigma), 0.01% (w/v) CaCl2 and 0.2% (w/v) K2 HPO4 , KH2 PO4 , and (NH4 )2 SO4 . The medium pH was adjusted to 3 and 5. Cultures were incubated at 29 and 37 ◦ C for 48 and 72 h at 200 rpm (Table 1). Alginate beads were recovered from the fermentation by filtration and rinsed with sterile 0.05 M CaCl2 , and afterwards were again incubated in fresh medium, repeating this procedure for two consecutive cycles. The free cell filtrates from each cycle were used to quantify endoPG activity. 2.5. Biomass measurements Entrapped biomass was measured by gently releasing it from the immobilized state by incubation in 0.2 M sodium citrate

EndoPG activity was determined by viscosity reduction of a pectin solution to 1% (w/v) at pH 4.2, and measured with an Ostwald viscometer, previously reported by Larios et al. [11]. EndoPG activity unit was defined as the amount of enzyme that produced a change of 0.1 per second in the relative viscosity at 30 ◦ C. 2.7. Bead mechanical resistance assessment Bead mechanical resistance assessment was performed according to a modified method described by Wang [10]. Fifty, alginate beads were placed on a shaking flask containing 62.5 ml of Na Cl (0.85%) with five glass beads and incubated at 35 ◦ C under shaking at 250 rpm for 6 h. After this, the flask contents were filtered by a stain steel sieve and the gel beads were observed under the microscope at 10× power. Mechanical resistance was expressed as fracture frequency (%) = [N/Nt ] × 100, where N is the number of fractured beads and Nt is the total number of gel beads. 2.8. Experimental design and statistical analysis Screening of relevant fermentation parameters for the immo7−3 bilized system was done using a 2IV factorial design in four blocks described by Montgomery [12], simultaneously evaluating seven factors (Table 1). Factors and levels were selected according to preliminary studies [8] and literature reports [2,13].

Table 1 7−3 Treatment combinations of 2IV factorial design Block

Temperature (◦ C)

Bead diameter (mm)

Alginate concentration (%)

Spore content (spores/ml gel)

Volume of beads (ml)

pH

Fermentation time (h)

