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Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling
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Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling Maria Papagianni a,∗ , Emmanuel M. Papamichael b a b
Department of Hygiene and Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece Department of Chemistry, University of Ioannina, Greece
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 19 September 2014 Accepted 22 September 2014 Available online xxx Keywords: Pediococcus pentosaceus Pediocin Fed-batch culture Modeling Sucrose
a b s t r a c t Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The antimicrobial peptide SM-1 is a 5.37 kDa bacteriocin produced by the food-grade lactic acid bacterium (LAB) Pediococcus pentosaceus Mees 1934 in fermentation [1]. Due to the presence of the characteristic consensus motif – YGNGV – in the N-terminus of its molecule (the “pediocin motif”), the antimicrobial peptide was identified as a pediocin or a class IIa bacteriocin. It is a heat and cold-stable peptide with inhibitory activity against several Grampositive food spoilage and food-born pathogens, including Listeria monocytogenes, which in general exhibits the characteristics of a potential biopreservative. Results of our earlier work [1] showed that the production of pediocin SM-1 in batch culture under, the previously identified as optimal, semi-aerobic (60% dissolved oxygen saturation) conditions, is characterized by primary metabolite kinetics. Commercial application of the bacteriocin however, would require increased productivities. This could result by the use of the appropriate complex media, well-controlled fermentation parameters such as
∗ Corresponding author. Tel.: +30 2310 999804; fax: +30 2310 999829. E-mail address: [email protected] (M. Papagianni).
temperature and pH, as well as growth of the producer microorganism using a suitable culture mode, e.g. batch or fed-batch culture. Research with other bacteriocins from LAB have shown that use of complex media and application of controlled culture conditions lead to increased process productivities [2–4]. Also, regulation of the source of carbon affects cell growth and product formation [5–7]. Increased initial sucrose or glucose concentrations were found to repress catabolism in Lactococcus lactis and inhibit nisin biosynthesis in batch culture [8,9], while controlled concentrations at the desirable levels through fed-batch strategies can eliminate substrate inhibitions [9]. Reports on the production of bacteriocins in fed-batch culture are rather limited compared to the significant volume of reports on bacteriocins from LAB and similarly limited are the proposed models that describe the respective processes. Regarding pediocins, most reports are dealing with bacteriocins from Pediococcus acidilactici strains [10] while fed-batch culture results are reported in the works of Guerra and co-workers with P. acidilactici NRRL B-5627 [11–13]. Using by-products of the food industry as substrates, e.g. whey or mussel processing wastes, the authors developed models that can be used to design feeding strategies for enhancing and controlling fed-batch pediocin production by P. acidilactici. In the present study, pediocin SM-1 production was investigated for
http://dx.doi.org/10.1016/j.procbio.2014.09.023 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023
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the first time in fed-batch cultures of P. pentosaceus using complex media and sucrose as the main carbon-source. A mathematical model was developed to describe the fermentation process. 2. Materials and methods 2.1. Fermentations P. pentosaceus Mees 1934, a pediocin producing strain, was grown in complex medium (CM) containing 30 g/l sucrose, 10 g/l yeast extract, 10 g/l soy peptone, 10 g/l KH2 PO4 , 2 g/l NaCl and 0.2 g/l MgSO4 ·7H2 O. Following growth until the mid logarithmic phase (OD600 1.4) the inoculum culture was transferred (2%, v/v) in a stirred tank bioreactor (BIOFLO 110, New Brunswick Scientific) with a working volume of 2.0 l. The agitation system of the bioreactor consisted of two 6-bladed Rushton type impellers (52 mm) operated at 150 rpm. The reactor was also equipped with baffles. Process temperature was maintained at 30 ◦ C. The dissolved oxygen tension (DOT) was maintained at 60% of saturation by sparging the reactor with a mixture of atmospheric air and nitrogen, adjusted by two mass flow controllers and was kept constant by feedback regulation of the ratio. The pH was controlled at 6.8 throughout fermentation by automatic addition of a 5 M NaOH solution. Fed-batch cultures were initiated as batch cultures with 10 g/l initial sucrose concentration. A concentrated sucrose solution (400 g/l) was added at various constant feeding rates (5, 7, 9, and 10 g/l/h) and feeding started when residual sucrose concentration was reduced to 5 g/l (off-line measurements) (∼the 5th hour of fermentation). Samples were withdrawn aseptically at regular intervals and analyzed. Experiments were carried out in triplicate and mean values and standard deviations are presented here.
