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Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037

Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037 Ann Microbiol (2011) 61:947–953 DOI 10.1007/s13213-011-0218-5 ORIGINAL ARTICLE Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037 Stanislav Obruca & Ivana Marova & Sona Melusova & Ludmila Mravcova Received: 18 June 2010 /Accepted: 28 January 2011 /Published online: 19 February 2011 Springer-Verlag and the University of Milan 2011 Abstract Poly(3-hydroxybutyrate) (PHB) is a polyester waste cheese whey. Nevertheless, experiments in belonging to the family of polyhydroxyalkanoates, which laboratory-scale and semi-productive fermentors are needed accumulate in a wide variety of bacterial strains. PHB to test performance under high cell density cultivation. appears to be a biodegradable alternative to traditional . . petrochemical polymers such as polypropylene and poly- Keywords Bacillus megaterium Polyhydroxyalkanoate . . ethylene. In this work, we tested direct conversion of cheap Poly(3-hydroxybutyrate) Cheese whey Exogenous stress waste cheese whey into PHB employing the bacterial strain Bacillus megaterium CCM 2037. Optimization of medium composition improved PHB yields about 50 fold (biomass Introduction −1 and PHB yields 2.82 and 1.05 g l , respectively) as compared to none-optimized whey. Furthermore, PHB Polyhydroxyalkanoates (PHA) are biopolymers produced and yields were improved by about 40% by introducing 1% accumulated in the form of intracellular granules by a number ethanol into the medium at the beginning of the stationary of bacterial strains. Of the large PHA family, a homopolymer −1 −1 phase of growth (biomass 2.87 g l , PHB 1.48 g l ). of 3-hydroxybutyrate, poly(3-hydroxybutyrate) (PHB), is the According to the results of experiments carried out in most widespread in nature and the best characterised PHA Erlenmeyer flasks, B. megaterium CCM 2037 can be compound. PHB has aroused much interest in industry and considered a candidate for direct PHB production from research thanks to its biocompatible, biodegradable, thermo- plastic and piezoelectric properties. Nowadays, PHB is considered to act as an alternative to common plastics derived S. Obruca (*) I. Marova from petrol (Kadouri et al. 2005). Faculty of Chemistry, Centre for Materials Research The high production cost is one of the main factors CZ.1.05/2.1.00/01.0012, Brno University of Technology, preventing wider use of PHB. Analysis and economic Purkynova 118, 612 00 Brno, Czech Republic evaluation of bacterial PHB production has suggested that e-mail: Stana.O@seznam.cz the cost of substrate (mainly carbon source) contributes most (up to 50%) to the overall production costs (Choi and : : S. Obruca I. Marova S. Melusova Lee 1997). Therefore, PHB could be produced more Faculty of Chemistry, Institute of Food Chemistry and Biotechnology, economically using cheap waste substrates. Brno University of Technology, Cheese whey is the major by-product from the Purkyňova 118, manufacture of cheese and casein, representing 80– 612 00 Brno, Czech Republic 90% of the volume of transformed milk. Cheese L. Mravcova production in the European Union produces a total of Faculty of Chemistry, Institute of Chemistry approximately 40,462,000 tons of whey per year. Most and Technology of Environmental Protection, of this whey is used for production of lactose and in Brno University of Technology, animal feed, but an annual amount of 13,462,000 tons Purkynova 118, 612 00 Brno, Czech Republic of whey per year, containing about 619,250 tons of 948 Ann Microbiol (2011) 61:947–953 lactose, constitutes a surplus product (Koller et al. culture was inoculated into a 250-ml Erlenmeyer flask 2008). For this reason, cheese whey represents a promis- containing 100 ml whey medium. ing substrate for cheap production of PHB in large Whey was obtained from the cheese manufacturer amounts. Pribina Pribyslav (Pribyslav, Czech Republic). The Although biotechnological production of PHB from whole whey was treated in order to remove excess different sugars via condensation of acetyl-CoA units stem- proteins. Whey was acidified to pH 4.0 with 1.0 M ming from hexose catabolism is well described (Kessler and H SO and heated (100°C for 20 min), cooled and 2 4 Wilholt 1999), only a limited number of bacterial strains centrifuged at 8,000 rpm for 5 min. The treated whey directly convert lactose into PHB. A few reports are was used in experiments after adjusting the pH to 7.0 with available on PHB production from lactose and whey by 1.0 M NaOH and MM medium components (except −1 recombinant Escherichia coli (Wong and Lee 1998;Ahn et lactose), and 0.1 g l yeast extract were added at the al. 2000). Methylobacterium sp. ZP24 (Yellore and Desai concentration used in MM unless otherwise indicated. 1998;Nathet al. 2007) and the thermophillic bacterium Thermus thermophilus HB8 (Pantazaki et al. 2009) are also Analytical methods able to utilize whey lactose for PHA biosynthesis. Recently, Pandian et al. (2010) reported PHA production from a Cell growth was monitored by measuring the absorbance of mixture of rice bran, non-specified diary waste and sea water culture broth at 630 nm on a Helios α instrument (Unicam, employing a Gram-positive bacterium isolated from brackish Leeds, UK) after suitable dilution with distilled water. Cell water. Based on morphological and physiological properties biomass was calculated using a calibration curve at A 630 nm and the nucleotide sequence of its 16S rRNA, it was and dry cell mass. For dry cell mass determination, suggested that the isolate was closely related to Bacillus harvested cells (centrifugation: 8,000 rpm, 10 min) were megaterium (Pandian et al. 2010). Pseudomonas hydro- dried at 105°C to constant weight. The supernatant obtained genovora was also used for PHA production using cheese by centrifugation