T1 T2 T3 T4

29 29 29 29

1.78 1.78 1.78 1.78

2 3 2 3

2 × 106 2 × 106 4 × 107 4 × 107

5 15 5 15

3 5 5 3

48 72 72 48

T1 T2 T3 T4

37 37 37 37

1.78 1.78 1.78 1.78

2 3 2 3

2 × 106 2 × 106 4 × 107 4 × 107

15 5 15 5

3 5 5 3

72 48 48 72

T1 T2 T3 T4

29 29 29 29

2.20 2.20 2.20 2.20

2 3 2 3

2 × 106 2 × 106 4 × 107 4 × 107

15 5 15 5

5 3 3 5

48 72 72 48

IV T1 T2 T3 T4

37 37 37 37

2.20 2.20 2.20 2.20

2 3 2 3

2 × 106 2 × 106 4 × 107 4 × 107

5 15 5 15

5 3 3 5

72 48 48 72

I

II

III

N. Reyes et al. / Biochemical Engineering Journal 32 (2006) 43–48

Each block was replicated twice and the results analyzed by analysis of variance (ANOVA) at 95% confidence using the Statgraphics Plus ver. 4.1 program. Means were compared by least significant difference [12]. 3. Results and discussion 3.1. Microorganism immobilization Volumetric production of endoPG by the hybrid HL cells was strongly dependent on temperature. The enzyme production at 37 ◦ C was significantly (P < 0.05) higher than at 29 ◦ C (Fig. 1A–E). Similar results were obtained with free cells of HL on lemon rind [8], thus the immobilization did not modify the temperature at which the greatest quantity of enzyme was produced. At 37 ◦ C greater spore loads within the beads (4 × 107 spores/ ml gel) produced approximately twice the volumetric and specific endoPG production than with 2 × 106 spores/ml gel (Fig. 1A). A similar phenomenon had been observed with immobilized A. niger 26 [2], where the increase of spore loads in calcium alginate beads up to 1 × 109 spores/ml gel resulted in a direct increase of polymethylgalacturonase (PMG), as a outcome of rapid growth on the surface of the beads, facilitating contact with the medium and excretion of products. A similar rise in productivity as an effect of an increment of bead densities was observed during the production of ␣-amylase [14] and ethanol [15] with immobilized cells of Bacillus and S. cerevisiae, respectively. Enzyme production increased after 48 h fermentation, and remained unchanged at 72 h (Fig. 1B), whereas with free cells the highest yield was obtained after 72 h [8]. Some results have shown that the difference in the speed of PMG production with Aspergillus cells depended on the growth phase of the culture, being greater in the exponential phase for free cells and in the linear phase for immobilized cells [16]. Therefore, it is possi-

45

ble that the production of endoPG in immobilized HL occurred logarithmically. Greater yields of endoPG were observed with 1.78 mm diameter beads (Fig. 1C). These results were consistent with those indicating that smaller diameters increase the superficial area of oxygen transfer and minimize the problems of mass exchange, thereby facilitating the production of metabolites. The sizes of 0.5 mm with Kluyveromyces fragilis NRRL-11109 increased the activity of ␤-galactosidase [17] and S. cerevisiae immobilized in beads of ≤2 mm diameter improved ethanol production over 5 mm diameter beads [15]. Although, it has been reported that calcium alginate beads with smaller bead diameters (0.25 mm) are up to 75% less susceptible to mechanical shear than those twice the size [6], we found that for the fungus HL size itself was not as significant (P > 0.05) in mechanical bead resistance as the interaction of bead diameter to spore concentration ratio (Fig. 2). At 2 × 106 spores/ml gel, fracture was similar for both diameters (73% for 1.78 mm; 71% for 2.2 mm), while at 4 × 107 spores/ml gel the 1.78 mm beads had a higher fracture index (85%) than the 2.2 mm beads (53%). The behavior of the hybrid HL at the high cellular density formed a weak gel matrix with low mechanical resistance, whereas by increasing the amount of gel, the denser gel mass on the surface improved abrasion resistance [18]. Spatial distribution of alginate within the bead could also favor bead mechanical resistance; since had been observed in 4 mm diameter beads, the edge of the alginate matrix was twice more dense than at its center [19] where most of the fungal biomass was found. Variation in pH also, produced different responses in endoPG productivity. At 37 ◦ C approximately 250 U/(l h−1 ) were excreted, independent of pH (Fig. 1D), whereas entrapped biomass at pH 5 was higher (306 mg) than at pH 3 (207 mg). The lower growth at pH 3 also led to minimum support cell detachment, with 48 mg at the latter pH versus 119 mg at the former. This means that the acidic stress of the culture medium affected

Fig. 1. Effect of temperature (29 and 37 ◦ C) and its interaction with other fermentation parameters on endoPG productivity by immobilized cells of Aspergillus HL: (A) spore concentration (, 2 × 106 /ml gel; , 2 × 107 /ml gel), (B) fermentation time (, 48 h; , 72 h), (C) bead diameter (, 1.78 mm; , 2.20 mm), (D) pH (, 3; , 5) and (E) volume of beads (, 5 ml; , 15 ml). Different letters above the bars (a, b, c, and d) indicate a significant difference (P < 0.05) of means from eight observations.