third term representing maintenance requirements can be ignored since experimentally determined maintenance coefficients are very low and at the same time may introduce a considerable degree of error. Therefore such a term was not included in Eq. (2). The rate of lactate production can be expressed by the Luedeking–Piret model [18] that introduces the type of product as follows: dx dL =˛ + ˇx dt dt
(3)
where, ˛ is the constant for growth-associated kinetics of lactate production in g L/g CDW and ˇ is the non-growth associated constant in g L/g CDW/h. Pediocin is produced mainly during the exponential phase of growth and its titer decreases after biomass has reached its maximum. Pediocin production can be described by the following: dP dx = qp dt dt
for
dx dP ≥ 0 or = −Rin x4 dt dt
for
dx ≤0 dt
(4)
where, qp is the specific pediocin production rate in AU/g CDW/h and Rin is the specific rate of pediocin inactivation in AU/g CDW/h. 2.3.2. Rate equations for fed-batch culture These are derived from the rate equations of the batch culture and the introduction of terms that describe the change in volume V, the feeding rate rfeed and the concentration of sucrose Sin in the feeding solution. The respective equations for growth, sucrose utilization, lactate production and pediocin production are the following:
d(Vx) X = max 1 − Xmax dt
− Kx Vx
(5)
where, V is the broth volume. 2.2. Analytical methods − Biomass concentration was monitored spectrophotometrically by measuring the optical density at 600 nm and correlating the optical density (OD) with cell dry weight (CDW). One unit of OD at 600 nm was equivalent to 0.25 g/l CDW. Bacteriocin activity was assessed in cell-free filtrates of culture broths as described earlier [14] using P. acidilactici ATCC 25740 as the indicator microorganism. To eliminate the antimicrobial effect of lactate, the pH of samples was adjusted to 6.0 using 1 M NaOH. Lactate concentration was determined using the EnzyPlus D/L Lactic Acid Kit by Diffchamb AB (Sweden). Sucrose concentration was determined according to the Roe’s method [15]. 2.3. Process modeling 2.3.1. Rate equations for batch culture Cell growth of P. pentosaceus Mees 1934 was described adequately by the logistic model of Mercier et al. [16] modified to include a term for cell death according to Lv et al. [17] as follows:
dx X = max 1 − Xmax dt
− Kx x
1 dL 1 dx dS = + YL/S dt Yx/S dt dt
(6)
where rfeed is the feeding rate in l/h and Sin is the sucrose concentration in the feeding solution in g/l. d(VL) d(Vx) =˛ + ˇVx dt dt
(7)
d(VP) d(Vx) = qp − Rin Vx dt dt
(8)
The culture volume evolution in fed-batch culture is given by dV = rfeed = rin − rout dt
(9)
where, rin and rout are the inflow and outflow rates, respectively. Non-linear regression analysis and modeling of fermentation data were carried out on Microsoft Excel 2010 [19]. The Runge–Kutta method was used for integrating the model equations while the Powell method was applied to obtain the minimum total residual sum of squares for achieving the best fit of the experimental data.
(1) 3. Results and discussion
where, x is the CDW in g/l, xmax is the maximum CDW in g/l, max is the maximum specific growth rate in h−1 , and Kx is the specific rate of cell death in h−1 . The rate of sucrose utilization was described by the equation: −
1 d(Vx) d(VS) 1 d(VL) = rfeed Sin − + YL/S dt Yx/S dt dt
(2)
where, S is the residual sucrose concentration in g/l, L is the concentration of the produced lactate in g/l, YL/S is the yield of lactate on sucrose consumed in g L/g S, and Yx/S is the yield of biomass on sucrose consumed in g CDW/g S. According to Mercier et al. [16] a
Fig. 1 shows the time courses of biomass, residual sucrose, lactate and pediocin concentrations (as points) of a standard batch fermentation at 150 rpm, 60% DOT, pH 6.8 and 30 g/l initial sucrose concentration. Sucrose was consumed within 12 h of fermentation. Biomass reached 3.5 g/l CDW, while pediocin production followed cell growth and reached a maximum of 67 AU/ml within 14 h of fermentation. Beyond that time-point production decreased while biomass production rates were almost zero. Pediocin production showed primary metabolite kinetics as has been observed earlier with the same microorganism during growth on glucose and under
Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023
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Pediocin
Biomass (g/l)
3
60
2,5
50
2
40
1,5
30
1
20
0,5
10
0
Sucrose
140
Lactate
10
Pediocin
120
8
100
6
80 60
4
40 2
20
0 0
2
4
6
8
10
12
14
16
18
0
Time (hours)
60% DOT [1]. Bacteriocins are generally considered to be primary metabolites, and as such, they are formed at a rate depending only on the growth rate of the producer organism [18]. Deviations however exist, e.g. as mixed kinetics, and seem to appear under stress conditions as reported in the works of Guerra at al. [20] with nisin and pediocin, Neysens et al. [21] with amylovorin, and Papagianni and Papamichael [7] with weissellin A. Examples of the strict primary metabolite production model include the cases of nisin [8,17], lactocin [22], and leucocin LA54A [23]. However, studies have considered pediocin AcH [24] and propionicin [25] to be secondary metabolites. Experimental data (Table 1) were used for model parameter values estimation using Eqs. (1)–(4) as shown in Section 2. The obtained values were plotted against time and are presented in Fig. 1 as lines along the experimentally obtained data points. It can be observed from the figure that model simulation for batch culture was very good for the four fermentation parameters tested. 160
12
140
Sucrose Lactate
120
Pediocin 8
100
6
80 60
4
40 2
20
0
Sucrose, Lactate (g/l), Pediocin (AU/ml)
Biomass 10
0 0
2
4
6
8
10
0 0
Fig. 1. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations in batch culture of P. pentosaceus Mees 1934 at 150 rpm, 60% DOT, pH 6.8 and 30 g/l initial sucrose concentration. Experimental data are denoted as points while the lines represent model-derived values.
Biomass (g/l)
160
Biomass
Sucrose, Lactate (g/l), Pediocin (AU/ml)
70
Lactate
Biomass (g/l)
Sucrose
3,5
12
80
Biomass
Sucrose, Lactate (g/l), Pediocin (AU/ml)
4
3
12
14
16
18
Time (hours) Fig. 2. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 5 g/l/h. The arrows indicate the period of feeding.