of the culture broth at 8,000 rpm for whey, but this bacterial strain is not able to utilize lactose 10 min was used for analysis of residual lactose by the directly, requiring hydrolysis of whey lactose prior to Somogyi-Nelson method (Deng and Tabatabai 1994). cultivation (Koller et al. 2008). The PHB content in dried cells was determined by gas We have previously reported that Bacillus megaterium chromatography (Finnigan Trace GC Ultra, Austin, TX, CCM 2037 is able to utilize lactose and accumulate PHB column DB-WAX 30 m by 0.25 mm) with mass spectrom- (Obruca et al. 2008). In this study, we focused on etry detection (Finnigan Trace DSQ) according to Brandl et optimization of the whey medium in order to enhance al. (1988). PHB and biomass yields. Moreover, application of exoge- nous stress was studied as a potential strategy to enhance Analysis of treated whey PHB biosynthesis in cells. Concentration of dry matter was estimated after drying (105°C) 10 ml whey to constant weight. Ash content was Materials and methods determined as the weight of solids after incubation of 2 ml whey at 800°C for 2 h. The phosphorus content was Microorganism, media and growth conditions measured by the molybdenum blue colorimetric method (A ) using whey ash. The concentration of soluble 610 nm Bacillus megaterium CCM 2037 was obtained from the protein was performed using the biuret method with bovine Czech Collection of Microorganisms (Brno, Czech serum albumin as a standard. Whey sugar content was Republic). estimated by HPLC (pump LCP 4020, thermostat LCO −1 CCM Bacillus medium, consisting of peptone 5 g l , 101, degasser DG-1210, refractometric detector RIDK 102; −1 −1 −1 yeast extract 3 g l , MnSO 0.01 g l and agar 20 g l , Ecom, Czech Republic) with a ZOBRAX NH column 4 2 was used for maintaining the culture. Mineral medium (150 cm×4,6 mm, 5 μm; Chromservis, Czech Republic), (MM) was used for inoculum preparation. MM contained chromatographic conditions were: 25°C; acetonitrile: water −1 −1 −1 lactose 8 g l , (NH ) SO 5g l ,Na HPO 2.5 g l , 75:25; mobile phase flow 1.0 ml/min. 4 2 4 2 4 −1 −1 −1 KH PO 2.5 g l , MgSO 0.2 g l and MnSO 0.01 g l . 2 4 4 4 The initial pH of the medium was adjusted to 7.0. Inoculum Media optimization using Placket-Burman experimental was developed in 250-mL Erlenmeyer flasks containing design 100 ml medium. MM medium was inoculated with bacterial culture and cultivated at an agitation speed of The dilution of whey (carbon source), and concentrations of 150 rpm, at 30°C for 24 h. Subsequently, 5 ml of the nitrogen source [(NH ) SO ], mineral salts and yeast 4 2 4 Ann Microbiol (2011) 61:947–953 949 Table 1 Range of factors Factor Name Level studied in the Placket-Burman experiment 1 −1 A Whey Not-diluted Diluted -1 −1 (Lactose 40 g l ) (Lactose 40 g l ) -1 −1 B (NH4) SO 5g l 1g l 2 4 -1 −1 CNa HPO ;KH PO (1:1) 5 g l 1g l 2 4 2 4 -1 −1 D MgSO 0.2 g l 0.04 g l -1 −1 E Yeast autolysate 1 g l 0.1 g l extract were tested using a Placket-Burman experimental addition, the whey could be expected to contain some design. Each parameter was tested at two levels, high (+1) minor components such as free amino acids and vitamins and low (−1) (Table 1). A design of 12 experiments was (not analyzed), which are likely to support bacterial formulated for five factors using Minitab software. The growth. experiments were performed in 250-ml Erlenmeyer flasks containing 100 ml whey medium at 150 rpm, 30°C for 50 h Supplementation of whey by salts in duplicate. Response was measured in terms of (1) biomass production, (2) PHB accumulation in cells (% w/w), and (3) In order to test whether whey itself contains all necessary PHB yields. elements for bacterial growth, a culture of Bacillus megaterium was inoculated into whey with and without added salts according to MM (Fig. 1). Biomass and PHB yields were analyzed after 48 h. Results and discussion In whey medium without added salts, bacterial growth and PHB production were rather low. Conversely, addition Analysis of whey composition of salts promoted the growth of bacterial culture and also improved PHB production (almost 10 times). An explana- Used cheese whey contains about 6.8% of dry matter, tion could be that, despite excess salts, whey itself lacks composed mainly of sugars and salts. Moreover, some nitrogen, phosphorus, magnesium and manganese. There- soluble proteins are still present in whey even after the fore, these components must be added in the form of treatment. In spite of the high concentration of salts mineral salts. (expressed as ash), which could result in high osmotic pressure influencing bacterial growth negatively, the Optimization of whey supplementation tested wastecheese wheyseems to beapromising complex substrate for PHB production. Whey sugars For multivariable processes such as biotechnological (lactose and glucose; galactose was not detected) could systems, in which numerous potentially influential be utilized by the bacterial culture as carbon sources, factors are involved, it is not always obvious to soluble proteins could serve as a complex carbon and determine which are the most important. Hence, it is nitrogen sources, and a low concentration of potential phosphorus sources was also observed (Table 2). In Table 2 Composition of cheese whey. Results given are average ± standard deviation; each analysis was performed in triplicate Substance Concentration Water 93% −1 Dry matter 68 g l −1 Ash 27.10±0.30 g l −1 Lactose 39.60±0.45 g l −1 Glucose 0.35±0.02 g l 3− −1 PO 63.0±1.2 mg l −1 Fig. 1 Biomass and poly(3-hydroxybutyrate) (PHB) yields by Soluble proteins 2.0±0.1 g l Bacillus megaterium on whey with and without addition of salts 950 Ann Microbiol (2011) 61:947–953 Table 3 Experimental design and responses of Placket-Burman study -1 2- -1 -1 -1 -1 -1 Whey (NH ) SO [g l ]PO [g l ] MgSO [g l ] Yeast Extract [g l ] Biomass [g l ] PHB [%] PHB [g l ] 4 2 4 4 4 1 