46

N. Reyes et al. / Biochemical Engineering Journal 32 (2006) 43–48 Table 2 Volumetric and specific endoPG productivity by immobilized Aspergillus HL Blocka

endoPG productivityb Volumetric (U/(100 ml h−1 ))

Specific (U/(mg DW h−1 ))

I T1 T2 T3 T4

7.87 25.90 8.91 3.36

± ± ± ±

2.48 3.40 1.27 1.43

0.055 0.054 0.028 0.014

± ± ± ±

0.020 0.006 0.003 0.004

T1 T2 T3 T4

19.26 17.87 42.44 40.67

± ± ± ±

0.86 1.12 0.61 2.69

0.081 0.090 0.114 0.210

± ± ± ±

0.014 0.016 0.087 0.183

T1 T2 T3 T4

12.37 4.58 3.51 6.06

± ± ± ±

5.26 2.14 0.04 0.22

0.046 0.020 0.011 0.028

± ± ± ±

0.016 0.005 0.004 0.014

IV T1 T2 T3 T4

12.28 13.74 29.46 24.27

± ± ± ±

0.90 3.33 5.81 0.88

0.060 0.090 0.254 0.068

± ± ± ±

0.001 0.030 0.109 0.023

II

III

Fig. 2. Interaction between bead diameter and spore concentration on bead fracture. Different letters above the bars (a, b, c) indicate a significant difference (P < 0.05) of means from eight observations.

HL growth and induced greater production of the enzymes. The inductor effect on endoPG synthesis of pH’s 2.5–3.0 has been reported in submerged fermentation with free Aspergillus sp. CH-Y-1043 [20]. The incubation of 5 and 15 ml of beads showed the same productivity of endoPG at 37 ◦ C (P > 0.05) (Fig. 1E). However, the concentration of biomass on completion of the culture was 16.5 and 7.68 mg biomass/ml bead, respectively. These results show that an increase in the quantity of beads reduced growth due to the competition for nutrients and did not favor an increase in enzyme production. This behavior coincides with A. niger 26, in which growth and PMG production decreased with the use of a greater quantity of spores [2]. Based on the above results the best conditions for HL immobilization with the studied variables corresponded to treatment 4 (T4) of block II (Table 2): 2% sodium alginate, 1.78 mm bead diameter, 4 × 107 spores/ml gel concentration, 5 ml of beads, pH 3.0, 37 ◦ C and 48 h. These conditions provided high volumetric and specific endoPG productivity. 3.2. Effect of temperature and pH In order to obtain a system exhibiting low biomass growth in the bead with minimal cell detachment, initial fermentation temperature and pH were studied in greater detail using a range of 27–39 ◦ C and 2.5–5, respectively. An increase in the incubation temperature of the beads did not affect the growth of the microorganism in the bead and the total biomass (entrapped and free) was approximately 2.2 ± 0.1 g/l in most fermentations (Fig. 3A). However, the ratio of entrapped and free mycelium remained higher at elevated temperatures. Retained and separated HL biomass was three times lower than was reported for A. niger 26 cells, producers of pectinases entrapped in calcium alginate at 30 ◦ C [2], which corroborates the reduced growth of HL under the conditions evaluated. EndoPG productivity (Fig. 3B) was 38.5 ± 1.4 U/(l h−1 ) in the range from 27 to 33 ◦ C, and subsequently increased 5, 4

DW, dry weight. a Within each block T1, T2, T3 and T4 represent different treatments according to the experimental design (Table 1). b Average endoPG obtained after two consecutive cycles. Results are triplicates of two experiments.

and 10 times for 35, 37 and 39 ◦ C, respectively. The increase in endoPG production was not associated with changes in the growth of microorganisms, or with variations in pH during the culture, thereby confirming that the high temperatures favored the production of these enzymes. While in other studies with HL free cells, highest production of EndoPG was obtained at 37 ◦ C [8]. Although there are few reports about the effect of the temperature on immobilized cells, it has been observed that Immobilization can modify the optimal temperature in comparison with free cells. In Lactobacillus amylovorus a raise of 3 ◦ C improved the concentration of lactic acid [21] and in Candida krusei an increase of 15 ◦ C was necessary to elevate the production of phytases in comparison to free cells [22]. As shown in Fig. 3C, mycelium attachment was also improved by the increase of the initial pH of the fermentation medium; at pH 2.5 the growth represented only 5% in comparison with the maximum obtained at pH 4.5 (3.2 g/l). However, with the pH increase the bead diameter also increased; at pH 4.5 the diameter increased to 3.7 mm, with a minimal separation of cells. In contrast, higher endoPG productivity was observed at acid pH’s between 2.8 and 3.5 but at higher pH’s it dropped drastically (Fig. 3D). These results demonstrate that acidic stress favors the synthesis and/or secretion of endoPG, while growth is seriously affected. In immobilized cells were observed an inductor effect at a pH of 5.5–6 [3], but not at lower pH’s similar to the results of this study.