2
4
6
8
10
12
14
16
18
Time (hours) Fig. 3. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 7 g/l/h. The arrows indicate the period of feeding.
Fig. 2 shows the time courses of biomass, residual sucrose, lactate and pediocin concentrations (as points) in the fed-batch fermentation with 10 g/l initial sucrose concentration and 5 g/l/h feeding rate of a 400 g/l sucrose solution. Feeding was carried out between the 4th and 12th hour of fermentation. Residual sucrose concentration remained at very low levels and no sucrose was detected beyond the 10th hour of fermentation. Biomass reached the maximum of 3.9 g/l CDW at 14 h (3.5 g/l in the batch culture) while lactate 23 g/l (16 g/l in the batch culture). Pediocin production rate was increased during feeding and its overall production reached 102 AU/ml, a significantly higher titer compared to the obtained in the standard batch fermentation run. The maximum titer of pediocin was obtained in the fed-batch culture of 7 g/l/h feeding rate (Fig. 3) and it was 145 AU/ml. Biomass reached 4.1 g/l and lactate 25 g/l. By increasing the feeding rate to 9 (not shown) and 10 g/l/h (Fig. 4), pediocin production decreased and lactate production increased, while maximum biomass levels were 4 and 3.8 g/l CDW, respectively. An increase in residual sucrose concentration was monitored in the last two runs. It is obvious that while biomass production is rather unaffected, pediocin biosynthesis is affected by the concentration of sucrose in the fermentation broth and an optimum area for sucrose concentration levels exist beyond which production decreases. The effect is also obvious on the kinetic parameter specific production rate qp (AU/g CDW/h) as shown in Table 1, where the fed-batch culture with 7 g/l/h feeding rate appears to give the highest values (max. 6.78 AU/g/h). The lines in the plots of Figs. 2–4 represent model derived values using Eqs. (5)–(8). Obviously, the model of Eq. (8) that describes growth-associated pediocin production fits very well the experimental data in all fed-batch experiments. Therefore, as a product of primary metabolism, pediocin biosynthesis is expected to be influenced by the type and concentration of the carbon source. Research carried out with L. lactis grown in media containing glucose has shown that the concentration of glucose regulates its uptake and improving its uptake could result in nisin yield improvements [9]. Maintaining elevated glucose levels in the medium (>10 g/l) in glucostat fed-batch fermentations resulted in decreased nisin yields. Similarly, again with L. lactis and sucrose as the carbon source, elevated concentrations in both batch and fed-batch fermentations resulted in decreased nisin yields in the works of De Vuyst and
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Table 1 Estimated kinetic parameters in batch and fed-batch cultures of P. pentosaceus Mees 1934: maximum specific growth rate max , maximum biomass production rate Xmax , specific rate of cell death Kx , yield of lactate on sucrose YL/S , constant for growth-associated kinetics of lactate production ˛, non-growth associated constant ˇ, specific production rate of pediocin qp and specific rate of pediocin inactivation Rin . Type of culture
Kinetic parameters max (h−1 )
Xmax (g/l)
Kx (h−1 )
YL/S (g L/g)
˛ (g L/g DCW)
ˇ (g L/g DCW/h)
qp (AU/g DCW/h)
Rin (AU/g DCW/h)
Batch
1.12 ± 0.05
3.5 ± 0.12
0.053 ± 0.002
0.53 ± 0.02
6.01 ± 0.6
0.24 ± 0.01
1.97 ± 0.08
2.6 ± 0.11
Fed-batch 5 g/l/h 7 g/l/h 9 g/l/h 10 g/l/h
1.35 1.37 1.36 1.32
0.05 0.04 0.05 0.03
3.9 4.1 4.0 3.8
± ± ± ±
0.14 0.12 0.16 0.11
0.082 0.054 0.063 0.080
± ± ± ±
0.001 0.002 0.002 0.002
0.73 0.78 0.91 0.92
Sucrose
140
Lactate Pediocin
120
8
Biomass (g/l)
0.02 0.02 0.03 0.03
160
Biomass
10
± ± ± ±
100 6
80 60
4 40 2
20
0
Sucrose, Lactate (g/l), Pediocin (AU/ml)
12
± ± ± ±
0 0
2
4
6
8
10
12
14
16
18
Tim e (hours) Fig. 4. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 10 g/l/h. The arrows indicate the period of feeding.