None-dilution 1.0 5.0 0.04 0.10 0.90 2.06 0.02 2 None-dilution 5.0 1.0 0.20 0.10 0.76 3.20 0.02 3 Diluted 5.0 5.0 0.04 1.00 2.56 16.26 0.42 4 None-dilution 1.0 5.0 0.20 0.10 0.92 1.90 0.02 5 None-dilution 5.0 1.0 0.20 1.00 0.77 5.08 0.04 6 None-dilution 5.0 5.0 0.04 1.00 0.75 1.95 0.01 7 Diluted 5.0 5.0 0.20 0.10 2.40 27.36 0.66 8 Diluted 1.0 5.0 0.20 1.00 2.02 12.72 0.26 9 Diluted 1.0 1.0 0.20 1.00 1.95 13.51 0.26 10 None-dilution 1.0 1.0 0.04 1.00 1.01 2.17 0.02 11 Diluted 5.0 1.0 0.04 0.10 2.47 35.48 0.87 12 Diluted 1.0 1.0 0.04 0.10 2.99 35.49 1.06 necessary to submit the process to an initial screening PHB production. This is probably due to the fact that yeast design prior to optimization. Plackett–Burman method- extract can serve as a nitrogen source, and PHB biosyn- ology could be a tool for this initial screening, because thesis is much more pronounced under nitrogen-limiting it makes it possible to determine the influence of conditions (Kessler and Wilholt 1999). Therefore, in various factors with only a small number of trials subsequent experiments only a low concentration −1 (Khanna and Srivastava 2005). (0.1 g l ) of yeast extract was added to the whey medium. We used Placket-Burman methodology to optimize the Conversely, whey dilution seems to be crucial for biomass composition of whey medium for PHB production. Five as well as for PHB production (P<0.05). A high factors were selected for optimization, and each factor was concentration of salts and lactose in undiluted whey tested at two levels, high and low (Table 1). PHB and medium probably caused high osmotic pressure, which biomass yields were analyzed after 50 h. The experiment consequently inhibited bacterial growth and PHB biosyn- was designed by Minitab software. thesis (t values<0). High concentrations of lactose could According to the results of the Placket-Burman study also induce substrate growth inhibition, hence whey (results summarized in Tables 3, 4), the concentrations of dilution was optimized in order to achieve maximal 3− (NH ) SO ,PO and MgSO are not statistically biomass and PHB yields (Fig. 2). 4 2 4 4 4 important either for biomass or for PHB production (P> The highest biomass and PHB yields were obtained 0.05). Nevertheless, in previous experiments addition of when whey was diluted to a lactose concentration of −1 salts had strongly enhanced biomass and PHB production; 20gl . Thereafter, biomass and PHB yields were −1 −1 therefore, in subsequent experiments salts were added at 2.51 g l and 0.79 g l , respectively. the concentration used as the lower level (−1) in the The growth and PHB production course of B. Placket-Burman analysis. The addition of yeast extract, megaterium in optimized whey medium were also deter- which was tested as a potential source of vitamins, amino mined. Growth was accompanied by lactose utilization acids etc., had a statistically significant negative impact on throughout the whole cultivation. Biomass formation Table 4 The results of data -1 -1 Biomass [g l ] PHB [%] PHB [g l ] analysis (t-values and P values) for the effect of medium compo- a a a t value P value t value P value t value P value nents on growth and poly(3- hydroxybutyrate) (PHB) Whey concentration -11.46 0.000 -7.53 0.000 -6.40 0.001 production (NH ) SO -0.10 0.923 1.30 0.241 0.73 0.493 4 2 4 2- PO -0.48 0.647 -1.98 0.095 -1.70 0.139 MgSO -2.31 0.051 -1.79 0.123 -2.17 0.073 t value is statistically significant Yeast extract -1.69 0.142 -3.26 0.017 -3.09 0.021 only if P<0.05 Ann Microbiol (2011) 61:947–953 951 Fig. 2 The effect of whey dilution (expressed as lactose concentration) on PHB and bio- mass production th reached a maximum after 28 h of cultivation; thereafter, ethanol and hydrogen peroxide at the 25 hour of th stationary phase occurred. This lasted until the 50 hour cultivation (Table 5). of cultivation, when biomass concentration started to Both hydrogen peroxide and ethanol increased PHB decrease. The highest PHB yields were observed at the biosynthesis in B. megaterium cells. The most effective th 50 hour (see Fig. 3). strategy was the application of 1% ethanol, which enhanced PHB yields about 41% as compared to the PHB production under stress conditions control culture (Table 5). In contrast, application of hydrogen peroxide enhanced PHB yields only very We have recently reported that the stress response of slightly. Cupriavidus necator H16 to ethanol and hydrogen peroxide The reason why ethanol enhances PHB yield is that is accompanied by enhanced PHB accumulation. However, ethanol is metabolized via oxidation to acetyl-CoA. the stress has to be applied at the beginning of the During these reactions, reduced coenzymes NAD(P)H stationary phase, and at an optimized level. This strategy stimulating flux of acetyl-CoA into PHB biosynthetic could be used in biotechnology as a simple, cheap and pathway are formed, and free CoA, which inhibits PHB effective tool to enhance total PHB yields (Obruca et al. biosynthesis, is used to build acetyl-CoA. Moreover, 2010a, b). In order to study whether this strategy could also acetyl-CoA, as the final product of ethanol metabolism, be used in Bacillus megaterium cultivated on cheap whey is the initial substrate of the PHB biosynthetic pathway medium, we decided to apply different concentrations of (Obruca et al. 2010b). Fig. 3 Growth and production characteristics of Bacillus megaterium in optimized whey medium 952 Ann Microbiol (2011) 61:947–953 Table 5 PHB and biomass yields after stress factor application at the environmentally friendly recovery of PHA using lytic th 25 hour of cultivation. Results given are average ± standard enzymes such as lysozyme or mutanolysin. Finally, Gram- deviation; each cultivation was performed and analyzed in triplicate negative bacteria, which are currently the only commercial -1 -1 Biomass [g l ] PHB [g l ] PHB [%] sources of PHA, accumulate lipopolysacharides that