N. Reyes et al. / Biochemical Engineering Journal 32 (2006) 43–48

47

Fig. 3. Effect of incubation temperature and pH on cell attachment (A and C) and volumetric endoPG productivity (B and D). The error bars in the figures indicate the standard deviations of triplicates from three independent replicates.

These results suggest the convenience of using a pH between 2.8 and 3.5 and a temperature of 39 ◦ C to obtain a high production of endoPG with a low growth of HL and a minimal separation of cells. 3.3. Comparison of free versus immobilized cell A comparison of endoPG production from free and immobilized cells of HL showed important differences (Fig. 4). In the free cells a delay in the production of endoPG was observed in comparison with the entrapped cells, since an important increase

was observed after 48 h and it was possible to produce 13 U/ml at 72 h of fermentation. On the other hand, in immobilized cells, activity occurred with 24 h of culture, reaching 21 U/ml in 72 h, which is almost double in comparison to the free system. This behavior can be explained by previous works [13,23] that showed that a strong catabolic repression appears on the parental strain (Aspergillus sp.) by presence of pectin hydrolysis products in the culture media, so as these can not easy reach the immobilized cell in within the gel, it is likely, that catabolic repression is reduced on immobilized cells. Other works [24,25] had also suggested that the protection of mechanical shear on submerged immobilized fungal cultures could trigger an enhanced metabolic performance in comparison to that presented by the free cell system. 4. Conclusions

Fig. 4. Comparison of endoPG production in free and immobilized cells of Aspergillus HL at 39 ◦ C and pH 2.8.

During the immobilization and culture of Aspergillus HL, the most important factor in the production of endoPG was temperature, exercising an influence by itself or in interaction with other variables, especially pH. High temperatures and low pH made it possible to decrease the growth and the separation of cells from the support and doubled the activity of endoPG in comparison with free cells. Bead size as well as spore/gel concentration ratio, are important variables that affect beads’ mechanical resistance. The potential use of this immobilized system in semi-continuous or continuous cultures could be of considerable economic interest in the production of endoPG.

48

N. Reyes et al. / Biochemical Engineering Journal 32 (2006) 43–48

Acknowledgements This work was financed by Consejo Nacional de Ciencia y Tecnolog´ıa (Conacyt, Mexico), and Consejo del Sistema Nacional de Educaci´on Tecnol´ogica (COSNET). References [1] D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari, Applications of pectinases in the commercial sector: a review, Bioresour. Technol. 77 (2001) 215–227. [2] M. Angelova, P. Sheremetska, M. Lekov, Enhanced polymethylgalacturonase production from Aspergillus niger 26 by calcium alginate immobilization, Process Biochem. 33 (1998) 299–305. [3] S. Pashova, L. Slokoska, P. Sheremetska, E. Krumova, L. Vasileva, M. Angelova, Physiological aspects of immobilized Aspergillus niger cells producing polymethylgalacturonase, Process Biochem. 35 (1999) 15–19. [4] A. Groboillot, D.K. Boadi, D. Poncelet, R.J. Neufeld, Immobilization of cells for application in the food industry, Crit. Rev. Biotechnol. 14 (1994) 75–104. [5] J. Guy-Alain, L. Coquet, S. Vilain, T. Jouenne, Immobilized-cell physiology: current data and the potentialities of proteomics, Enzyme Microb. Technol. 31 (2002) 201–212. [6] D. Poncelet, R.J. Neufeld, Shear breakage of nylon membrane microcapsules in a turbine reactor, Biotechnol. Bioeng. 33 (1989) 95–103. [7] M.G. Quintana, H. Dalton, Production of toluene cis-diol by immobilized Pseudomonas putida UV4 cells in barium alginate beads, Enzyme Microb. Technol. 22 (1998) 713–720. [8] S. Sol´ıs, M.E. Flores, C. Huitr´on, Improvement of pectinase production by interspecific hybrids of Aspergillus strains, Lett. Appl. Microbiol. 24 (1997) 77–81. [9] P. Sriamornsak, Preliminary investigation of some polysaccharides as a carrier for cell entrapment, Eur. J. Pharm. Biopharm. 46 (1998) 233–236. [10] Y.J. Wang, Development of new polycations for cell encapsulation with alginate, Mater. Sci. Eng. C 13 (2000) 59–63. [11] G. Larios, J.M. Garc´ıa, C. Huitr´on, Endopolygalacturonase production from untreated lemon peel by Aspergillus sp. CH-Y-1043, Biotechnol. Lett. 11 (1989) 729–734. [12] D.C. Montgomery, Design and Analysis of Experiments, 2nd ed., John Wiley and Sons Inc., New York, 2003.