Vandamme [8], James and Larry [26] and Lv et al. [17]. Working with sucrose in the present case, the results show that the productivity of pediocin is greatly influenced by the concentration of sucrose and by supplying sucrose at an increased rate (>7 g/l/h for the particular system) pediocin production is restricted while large amounts of lactate are produced. Maintaining the feeding rate of sucrose at 7 g/l/h resulted in a 119% increase of the pediocin titer compared to that of the standard batch culture. However, further research into the particular production system would include sucrostat fedbatch culture experiments and sucrose transport studies in order to determine the dynamics of the sucrose transport system in the particular microorganism and investigate the relationship between the sucrose uptake rate (sucrose regulation) and pediocin production. 4. Conclusions Our work aimed to provide information on the effects of sucrose (as the main source of carbon in a complex medium) concentration on P. pentosaceus Mees 1934 growth and pediocin production in semi-aerobic, pH-controlled, fed-batch fermentation. From the presented results it becomes clear that the feeding rate and therefore the residual sucrose concentration influenced greatly the outcome of the pediocin fermentation. The maximum titer of pediocin (145 AU/ml) was obtained in the fed-batch culture with 7 g/l/h feeding rate and was increased by 119% compared to the titer obtained in batch culture, while further increased feeding rates
4.32 4.83 4.98 5.12
± ± ± ±
0.5 0.5 0.5 0.6
0.68 0.44 0.58 0.59
± ± ± ±
0.01 0.01 0.02 0.02
3.02 4.16 3.00 2.83
± ± ± ±
0.12 0.12 0.13 0.14
2.1 1.8 1.5 1.2
± ± ± ±
0.10 0.09 0.009 0.008
(9 and 10 g/l/h) resulted in decreased pediocin yields. The specific rate of pediocin formation appears to be sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known kinetic models for batch and fed-batch cultures and growth associated production, simulated successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture. The obtained data can be exploited in further studies on process optimization and as a reference in studies with various feeding regimes, e.g. pH feedback controlled fed-batch cultures, step-increasing strategies or others. References [1] Anastasiadou S, Papagianni M, Filiousis G, Ambrosiadis I, Koidis P. Growth and metabolism of a meat isolated strain of Pediococcus pentosaceus in fermentation. Purification, characterization and properties of the produced pediocin SM1. Enzyme Microb Technol 2008;43:448–54. [2] De Vuyst L, Gallewaert R, Crabbe K. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology 1996;142:817–27. [3] Papagianni M, Papamichael EM. Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX. Bioresour Technol 2011;102:6730–4. [4] Papagianni M. Effects of dissolved oxygen and pH levels on weissellin A production by Weissella paramesenteroides DX in fermentation. Bioprocess Biosyst Eng 2012;35:1035–41. [5] Vant Hul JS, Gibbons WR. Neutralization/recovery of lactic acid from Lactococcus lactis: effects on biomass, lactic acid, and nisin production. World J Microbiol Biotechnol 1997;13:527–32. [6] Lv W, Cong W, Cai Z. Effect of sucrose on nisin production in batch and fed-batch culture by Lactococcus lactis. J Chem Technol Biotechnol 2005;80:511–4. [7] Papagianni M, Papamichael EM. Production of the antimicrobial protein weissellin A by Weissella paramesenteroides DX in batch fermentations: the type of carbohydrate used as the C-source in the substrate affects the association of production with growth. Appl Biochem Biotechnol 2012;168:1212–22. [8] De Vuyst L, Vandamme EJ. Influence of the carbon source on nisin production by Lactococcus lactis subsp. lactis batch fermentations. J Gen Microbiol 1992;138:571–8. [9] Papagianni M, Avramidis N, Filiousis G. Investigating the relationship between the specific glucose uptake rate and nisin production in aerobic batch and fed-batch glucostat cultures of Lactococcus lactis. Enzyme Microb Technol 2007;40:1557–63. [10] Papagianni M, Anastasiadou S. Pediocins: the bacteriocins of pediococci. Microb Cell Fact 2009;8:3. [11] Guerra NP, Pastrana L. Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources. Lett Appl Microbiol 2002;34:114–8. [12] Guerra NP, Bernardez PF, Agrasar AT, Macias CL, Pastrana L. Fed-batch pediocin production by Pediococcus acidilactici NRRL B5627 on whey. Biotechnol Appl Biochem 2005;42:17–23. [13] Guerra NP, Bernardez PF, Pastrana L. Modelling the stress inducing biphasic growth and pediocin production by Pediococcus acidilactici NRRL B5627 in realkalized fed-batch cultures. Biochem Eng J 2008;40:465–72. [14] Papagianni M, Avramidis N, Filiousis G, Dasiou D, Ambrosiadis I. Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor indicator microorganism. Microb Cell Fact 2006;5:30. [15] Oardini CE, Leloir LF, Ohiriboga J. The biosynthesis of sucrose. J Biol Chem 1955:214–49. [16] Mercier P, Yerushalmi L, Rouleau D, Dochain D. Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus. J Chem Technol Biotechnol 1992;55:111–21. [17] Lv W, Zhang X, Cong W. Modelling the production of nisin by Lactococcus lactis in fed-batch culture. Appl Microbiol Biotechnol 2005;68:322–6.
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[18] Luedeking R, Piret EL. A kinetic study of the lactic acid fermentation. Batch process at controlled pH. J Biochem Microbiol Technol Eng 1959;1:393–412. [19] Brown AM. A step-by-step guide to non-linear regression analysis of experimental data using a Microsoft Excel spreadsheet. Comput Methods Programs Biomed 2011;65:191–200. [20] Guerra NP, Luisa Rua M, Pastrana L. Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey. Int J Food Microbiol 2001;70:267–81. [21] Neysens P, Messens W, Gevers D, Swings J, De Vuyst L. Biphasic kinetics of growth and bacteriocin production with Lactobacillus amylovorus DCE 471 occur under stress conditions. Microbiology 2003;149:1073–82. [22] Vignolo GM, De Kairuz MN, De Ruiz AAP, Oliver G. Influence of growth conditions on the production of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL705. J Appl Bacteriol 1995;78:5–10.