co- purify with PHA and cause immunogenic reactions. This Control 2.82±0.11 1.05±0.05 37.23 complicates application of PHA in medicine. On the other EtOH 0.5% 2.79±0.08 1.43±0.07 51.23 hand, Gram-positive bacterial strains, such as Bacillus EtOH 1 % 2.87±0.08 1.48±0.08 51.57 megaterium CCM 2037, lack lipopolysacharides, which EtOH 1.5 % 2.68±0.03 1.22±0.12 45.52 makes them a more attractive source of PHA (Valappil et al. H O 1 mM 2.62±0.12 1.18±0.16 45.04 2 2 2007). H O 3 mM 2.75±0.06 1.09±0.03 39.61 2 2 Further experiments should focus on fermentor cultiva- H O 5 mM 2.77±0.09 1.15±0.05 41.53 2 2 tion to reach high cell density and improve PHB yields. Nevertheless, we have proved that application of controlled stress conditions (ethanol) is a promising strategy for improving the process of PHB production from cheese Table 6 summarizes yields of PHA production whey using B. megaterium. employing wild type bacterial strains from cheese whey that have been reported recently in the literature. Total PHB yields obtained in this work are relatively low as Conclusions compared to those obtained in fermentor in fed-batch mode (Nath et al. 2007;Kolleretal 2007). This is due In this work, we tested Bacillus megaterium CCM 2037 predominantly to the relatively low growth of bacterial as a bacterial strain able to utilize waste cheese whey and culture in Erlenmeyer flasks. On the other hand, Pseudo- produce PHB. Because supplementation of cheese whey monas hydrogenovora was employed for PHA production medium with salts is necessary to reach higher PHB from whey in fermentors in fed-batch mode with highest yields, optimization of whey media composition was −1 PHA yields of 1.4 g l (Koller et al. 2008). This is performed. In our experiments, PHB production was comparable to the yield reached in batch mode in our enhanced about 50 times by optimization of cheese −1 flasks experiments (1.5 g l ). medium as compared to cheese whey alone. Furthermore, Optimization of medium composition as well as con- even higher PHB yields can be obtained if the bacterial trolled introduction of stress factors significantly enhanced culture is exposed to 1% ethanol as an exogenous stress PHB content in cells to relatively high levels, even in factor applied at the beginning of stationary phase. This comparison with results reported in the literature (Table 6). novel strategy enhanced PHB production by about 41%. This is beneficial in terms of total PHB yields but, Our results indicate the potential of Bacillus megaterium furthermore, is also likely to reduce the cost of PHA for industrial PHB production from cheap whey substrate. recovery because the PHB content in cells strongly affects Nevertheless, further experiments carried out in laboratory the efficiency and cost of downstream processing (Lee and and semi-productive bioreactors are needed to obtain high Choi 1999). Bacillus megaterium CCM 2032 is a Gram- cell density and improve production parameters still positive strain that could also facilitate simple and further. Table 6 Biomass and polyhydroxyalkanoate (PHA) yield from cheese whey reported in the literature -1 -1 Reference Biomass [g l ] PHA [%] PHA [g l ] Microorganism Cultivation device Nath et al. 2007 3.9 Methylobacterium sp. ZP24 Fermentor Koller et al. 2008 12 1.4 Pseudomonas hydrogenovora Fermentor Koller et al. 2007 12 1.3 Pseudomonas hydrogenovora Fermentor Koller et al. 2007 40 2.7 Hydrogenophaga pseudoflava Fermentor Koller et al. 2007 50 5.5 Haloferax meditarranei Fermentor Yellore and Desai 1998 9.9 59 5.9 Methylobacterium sp. ZP24 Flasks Pantazaki et al. 2009 1.6 35 0.5 Thermus thermophilus HB8 Flasks This work 2.9 51 1.5 Bacillus megaterium CCM 2037 Flasks Ann Microbiol (2011) 61:947–953 953 Acknowledgments This work was supported by projects MSM from whey by Pseudomonas hydrogenovova. Bioresour Technol 0021630501 and BD 16001004 of The Czech Ministry of Education 99:4854–4863 and by the project “Centre for Materials Research at FCH BUT” Lee SY, Choi J (1999) Effect of fermentation performance on the No. CZ.1.05/2.1.00/01.0012 from ERDF. economics of poly-(3-hydroxybutyrate) production by Alcali- genes latus. Polymer Degrad Stabil 59:387–393 Nath A, Dixit M, Bandiya A, Chavda S, Desai AJ (2007) Enhanced production and scale up studies using cheese whey in fed batch References culture of Methylobacterium sp. ZP24. Bioresour Technol 99:5749–5755 Ahn WS, Park SJ, Lee SY (2000) Production of poly(3-hydroxybutyrate) Obruca S, Melusova S, Marova I, Svoboda Z (2008) Strategies for by fed-batch culture of recombinant Escherichia coli with enhancing poly(3-hydroxybutyrate) production in selected bacte- highly concentrated whey solution. Appl Environ Microbiol rial strains. Chem Listy 102:1255–1256 102:61–68 Obruca S, Marova I, Svoboda Z, Mikulikova R (2010a) Use of Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas controlled exogenous stress for improvement of poly(3-hydrox- oleovorans as a source of Poly(beta-hydroxyalkanoates) for ybutyrate) production in Cupriavidus necator. Folia Microbiol potential application as a biodegradable polyester. Appl Environ 55:17–22 Microbiol 54:1977–1982 Obruca S, Marova I, Stankova M, Mravcova L, Svoboda Z (2010b) Effect Choi JI, Lee SY (1997) Process analysis and economical evaluation of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) for poly(3-hydroxybutyrate) production by fermentation. Bio- biosynthetic pathway in Cupriavidus necator H16. World J Microb process Eng 17:335–342 Biotechnol 26:1261–1267 Deng SP, Tabatabai MA (1994) Colorimetric determination of Pandian SR, Deepak V, Kalishwaralal K, Rameskrumar N, Jeyaraj M, reducing sugar in soils. Soil Biol Biochem 26:473–477 Gurunathan S (2010) Optimization and fed-batch production of Kadouri D, Jurkevitch E, Okon Y, Castro-Sowinski S (2005) PHB utilizing dairy waste and sea water as nutrient sources by Ecological and agricultural significance of