[13] G. Aguilar, C. Huitron, Stimulation of the production of extracellular pectinolytic activities of Aspergillus sp. by galacturonic acid and glucose addition, Enzyme Microb. Technol. 9 (1987) 690–696. [14] R. Jamuna, S.V. Ramakrishna, Continuous synthesis of thermostable ␣amylase by Bacillus cells immoblilized in calcium alginate, Enzyme Microb. Technol. 14 (1992) 36–41. [15] J. Gutenwik, B. Nilsson, A. Axelsson, Mass transfer effects on the reaction rate for heterogeneously distributed immobilized yeast cells, Biotechnol. Bioeng. 79 (2002) 664–673. [16] S. Pashova, L. Slokoska, E. Krumova, M. Angelova, Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26, Enzyme Microb. Technol. 24 (1999) 535–540. [17] E. Castillo, D. Ram´ırez, L. Casas, A. L´opez-Mungu´ıa, A two-phase method to produce gel beads, Appl. Biochem. Biotechnol. 34–35 (1992) 477– 486. [18] G. Orive, R.M. Hern´andez, A.R. Gasc´on, M. Igartua, J.L. Pedraz, Development and optimisation of alginate–PMCG–alginate microcapsules for cell immobilization, Int. J. Pharm. 259 (2003) 57–68. [19] M. Heinemann, H. Meinberg, J. B¨uchs, H.J. Kob, B. AnsorageSchumacher, Method for quantitative determination of spatial polymer distribution in alginate beads using Raman spectroscopy, Appl. Spectrosc. 59 (2005) 280–285. [20] G. Aguilar, B.A. Trejo, J.M. Garc´ıa, C. Huitr´on, Influence of pH on endoand exo-pectinase production by Aspergillus sp. CH-Y-1043, Can. J. Microbiol. 37 (1991) 912–917. [21] J. Yan, R. Bajpai, E. Iannotti, M. Popovic, R. Mueller, Lactic acid fermentation from enzyme-thinned starch with immobilized Lactobacillus amylovorus, Chem. Biochem. Eng. Quart. 15 (2001) 59–63. [22] C.S. Quan, S.D. Fan, Y. Ohta, Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates, Appl. Microb. Biotechnol. 62 (2003) 41–47. [23] G. Aguilar, C. Huitron, Constitutive exo-pectinase produced by Aspergillus sp. CH-Y-1043 on different carbon sources, Biotechnol. Lett. 12 (1990) 655–660. [24] R. Dom´ınguez-Espinosa, C. Webb, Production of monascus pigments in wheat based substrates, World J. Microbiol. Biotechnol. 19 (2003) 329–336. [25] L.M. Harvey, B. McNeil, Liquid fermentation systems and product recovery of Aspergillus, in: J.E. Smith (Ed.), Biotechnology Handbooks, vol. 7, Plenum Press, New York, NY, 1994, pp. 141–176.