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[23] Geisen R, Becker B, Holzapfel WH. Bacteriocin production of Leuconostoc carnosum, LA54A at different combinations of pH and temperature. J Ind Microbiol 1993;12:337–40. [24] Ray B. Pediocin(s) of Pediococcus acidilactici as a food biopreservative. In: Ray B, Daeschel M, editors. Food biopreservatives of microbial origin. New York: CRC Press; 1992. p. 265. [25] Paik HD, Glatz BA. Enhanced bacteriocin production by Propionicterium thoenii in fed-batch fermentation. J Food Prot 1997;60: 1529–33. [26] James LS, Larry LM. Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis. Appl Environ Microbiol 1986;51:57–64.
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Short communication
Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling Maria Papagianni a,∗ , Emmanuel M. Papamichael b a b
Department of Hygiene and Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece Department of Chemistry, University of Ioannina, Greece
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 19 September 2014 Accepted 22 September 2014 Available online xxx Keywords: Pediococcus pentosaceus Pediocin Fed-batch culture Modeling Sucrose
a b s t r a c t Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The antimicrobial peptide SM-1 is a 5.37 kDa bacteriocin produced by the food-grade lactic acid bacterium (LAB) Pediococcus pentosaceus Mees 1934 in fermentation [1]. Due to the presence of the characteristic consensus motif – YGNGV – in the N-terminus of its molecule (the “pediocin motif”), the antimicrobial peptide was identified as a pediocin or a class IIa bacteriocin. It is a heat and cold-stable peptide with inhibitory activity against several Grampositive food spoilage and food-born pathogens, including Listeria monocytogenes, which in general exhibits the characteristics of a potential biopreservative. Results of our earlier work [1] showed that the production of pediocin SM-1 in batch culture under, the previously identified as optimal, semi-aerobic (60% dissolved oxygen saturation) conditions, is characterized by primary metabolite kinetics. Commercial application of the bacteriocin however, would require increased productivities. This could result by the use of the appropriate complex media, well-controlled fermentation parameters such as
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temperature and pH, as well as growth of the producer microorganism using a suitable culture mode, e.g. batch or fed-batch culture. Research with other bacteriocins from LAB have shown that use of complex media and application of controlled culture conditions lead to increased process productivities [2–4]. Also, regulation of the source of carbon affects cell growth and product formation [5–7]. Increased initial sucrose or glucose concentrations were found to repress catabolism in Lactococcus lactis and inhibit nisin biosynthesis in batch culture [8,9], while controlled concentrations at the desirable levels through fed-batch strategies can eliminate substrate inhibitions [9]. Reports on the production of bacteriocins in fed-batch culture are rather limited compared to the significant volume of reports on bacteriocins from LAB and similarly limited are the proposed models that describe the respective processes. Regarding pediocins, most reports are dealing with bacteriocins from Pediococcus acidilactici strains [10] while fed-batch culture results are reported in the works of Guerra and co-workers with P. acidilactici NRRL B-5627 [11–13]. Using by-products of the food industry as substrates, e.g. whey or mussel processing wastes, the authors developed models that can be used to design feeding strategies for enhancing and controlling fed-batch pediocin production by P. acidilactici. In the present study, pediocin SM-1 production was investigated for
http://dx.doi.org/10.1016/j.procbio.2014.09.023 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023
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the first time in fed-batch cultures of P. pentosaceus using complex media and sucrose as the main carbon-source. A mathematical model was developed to describe the fermentation process. 2. Materials and methods 2.1. Fermentations P. pentosaceus Mees 1934, a pediocin producing strain, was grown in complex medium (CM) containing 30 g/l sucrose, 10 g/l yeast extract, 10 g/l soy peptone, 10 g/l KH2 PO4 , 2 g/l NaCl and 0.2 g/l MgSO4 ·7H2 O. Following growth until the mid logarithmic phase (OD600 1.4) the inoculum culture was transferred (2%, v/v) in a stirred tank bioreactor (BIOFLO 110, New Brunswick Scientific) with a working volume of 2.0 l. The agitation system of the bioreactor consisted of two 6-bladed Rushton type impellers (52 mm) operated at 150 rpm. The reactor was also equipped with baffles. Process temperature was maintained at 30 ◦ C. The dissolved oxygen tension (DOT) was maintained at 60% of saturation by sparging the reactor with a mixture of atmospheric air and nitrogen, adjusted by two mass flow controllers and was kept constant by feedback regulation of the ratio. The pH was controlled at 6.8 throughout fermentation by automatic addition of a 5 M NaOH solution. Fed-batch cultures were initiated as batch cultures with 10 g/l initial sucrose concentration. A concentrated sucrose solution (400 g/l) was added at various constant feeding rates (5, 7, 9, and 10 g/l/h) and feeding started when residual sucrose concentration was reduced to 5 g/l (off-line measurements) (∼the 5th hour of fermentation). Samples were withdrawn aseptically at regular intervals and analyzed. Experiments were carried out in triplicate and mean values and standard deviations are presented here.