bacterial polyhydrox- Bacillus megaterium SRKP-3. Bioresour Technol 101:705–711 yalkanoates. Crit Rev Microbiol 31:55–67 Pantazaki AA, Papaneophytou CP, Pritsa AG, Liakopoulus-Kyriakides Kessler B, Wilholt B (1999) Poly(3-hydroxyalkanoates). In: Flickinger M, Kyriakidis DA (2009) Production of polyhydroxyalkanoates MC, Drew SW (eds) Encyclopedia of bioprocess technology— from whey by Thermus thermophilus HB8. Process Biochem fermentation, biocatalysis and bioseparation. Wiley, New York, 44:847–853 pp 2024–2040 Valappil SP, Boccaccini AR, Bucke C, Roy I (2007) Polyhydrox- Khanna S, Srivastava AK (2005) Statistical media optimization yalkanoates in Gram-positive bacteria: insight from the genera studies for growth and PHB production by Ralstonia eutropha. Bacillus and Streptomyces. Antonie Van Leeuwenhoek J G 91:1– Process Biochem 40:2173–2182 17 Koller M, Hesse P, Bona R, Kutschera C, Atlic A, Braunegg G (2007) Wong HH, Lee YY (1998) Poly(3-hydroxybutyrate) production from Potential of various archae- and eubacterial strains as industrial whey by high-density cultivation of recombinant Escherichia polyhydroxyalkanoates producers from whey. Macromol Biosci coli. Appl Microbiol Biotechnol 50:30–33 7:218–226 Yellore V, Desai A (1998) Production of poly-3-hydroxybutyrate from Koller M, Bona R, Chiellini E, Fernandes EG, Horvat P, Kutschera C, lactose and whey by Methylobacterium sp. ZP24. Lett Appl Hesse P, Braunegg G (2008) Polyhydroxyalkanoate production Microbiol 26:391–394 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037

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Springer Journals
Copyright
Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-011-0218-5
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Abstract

Ann Microbiol (2011) 61:947–953 DOI 10.1007/s13213-011-0218-5 ORIGINAL ARTICLE Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037 Stanislav Obruca & Ivana Marova & Sona Melusova & Ludmila Mravcova Received: 18 June 2010 /Accepted: 28 January 2011 /Published online: 19 February 2011 Springer-Verlag and the University of Milan 2011 Abstract Poly(3-hydroxybutyrate) (PHB) is a polyester waste cheese whey. Nevertheless, experiments in belonging to the family of polyhydroxyalkanoates, which laboratory-scale and semi-productive fermentors are needed accumulate in a wide variety of bacterial strains. PHB to test performance under high cell density cultivation. appears to be a biodegradable alternative to traditional . . petrochemical polymers such as polypropylene and poly- Keywords Bacillus megaterium Polyhydroxyalkanoate . . ethylene. In this work, we tested direct conversion of cheap Poly(3-hydroxybutyrate) Cheese whey Exogenous stress waste cheese whey into PHB employing the bacterial strain Bacillus megaterium CCM 2037. Optimization of medium composition improved PHB yields about 50 fold (biomass Introduction −1 and PHB yields 2.82 and 1.05 g l , respectively) as compared to none-optimized whey. Furthermore, PHB Polyhydroxyalkanoates (PHA) are biopolymers produced and yields were improved by about 40% by introducing 1% accumulated in the form of intracellular granules by a number ethanol into the medium at the beginning of the stationary of bacterial strains. Of the large PHA family, a homopolymer −1 −1 phase of growth (biomass 2.87 g l , PHB 1.48 g l ). of 3-hydroxybutyrate, poly(3-hydroxybutyrate) (PHB), is the According to the results of experiments carried out in most widespread in nature and the best characterised PHA Erlenmeyer flasks, B. megaterium CCM 2037 can be compound. PHB has aroused much interest in industry and considered a candidate for direct PHB production from research thanks to its biocompatible, biodegradable, thermo- plastic and piezoelectric properties. Nowadays, PHB is considered to act as an alternative to common plastics derived S. Obruca (*) I. Marova from petrol (Kadouri et al. 2005). Faculty of Chemistry, Centre for Materials Research The high production cost is one of the main factors CZ.1.05/2.1.00/01.0012, Brno University of Technology, preventing wider use of PHB. Analysis and economic Purkynova 118, 612 00 Brno, Czech Republic evaluation of bacterial PHB production has suggested that e-mail: Stana.O@seznam.cz the cost of substrate (mainly carbon source) contributes most (up to 50%) to the overall production costs (Choi and : : S. Obruca I. Marova S. Melusova Lee 1997). Therefore, PHB could be produced more Faculty of Chemistry, Institute of Food Chemistry and Biotechnology, economically using cheap waste substrates. Brno University of Technology, Cheese whey is the major by-product from the Purkyňova 118, manufacture of cheese and casein, representing 80– 612 00 Brno, Czech Republic 90% of the volume of transformed milk. Cheese L. Mravcova production in the European Union produces a total of Faculty of Chemistry, Institute of Chemistry approximately 40,462,000 tons of whey per year. Most and Technology of Environmental Protection, of this whey is used for production of lactose and in Brno University of Technology, animal feed, but an annual amount of 13,462,000 tons Purkynova 118, 612 00 Brno, Czech Republic of whey per year, containing about 619,250 tons of 948 Ann Microbiol (2011) 61:947–953 lactose, constitutes a surplus product (Koller et al. culture was inoculated into a 250-ml Erlenmeyer flask 2008). For this reason, cheese whey represents a promis- containing 100 ml whey medium. ing substrate for cheap production of PHB in large Whey was obtained from the cheese manufacturer amounts. Pribina Pribyslav (Pribyslav, Czech Republic). The Although biotechnological production of PHB from whole whey was treated in order to remove excess different sugars via condensation of acetyl-CoA units stem- proteins. Whey was acidified to pH 4.0 with 1.0 M ming from hexose catabolism is well described (Kessler and H SO and heated (100°C for 20 min), cooled and 2 4 Wilholt 1999), only a limited number of bacterial strains centrifuged at 8,000 rpm for 5 min. The treated whey directly convert lactose into PHB. A few reports are was used in experiments after adjusting the pH to 7.0 with available on PHB production from lactose and whey by 1.0 M NaOH and MM medium components (except −1 recombinant Escherichia coli (Wong and Lee 1998;Ahn et lactose), and 0.1 g l yeast extract were added at the al. 2000). Methylobacterium sp. ZP24 (Yellore and Desai concentration used in MM unless otherwise indicated. 1998;Nathet al. 2007) and the thermophillic bacterium Thermus thermophilus HB8 (Pantazaki et al. 2009) are also Analytical methods able to utilize whey lactose for PHA biosynthesis. Recently, Pandian et al. (2010) reported PHA production from a Cell growth was monitored by measuring the absorbance of mixture of rice bran, non-specified diary waste and sea water culture broth at 630 nm on a Helios α instrument (Unicam, employing a Gram-positive bacterium isolated from brackish Leeds, UK) after suitable dilution with distilled water. Cell water. Based on morphological and physiological properties biomass was calculated using a calibration curve at A 630 nm and the nucleotide sequence of its 16S rRNA, it was and dry cell mass. For dry cell mass determination, suggested that the isolate was closely related to Bacillus harvested cells (centrifugation: 8,000 rpm, 10 min) were megaterium (Pandian et al. 2010). Pseudomonas hydro- dried at 105°C to constant weight. The supernatant obtained genovora was also used for PHA production using cheese by centrifugation of the culture broth at 8,000 rpm for whey, but this bacterial strain is not able to utilize lactose 10 min was used for analysis of residual lactose by the directly, requiring hydrolysis of whey lactose prior to Somogyi-Nelson method (Deng and Tabatabai 1994). cultivation (Koller et al. 2008). The PHB content in dried cells was determined by gas We have previously reported that Bacillus megaterium chromatography (Finnigan Trace GC Ultra, Austin, TX, CCM 2037 is able to utilize lactose and accumulate PHB column DB-WAX 30 m by 0.25 mm) with mass spectrom- (Obruca et al. 2008). In this study, we focused on etry detection (Finnigan Trace DSQ) according to Brandl et optimization of the whey medium in order to enhance al. (1988). PHB and biomass yields. Moreover, application of exoge- nous stress was studied as a potential strategy to enhance Analysis of treated whey PHB biosynthesis in cells. Concentration of dry matter was estimated after drying (105°C) 10 ml whey to constant weight. Ash content was Materials and methods determined as the weight of solids after incubation of 2 ml whey at 800°C for 2 h. The phosphorus content was Microorganism, media and growth conditions measured by the molybdenum blue colorimetric method (A ) using whey ash. The concentration of soluble 610 nm Bacillus megaterium CCM 2037 was obtained from the protein was performed using the biuret method with bovine Czech Collection of Microorganisms (Brno, Czech serum albumin as a standard. Whey sugar content was Republic). estimated by HPLC (pump LCP 4020, thermostat LCO −1 CCM Bacillus medium, consisting of peptone 5 g l , 101, degasser DG-1210, refractometric detector RIDK 102; −1 −1 −1 yeast extract 3 g l , MnSO 0.01 g l and agar 20 g l , Ecom, Czech Republic) with a ZOBRAX NH column 4 2 was used for maintaining the culture. Mineral medium (150 cm×4,6 mm, 5 μm; Chromservis, Czech Republic), (MM) was used for inoculum preparation. MM contained chromatographic conditions were: 25°C; acetonitrile: water −1 −1 −1 lactose 8 g l , (NH ) SO 5g l ,Na HPO 2.5 g l , 75:25; mobile phase flow 1.0 ml/min. 4 2 4 2 4 −1 −1 −1 KH PO 2.5 g l , MgSO 0.2 g l and MnSO 0.01 g l . 2 4 4 4 The initial pH of the medium was adjusted to 7.0. Inoculum Media optimization using Placket-Burman experimental was developed in 250-mL Erlenmeyer flasks containing design 100 ml medium. MM medium was inoculated with bacterial culture and cultivated at an agitation speed of The dilution of whey (carbon source), and concentrations of 150 rpm, at 30°C for 24 h. Subsequently, 5 ml of the nitrogen source [(NH ) SO ], mineral salts and yeast 4 2 4 Ann Microbiol (2011) 61:947–953 949 Table 1 Range of factors Factor Name Level studied in the Placket-Burman experiment 1 −1 A Whey Not-diluted Diluted -1 −1 (Lactose 40 g l ) (Lactose 40 g l ) -1 −1 B (NH4) SO 5g l 1g l 2 4 -1 −1 CNa HPO ;KH PO (1:1) 5 g l 1g l 2 4 2 4 -1 −1 D MgSO 0.2 g l 0.04 g l -1 −1 E Yeast autolysate 1 g l 0.1 g l extract were tested using a Placket-Burman experimental addition, the whey could be expected to contain some design. Each parameter was tested at two levels, high (+1) minor components such as free amino acids and vitamins and low (−1) (Table 1). A design of 12 experiments was (not analyzed), which are likely to support bacterial formulated for five factors using Minitab software. The growth. experiments were performed in 250-ml Erlenmeyer flasks containing 100 ml whey medium at 150 rpm, 30°C for 50 h Supplementation of whey by salts in duplicate. Response was measured in terms of (1) biomass production, (2) PHB accumulation in cells (% w/w), and (3) In order to test whether whey itself contains all necessary PHB yields. elements for bacterial growth, a culture of Bacillus megaterium was inoculated into whey with and without added salts according to MM (Fig. 1). Biomass and PHB yields were analyzed after 48 h. Results and discussion In whey medium without added salts, bacterial growth and PHB production were rather low. Conversely, addition Analysis of whey composition of salts promoted the growth of bacterial culture and also improved PHB production (almost 10 times). An explana- Used cheese whey contains about 6.8% of dry matter, tion could be that, despite excess salts, whey itself lacks composed mainly of sugars and salts. Moreover, some nitrogen, phosphorus, magnesium