third term representing maintenance requirements can be ignored since experimentally determined maintenance coefficients are very low and at the same time may introduce a considerable degree of error. Therefore such a term was not included in Eq. (2). The rate of lactate production can be expressed by the Luedeking–Piret model [18] that introduces the type of product as follows: dx dL =˛ + ˇx dt dt
(3)
where, ˛ is the constant for growth-associated kinetics of lactate production in g L/g CDW and ˇ is the non-growth associated constant in g L/g CDW/h. Pediocin is produced mainly during the exponential phase of growth and its titer decreases after biomass has reached its maximum. Pediocin production can be described by the following: dP dx = qp dt dt
for
dx dP ≥ 0 or = −Rin x4 dt dt
for
dx ≤0 dt
(4)
where, qp is the specific pediocin production rate in AU/g CDW/h and Rin is the specific rate of pediocin inactivation in AU/g CDW/h. 2.3.2. Rate equations for fed-batch culture These are derived from the rate equations of the batch culture and the introduction of terms that describe the change in volume V, the feeding rate rfeed and the concentration of sucrose Sin in the feeding solution. The respective equations for growth, sucrose utilization, lactate production and pediocin production are the following:
d(Vx) X = max 1 − Xmax dt
− Kx Vx
(5)
where, V is the broth volume. 2.2. Analytical methods − Biomass concentration was monitored spectrophotometrically by measuring the optical density at 600 nm and correlating the optical density (OD) with cell dry weight (CDW). One unit of OD at 600 nm was equivalent to 0.25 g/l CDW. Bacteriocin activity was assessed in cell-free filtrates of culture broths as described earlier [14] using P. acidilactici ATCC 25740 as the indicator microorganism. To eliminate the antimicrobial effect of lactate, the pH of samples was adjusted to 6.0 using 1 M NaOH. Lactate concentration was determined using the EnzyPlus D/L Lactic Acid Kit by Diffchamb AB (Sweden). Sucrose concentration was determined according to the Roe’s method [15]. 2.3. Process modeling 2.3.1. Rate equations for batch culture Cell growth of P. pentosaceus Mees 1934 was described adequately by the logistic model of Mercier et al. [16] modified to include a term for cell death according to Lv et al. [17] as follows:
dx X = max 1 − Xmax dt
− Kx x
1 dL 1 dx dS = + YL/S dt Yx/S dt dt
(6)
where rfeed is the feeding rate in l/h and Sin is the sucrose concentration in the feeding solution in g/l. d(VL) d(Vx) =˛ + ˇVx dt dt
(7)
d(VP) d(Vx) = qp − Rin Vx dt dt
(8)
The culture volume evolution in fed-batch culture is given by dV = rfeed = rin − rout dt
(9)
where, rin and rout are the inflow and outflow rates, respectively. Non-linear regression analysis and modeling of fermentation data were carried out on Microsoft Excel 2010 [19]. The Runge–Kutta method was used for integrating the model equations while the Powell method was applied to obtain the minimum total residual sum of squares for achieving the best fit of the experimental data.
(1) 3. Results and discussion
where, x is the CDW in g/l, xmax is the maximum CDW in g/l, max is the maximum specific growth rate in h−1 , and Kx is the specific rate of cell death in h−1 . The rate of sucrose utilization was described by the equation: −
1 d(Vx) d(VS) 1 d(VL) = rfeed Sin − + YL/S dt Yx/S dt dt
(2)
where, S is the residual sucrose concentration in g/l, L is the concentration of the produced lactate in g/l, YL/S is the yield of lactate on sucrose consumed in g L/g S, and Yx/S is the yield of biomass on sucrose consumed in g CDW/g S. According to Mercier et al. [16] a
Fig. 1 shows the time courses of biomass, residual sucrose, lactate and pediocin concentrations (as points) of a standard batch fermentation at 150 rpm, 60% DOT, pH 6.8 and 30 g/l initial sucrose concentration. Sucrose was consumed within 12 h of fermentation. Biomass reached 3.5 g/l CDW, while pediocin production followed cell growth and reached a maximum of 67 AU/ml within 14 h of fermentation. Beyond that time-point production decreased while biomass production rates were almost zero. Pediocin production showed primary metabolite kinetics as has been observed earlier with the same microorganism during growth on glucose and under
Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023
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Pediocin
Biomass (g/l)
3
60
2,5
50
2
40
1,5
30
1
20
0,5
10
0
Sucrose
140
Lactate
10
Pediocin
120
8
100
6
80 60
4
40 2
20
0 0
2
4
6
8
10
12
14
16
18
0
Time (hours)
60% DOT [1]. Bacteriocins are generally considered to be primary metabolites, and as such, they are formed at a rate depending only on the growth rate of the producer organism [18]. Deviations however exist, e.g. as mixed kinetics, and seem to appear under stress conditions as reported in the works of Guerra at al. [20] with nisin and pediocin, Neysens et al. [21] with amylovorin, and Papagianni and Papamichael [7] with weissellin A. Examples of the strict primary metabolite production model include the cases of nisin [8,17], lactocin [22], and leucocin LA54A [23]. However, studies have considered pediocin AcH [24] and propionicin [25] to be secondary metabolites. Experimental data (Table 1) were used for model parameter values estimation using Eqs. (1)–(4) as shown in Section 2. The obtained values were plotted against time and are presented in Fig. 1 as lines along the experimentally obtained data points. It can be observed from the figure that model simulation for batch culture was very good for the four fermentation parameters tested. 160
12
140
Sucrose Lactate
120
Pediocin 8
100
6
80 60
4
40 2
20
0
Sucrose, Lactate (g/l), Pediocin (AU/ml)
Biomass 10
0 0
2
4
6
8
10
0 0
Fig. 1. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations in batch culture of P. pentosaceus Mees 1934 at 150 rpm, 60% DOT, pH 6.8 and 30 g/l initial sucrose concentration. Experimental data are denoted as points while the lines represent model-derived values.