and manganese. There- soluble proteins are still present in whey even after the fore, these components must be added in the form of treatment. In spite of the high concentration of salts mineral salts. (expressed as ash), which could result in high osmotic pressure influencing bacterial growth negatively, the Optimization of whey supplementation tested wastecheese wheyseems to beapromising complex substrate for PHB production. Whey sugars For multivariable processes such as biotechnological (lactose and glucose; galactose was not detected) could systems, in which numerous potentially influential be utilized by the bacterial culture as carbon sources, factors are involved, it is not always obvious to soluble proteins could serve as a complex carbon and determine which are the most important. Hence, it is nitrogen sources, and a low concentration of potential phosphorus sources was also observed (Table 2). In Table 2 Composition of cheese whey. Results given are average ± standard deviation; each analysis was performed in triplicate Substance Concentration Water 93% −1 Dry matter 68 g l −1 Ash 27.10±0.30 g l −1 Lactose 39.60±0.45 g l −1 Glucose 0.35±0.02 g l 3− −1 PO 63.0±1.2 mg l −1 Fig. 1 Biomass and poly(3-hydroxybutyrate) (PHB) yields by Soluble proteins 2.0±0.1 g l Bacillus megaterium on whey with and without addition of salts 950 Ann Microbiol (2011) 61:947–953 Table 3 Experimental design and responses of Placket-Burman study -1 2- -1 -1 -1 -1 -1 Whey (NH ) SO [g l ]PO [g l ] MgSO [g l ] Yeast Extract [g l ] Biomass [g l ] PHB [%] PHB [g l ] 4 2 4 4 4 1 None-dilution 1.0 5.0 0.04 0.10 0.90 2.06 0.02 2 None-dilution 5.0 1.0 0.20 0.10 0.76 3.20 0.02 3 Diluted 5.0 5.0 0.04 1.00 2.56 16.26 0.42 4 None-dilution 1.0 5.0 0.20 0.10 0.92 1.90 0.02 5 None-dilution 5.0 1.0 0.20 1.00 0.77 5.08 0.04 6 None-dilution 5.0 5.0 0.04 1.00 0.75 1.95 0.01 7 Diluted 5.0 5.0 0.20 0.10 2.40 27.36 0.66 8 Diluted 1.0 5.0 0.20 1.00 2.02 12.72 0.26 9 Diluted 1.0 1.0 0.20 1.00 1.95 13.51 0.26 10 None-dilution 1.0 1.0 0.04 1.00 1.01 2.17 0.02 11 Diluted 5.0 1.0 0.04 0.10 2.47 35.48 0.87 12 Diluted 1.0 1.0 0.04 0.10 2.99 35.49 1.06 necessary to submit the process to an initial screening PHB production. This is probably due to the fact that yeast design prior to optimization. Plackett–Burman method- extract can serve as a nitrogen source, and PHB biosyn- ology could be a tool for this initial screening, because thesis is much more pronounced under nitrogen-limiting it makes it possible to determine the influence of conditions (Kessler and Wilholt 1999). Therefore, in various factors with only a small number of trials subsequent experiments only a low concentration −1 (Khanna and Srivastava 2005). (0.1 g l ) of yeast extract was added to the whey medium. We used Placket-Burman methodology to optimize the Conversely, whey dilution seems to be crucial for biomass composition of whey medium for PHB production. Five as well as for PHB production (P<0.05). A high factors were selected for optimization, and each factor was concentration of salts and lactose in undiluted whey tested at two levels, high and low (Table 1). PHB and medium probably caused high osmotic pressure, which biomass yields were analyzed after 50 h. The experiment consequently inhibited bacterial growth and PHB biosyn- was designed by Minitab software. thesis (t values<0). High concentrations of lactose could According to the results of the Placket-Burman study also induce substrate growth inhibition, hence whey (results summarized in Tables 3, 4), the concentrations of dilution was optimized in order to achieve maximal 3− (NH ) SO ,PO and MgSO are not statistically biomass and PHB yields (Fig. 2). 4 2 4 4 4 important either for biomass or for PHB production (P> The highest biomass and PHB yields were obtained 0.05). Nevertheless, in previous experiments addition of when whey was diluted to a lactose concentration of −1 salts had strongly enhanced biomass and PHB production; 20gl . Thereafter, biomass and PHB yields were −1 −1 therefore, in subsequent experiments salts were added at 2.51 g l and 0.79 g l , respectively. the concentration used as the lower level (−1) in the The growth and PHB production course of B. Placket-Burman analysis. The addition of yeast extract, megaterium in optimized whey medium were also deter- which was tested as a potential source of vitamins, amino mined. Growth was accompanied by lactose utilization acids etc., had a statistically significant negative impact on throughout the whole cultivation. Biomass formation Table 4 The results of data -1 -1 Biomass [g l ] PHB [%] PHB [g l ] analysis (t-values and P values) for the effect of medium compo- a a a t value P value t value P value t value P value nents on growth and poly(3- hydroxybutyrate) (PHB) Whey concentration -11.46 0.000 -7.53 0.000 -6.40 0.001 production (NH ) SO -0.10 0.923 1.30 0.241 0.73 0.493 4 2 4 2- PO -0.48 0.647 -1.98 0.095 -1.70 0.139 MgSO -2.31 0.051 -1.79 0.123 -2.17 0.073 t value is statistically significant Yeast extract -1.69 0.142 -3.26 0.017 -3.09 0.021 only if P<0.05 Ann Microbiol (2011) 61:947–953 951 Fig. 2 The effect of whey dilution (expressed as lactose concentration) on PHB and bio- mass production th reached a maximum after 28 h of cultivation; thereafter, ethanol and hydrogen peroxide at the 25 hour of th stationary phase occurred. This lasted until the 50 hour cultivation (Table 5). of cultivation, when biomass concentration started to Both hydrogen peroxide and ethanol increased PHB decrease. The highest PHB yields were observed at the biosynthesis in B. megaterium cells. The most effective th 50 hour (see Fig. 3). strategy was the application of 1% ethanol, which enhanced PHB yields about 41% as compared to the PHB production under stress conditions control culture (Table 5). In contrast, application of hydrogen peroxide enhanced PHB yields only very We have recently reported that the stress response of slightly. Cupriavidus necator H16 to ethanol and hydrogen peroxide The reason why ethanol enhances PHB yield is that is accompanied by enhanced PHB accumulation. However, ethanol is metabolized via oxidation to acetyl-CoA. the stress has to be applied at the beginning of the During these reactions, reduced coenzymes NAD(P)H stationary phase, and at an optimized level. This strategy stimulating flux of acetyl-CoA into PHB biosynthetic could be used in biotechnology as a simple, cheap and pathway are formed, and free CoA, which inhibits PHB effective tool to enhance total PHB yields (Obruca et al. biosynthesis, is used to build acetyl-CoA. Moreover, 2010a, b). In order to study whether this strategy could also acetyl-CoA, as the final product of ethanol metabolism, be used in Bacillus megaterium cultivated on cheap whey is the initial substrate of the PHB biosynthetic pathway medium, we decided to apply different concentrations of (Obruca et al. 2010b). Fig. 3 Growth and production characteristics of Bacillus megaterium in optimized whey medium 952 Ann Microbiol (2011) 61:947–953 Table 5 PHB and biomass yields after stress factor application at the environmentally friendly recovery of PHA using lytic th 25 hour of cultivation. Results given are average ± standard enzymes such as lysozyme or mutanolysin. Finally, Gram- deviation; each cultivation was performed and analyzed in triplicate negative bacteria, which are currently the only commercial -1 -1 Biomass [g l ] PHB [g l ] PHB [%] sources of PHA, accumulate lipopolysacharides that co- purify with PHA and cause immunogenic reactions. This Control 2.82±0.11 1.05±0.05 37.23 complicates application of PHA in medicine. On the other EtOH 0.5% 2.79±0.08 1.43±0.07 51.23 hand, Gram-positive bacterial strains, such as Bacillus EtOH 1 % 2.87±0.08 1.48±0.08 51.57 megaterium CCM 2037, lack lipopolysacharides, which EtOH 1.5 % 2.68±0.03 1.22±0.12 45.52 makes them a more attractive source of PHA (Valappil et al. H O 1 mM 2.62±0.12 1.18±0.16 45.04 2 2 2007). H O 3 mM 2.75±0.06 1.09±0.03 39.61 2 2 Further experiments should focus on fermentor cultiva- H O 5 mM 2.77±0.09 1.15±0.05 41.53 2 2 tion to reach high cell density and improve PHB yields. Nevertheless, we have proved that application of controlled stress conditions (ethanol) is a promising strategy for improving the process of PHB production from cheese Table 6 summarizes yields of PHA production whey using B. megaterium. employing wild type bacterial strains from cheese whey that have been reported recently in the literature. Total PHB yields obtained in this work are relatively low as Conclusions compared to those obtained in fermentor in fed-batch mode (Nath et al. 2007;Kolleretal 2007). This is due In this work, we tested Bacillus megaterium CCM 2037 predominantly to the relatively low growth of bacterial as a bacterial strain able to utilize waste cheese whey and culture in Erlenmeyer flasks. On the other hand, Pseudo- produce PHB. Because supplementation of cheese whey monas hydrogenovora was employed for PHA production medium with salts is necessary to reach higher PHB from whey in fermentors in fed-batch mode with highest yields, optimization of whey media composition was −1 PHA yields of 1.4 g l (Koller et al. 2008). This is performed. In our experiments, PHB production was comparable to the yield reached in batch mode in our enhanced about 50 times by optimization of cheese −1 flasks experiments (1.5 g l ). medium as compared to cheese whey alone. Furthermore, Optimization of medium composition as well as con- even higher PHB yields can be obtained if the bacterial trolled introduction of stress factors significantly enhanced culture is exposed to 1% ethanol as an exogenous stress PHB content in cells to relatively high levels, even in factor applied at the beginning of stationary phase. This comparison with results reported in the literature (Table 6). novel strategy enhanced PHB production by about 41%. This is beneficial in terms of total PHB yields but, Our results indicate the potential of Bacillus megaterium furthermore, is also likely to reduce the cost of PHA for industrial PHB production from cheap whey substrate. recovery because the PHB content in cells strongly affects Nevertheless, further experiments carried out in laboratory the efficiency and cost of downstream processing (Lee and and semi-productive bioreactors are needed to obtain high Choi 1999). Bacillus megaterium CCM 2032 is a Gram- cell density and improve production parameters still positive strain that could also facilitate simple and further. Table 6 Biomass and polyhydroxyalkanoate (PHA) yield from cheese whey reported in the literature -1 -1 Reference Biomass [g l ] PHA [%] PHA [g l ] Microorganism Cultivation device Nath et al. 2007 3.9 Methylobacterium sp. ZP24 Fermentor Koller et al. 2008 12 1.4 Pseudomonas hydrogenovora Fermentor Koller et al. 2007 12 1.3 Pseudomonas hydrogenovora Fermentor Koller et al. 2007 40 2.7 Hydrogenophaga pseudoflava Fermentor Koller et al. 2007 50 5.5 Haloferax meditarranei Fermentor Yellore and Desai 1998 9.9 59 5.9 Methylobacterium sp. ZP24 Flasks Pantazaki et al. 2009 1.6 35 0.5 Thermus thermophilus HB8 Flasks This work 2.9 51 1.5 Bacillus megaterium CCM 2037 Flasks Ann Microbiol (2011) 61:947–953 953 Acknowledgments This work was supported by projects MSM from whey by Pseudomonas hydrogenovova. Bioresour Technol 0021630501 and BD 16001004 of The Czech Ministry of Education 99:4854–4863 and by the project “Centre for Materials Research at FCH BUT” Lee SY, Choi J (1999) Effect of fermentation performance on the No. CZ.1.05/2.1.00/01.0012 from ERDF. economics of poly-(3-hydroxybutyrate) production by Alcali- genes latus. 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Annals of MicrobiologySpringer Journals

Published: Feb 19, 2011

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