Biomass (g/l)
160
Biomass
Sucrose, Lactate (g/l), Pediocin (AU/ml)
70
Lactate
Biomass (g/l)
Sucrose
3,5
12
80
Biomass
Sucrose, Lactate (g/l), Pediocin (AU/ml)
4
3
12
14
16
18
Time (hours) Fig. 2. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 5 g/l/h. The arrows indicate the period of feeding.
2
4
6
8
10
12
14
16
18
Time (hours) Fig. 3. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 7 g/l/h. The arrows indicate the period of feeding.
Fig. 2 shows the time courses of biomass, residual sucrose, lactate and pediocin concentrations (as points) in the fed-batch fermentation with 10 g/l initial sucrose concentration and 5 g/l/h feeding rate of a 400 g/l sucrose solution. Feeding was carried out between the 4th and 12th hour of fermentation. Residual sucrose concentration remained at very low levels and no sucrose was detected beyond the 10th hour of fermentation. Biomass reached the maximum of 3.9 g/l CDW at 14 h (3.5 g/l in the batch culture) while lactate 23 g/l (16 g/l in the batch culture). Pediocin production rate was increased during feeding and its overall production reached 102 AU/ml, a significantly higher titer compared to the obtained in the standard batch fermentation run. The maximum titer of pediocin was obtained in the fed-batch culture of 7 g/l/h feeding rate (Fig. 3) and it was 145 AU/ml. Biomass reached 4.1 g/l and lactate 25 g/l. By increasing the feeding rate to 9 (not shown) and 10 g/l/h (Fig. 4), pediocin production decreased and lactate production increased, while maximum biomass levels were 4 and 3.8 g/l CDW, respectively. An increase in residual sucrose concentration was monitored in the last two runs. It is obvious that while biomass production is rather unaffected, pediocin biosynthesis is affected by the concentration of sucrose in the fermentation broth and an optimum area for sucrose concentration levels exist beyond which production decreases. The effect is also obvious on the kinetic parameter specific production rate qp (AU/g CDW/h) as shown in Table 1, where the fed-batch culture with 7 g/l/h feeding rate appears to give the highest values (max. 6.78 AU/g/h). The lines in the plots of Figs. 2–4 represent model derived values using Eqs. (5)–(8). Obviously, the model of Eq. (8) that describes growth-associated pediocin production fits very well the experimental data in all fed-batch experiments. Therefore, as a product of primary metabolism, pediocin biosynthesis is expected to be influenced by the type and concentration of the carbon source. Research carried out with L. lactis grown in media containing glucose has shown that the concentration of glucose regulates its uptake and improving its uptake could result in nisin yield improvements [9]. Maintaining elevated glucose levels in the medium (>10 g/l) in glucostat fed-batch fermentations resulted in decreased nisin yields. Similarly, again with L. lactis and sucrose as the carbon source, elevated concentrations in both batch and fed-batch fermentations resulted in decreased nisin yields in the works of De Vuyst and
Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023
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Table 1 Estimated kinetic parameters in batch and fed-batch cultures of P. pentosaceus Mees 1934: maximum specific growth rate max , maximum biomass production rate Xmax , specific rate of cell death Kx , yield of lactate on sucrose YL/S , constant for growth-associated kinetics of lactate production ˛, non-growth associated constant ˇ, specific production rate of pediocin qp and specific rate of pediocin inactivation Rin . Type of culture
Kinetic parameters max (h−1 )
Xmax (g/l)
Kx (h−1 )
YL/S (g L/g)
˛ (g L/g DCW)
ˇ (g L/g DCW/h)
qp (AU/g DCW/h)
Rin (AU/g DCW/h)
Batch
1.12 ± 0.05
3.5 ± 0.12
0.053 ± 0.002
0.53 ± 0.02
6.01 ± 0.6
0.24 ± 0.01
1.97 ± 0.08
2.6 ± 0.11
Fed-batch 5 g/l/h 7 g/l/h 9 g/l/h 10 g/l/h
1.35 1.37 1.36 1.32
0.05 0.04 0.05 0.03
3.9 4.1 4.0 3.8
± ± ± ±
0.14 0.12 0.16 0.11
0.082 0.054 0.063 0.080
± ± ± ±
0.001 0.002 0.002 0.002
0.73 0.78 0.91 0.92
Sucrose
140
Lactate Pediocin
120
8
Biomass (g/l)
0.02 0.02 0.03 0.03
160
Biomass
10
± ± ± ±
100 6
80 60
4 40 2
20
0
Sucrose, Lactate (g/l), Pediocin (AU/ml)
12
± ± ± ±
0 0
2
4
6
8
10
12
14
16
18
Tim e (hours) Fig. 4. Time-courses of biomass (CDW), lactate, residual sucrose and pediocin concentrations (experimental data as points and model simulation as lines) in fed-batch culture of P. pentosaceus Mees 1934. Sucrose was supplied at the stable feeding rate of 10 g/l/h. The arrows indicate the period of feeding.
Vandamme [8], James and Larry [26] and Lv et al. [17]. Working with sucrose in the present case, the results show that the productivity of pediocin is greatly influenced by the concentration of sucrose and by supplying sucrose at an increased rate (>7 g/l/h for the particular system) pediocin production is restricted while large amounts of lactate are produced. Maintaining the feeding rate of sucrose at 7 g/l/h resulted in a 119% increase of the pediocin titer compared to that of the standard batch culture. However, further research into the particular production system would include sucrostat fedbatch culture experiments and sucrose transport studies in order to determine the dynamics of the sucrose transport system in the particular microorganism and investigate the relationship between the sucrose uptake rate (sucrose regulation) and pediocin production. 4. Conclusions Our work aimed to provide information on the effects of sucrose (as the main source of carbon in a complex medium) concentration on P. pentosaceus Mees 1934 growth and pediocin production in semi-aerobic, pH-controlled, fed-batch fermentation. From the presented results it becomes clear that the feeding rate and therefore the residual sucrose concentration influenced greatly the outcome of the pediocin fermentation. The maximum titer of pediocin (145 AU/ml) was obtained in the fed-batch culture with 7 g/l/h feeding rate and was increased by 119% compared to the titer obtained in batch culture, while further increased feeding rates
4.32 4.83 4.98 5.12
± ± ± ±
0.5 0.5 0.5 0.6
0.68 0.44 0.58 0.59
± ± ± ±
0.01 0.01 0.02 0.02
3.02 4.16 3.00 2.83
± ± ± ±
0.12 0.12 0.13 0.14
2.1 1.8 1.5 1.2
± ± ± ±
0.10 0.09 0.009 0.008
(9 and 10 g/l/h) resulted in decreased pediocin yields. The specific rate of pediocin formation appears to be sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known kinetic models for batch and fed-batch cultures and growth associated production, simulated successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture. The obtained data can be exploited in further studies on process optimization and as a reference in studies with various feeding regimes, e.g. pH feedback controlled fed-batch cultures, step-increasing strategies or others. References [1] Anastasiadou S, Papagianni M, Filiousis G, Ambrosiadis I, Koidis P. Growth and metabolism of a meat isolated strain of Pediococcus pentosaceus in fermentation. Purification, characterization and properties of the produced pediocin SM1. Enzyme Microb Technol 2008;43:448–54. [2] De Vuyst L, Gallewaert R, Crabbe K. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology 1996;142:817–27. [3] Papagianni M, Papamichael EM. Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX. Bioresour Technol 2011;102:6730–4. [4] Papagianni M. Effects of dissolved oxygen and pH levels on weissellin A production by Weissella paramesenteroides DX in fermentation. Bioprocess Biosyst Eng 2012;35:1035–41. [5] Vant Hul JS, Gibbons WR. Neutralization/recovery of lactic acid from Lactococcus lactis: effects on biomass, lactic acid, and nisin production. World J Microbiol Biotechnol 1997;13:527–32. [6] Lv W, Cong W, Cai Z. Effect of sucrose on nisin production in batch and fed-batch culture by Lactococcus lactis. J Chem Technol Biotechnol 2005;80:511–4. [7] Papagianni M, Papamichael EM. Production of the antimicrobial protein weissellin A by Weissella paramesenteroides DX in batch fermentations: the type of carbohydrate used as the C-source in the substrate affects the association of production with growth. Appl Biochem Biotechnol 2012;168:1212–22. [8] De Vuyst L, Vandamme EJ. Influence of the carbon source on nisin production by Lactococcus lactis subsp. lactis batch fermentations. J Gen Microbiol 1992;138:571–8. [9] Papagianni M, Avramidis N, Filiousis G. Investigating the relationship between the specific glucose uptake rate and nisin production in aerobic batch and fed-batch glucostat cultures of Lactococcus lactis. Enzyme Microb Technol 2007;40:1557–63. [10] Papagianni M, Anastasiadou S. Pediocins: the bacteriocins of pediococci. Microb Cell Fact 2009;8:3. [11] Guerra NP, Pastrana L. Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources. Lett Appl Microbiol 2002;34:114–8. [12] Guerra NP, Bernardez PF, Agrasar AT, Macias CL, Pastrana L. Fed-batch pediocin production by Pediococcus acidilactici NRRL B5627 on whey. Biotechnol Appl Biochem 2005;42:17–23. [13] Guerra NP, Bernardez PF, Pastrana L. Modelling the stress inducing biphasic growth and pediocin production by Pediococcus acidilactici NRRL B5627 in realkalized fed-batch cultures. Biochem Eng J 2008;40:465–72. [14] Papagianni M, Avramidis N, Filiousis G, Dasiou D, Ambrosiadis I. Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor indicator microorganism. Microb Cell Fact 2006;5:30. [15] Oardini CE, Leloir LF, Ohiriboga J. The biosynthesis of sucrose. J Biol Chem 1955:214–49. [16] Mercier P, Yerushalmi L, Rouleau D, Dochain D. Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus. J Chem Technol Biotechnol 1992;55:111–21. [17] Lv W, Zhang X, Cong W. Modelling the production of nisin by Lactococcus lactis in fed-batch culture. Appl Microbiol Biotechnol 2005;68:322–6.
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Please cite this article in press as: Papagianni M, Papamichael EM. Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.09.023