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Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum
Process Biochemistry 44 (2009) 369–372
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum Irina Krallish *, Svetlana Gonta, Ludmila Savenkova Laboratory of Microbial Storage Product Research, Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda blvd. 4, Riga LV-1586, Latvia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2008 Received in revised form 9 September 2008 Accepted 1 December 2008
The effect of aeration level and iron concentration on Azotobacter chroococcum 23 growth, PHB accumulation and antioxidative enzyme activities was investigated in shake flask experiments. Biomass yield and carbon source conversation coefficients increased in the presence of iron in the growth medium and under decreased aeration. The highest biomass production was observed for the culture grown in a medium with 36 mM of initial iron concentration and moderate aeration level. The highest PHB accumulation level (70–72% from cell dry weight) under our experimental conditions was observed at decreased aeration in the growth medium with 180 mM of initial iron concentration. Results obtained prove that both aeration level and iron supply have a marked influence on the activity of SOD and catalase. Bearing in mind the necessity of iron for the synthesis of both enzymes, only catalase showed a specific dependence on the intracellular iron accumulation level. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Azotobacter chroococcum 23 Poly-b-hydroxybutyrate (PHB) Iron accumulation Oxygen Catalase Superoxide dismutase (SOD)
1. Introduction Poly-b-hydroxybutyrate (PHB) is an energy and carbon storage material accumulated intracellularly by numerous microorganisms. Conditions for optimal production of PHB usually include an excess of carbon source and exhaustion of a single nutrient such as nitrogen, sulphur, phosphate, iron, magnesium, potassium or oxygen [1–3]. Cultivation of Azotobacter spp. strains under nitrogen fixation and oxygen limited conditions resulted in low cells and PHB yields [4,5]. This is due to the large amount of ATP and reducing power such as from NADPH, required for nitrogen fixation which are also required for the synthesis of cellular components, including PHB. In a strain of Azotobacter chroococcum 23 the addition of ammonium, sufficient air and regulation of glucose and phosphate supply increased both cell growth and PHB accumulation and a 36 h fed-batch fermentation resulted in a biomass yield 110 g/l with a PHB cellular concentration of 75% dry weight [5,6]. Iron limitation did not induce accumulation of PHB in A. chroococcum 23 as was demonstrated [1] for Alcaligenes eutrophus (also known as Ralstonia eutropha or Cupriavidus necator), moreover, A. chroococcum 23 is able to grow under concentrations of iron in the medium as high as 900 mM [7]. Iron is an essential nutrient for Azotobacter species and is required for optimal growth, respiratory protection, DNA synthesis, for nitrogenase activity and for protection against toxic oxygen
* Corresponding author. Tel.: +371 67034889; fax: +371 67034885. E-mail address: [email protected] (I. Krallish). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.12.003
products generated by active respiration [8]. Usually incorporated in different iron-containing enzymes and proteins or iron-storage proteins, ‘‘free’’ iron is also present within the cell forming a mobile, predominantly Fe2+ pool in the range 10 6 to 10 8 M [9]. The Fe2+ is generally considered as the metabolically active form of iron and is able to enhance the oxygen toxicity by interacting with oxygen and oxygen-reducing products to generate highly reactive and toxic free radicals [9]. Electron leaking from the respiratory chain at the levels of NADH dehydrogenase and ubiquinone sites gives rise to unspecific oxygen reduction and such formation of radicals such as superoxide (O2 ) and H2O2, as the superoxide radical’s dismutation product. Therefore, the rate of respiration can have a significant impact on above mentioned reactive oxygen species (ROS) production [10,11]. One of the most reactive oxygen species – hydroxyl radicals – are generated from H2O2 via Fenton reaction in the presence of free Fe2+ which catalyses this reaction and needs a source of reducing equivalents to regenerate the metal. Cells protect themselves against ROS toxicity via ROS-scavenging enzymes, including SOD, catalase, glutathione/thioredoxine peroxidase or reductase, glucose-6-phosphate dehydrogenase and a number of small molecules with antioxidative function. Among the latter having non-enzymatic antioxidative functionality, especially NADPH, plays an important role in maintaining the strong reducing environment in the cells [10,12]. NADPH is generated during the metabolism of glucose and is required for the proper operation of a variety of synthetic pathways, as well as for scavenging ROS generated by internal or external environmental stress conditions [10]. For Escherichia coli it was shown that NADPH accumulation resulted in down regulating the soxRS (superoxide response)
I. Krallish et al. / Process Biochemistry 44 (2009) 369–372
370
system by keeping the SoxR sensor in a reduced state or in stimulation of Fenton reaction by redox cycling the free iron in cell cytoplasm [13–15]. In PHB-producing bacteria under growthlimited conditions the excess of NADPH is redirected to the PHB synthesis as the cofactor of acetoacetyl-CoA reductase reaction. In that case, synthesis of PHB is used as an alternative electron sink to consume excess NADPH [3]. In this work, the effect of aeration level and iron concentration in growth medium on A. chroococcum 23 growth and PHB biosynthesis were examined. Under the same conditions the activities of SOD and catalase, the enzymes responsible for H2O2 production and decomposition, were also studied.
intracellular iron content was detected after removal of adsorbed iron by treatment of 10 ml of cell suspension (vortex; 1 min, maximal speed), followed by cell sedimentation using centrifugation. The sediment was subsequently treated with 2.5 ml of 7% perchloric acid overnight at room temperature and for 4 h at 90 8C [24] before iron determination using the bipyridyl method. Protein concentration in cell-free extracts was determined according to Bradford assay [25] with bovine serum albumin as the standard. 2.5. Reproducibility of results All data were generated in three or four independent experiments giving consistent results for every type of culture conditions. Representative results are expressed as the mean S.D.
3. Results and discussion
2. Materials and methods 2.1. Bacterial strain and media
3.1. Carbon source and iron consumption, product formation
A. chroococcum 23 strain was grown in a basic nitrogen Burk’s medium supplemented with 20 g/l glucose as carbon source [6]. The Fe-replete medium contained 36 mM or 5 times higher – 180 mM of iron added as soluble FeSO47H2O salt. The medium, made from reagent grade chemicals without addition of iron salts, contained about 0.2 mM Fe (Fe-limited medium). The total PO43 concentration in growth medium was 0.5 g/l which was shown to be optimal for obtaining both high PHB cellular concentration and high total PHB yield in the flask experiments [6]. The pH of the medium was adjusted to 6.9. Sugar solution was autoclaved separately. 2.2. Growth conditions Cells, maintained on slants of Burk’s medium, were used as inoculum (1% v/v) for liquid cultures (initial optical density at 540 nm, 0.07–0.09). Flasks experiments were carried out in 750 ml capacity Erlenmeyer flasks at 30 8C and 190 rpm on a rotary shaker. Aeration was decreased by increasing the culture volume per flask (CVF) [4,16,17]. Samples for determination of substrate consumption, biomass yields and PHB production were taken after 24 h of cultivation; enzyme activity determinations after 18 h of culture growth. At the end of cultivation the cells were harvested by centrifugation (4000 g at 4 8C for 20 min) and washed twice with ice-cold 0.9% NaCl solution. 2.3. Preparation of cell-free extract and enzyme activity measurement A. chroococcum 23 biomass was suspended in 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM EDTA and the cells were ruptured by sonication (UP 200S, Germany). The cell-free extract was obtained by centrifugation (15000 g at 4 8C for 20 min) and was stored at 30 8C until analysed. Catalase (EC 1.11.1.6) activity was determined as described by Aebi [18]. Superoxide dismutase (EC 1.15.1.1.) was assayed according to the method of Halliwell [19]. Calculation of the direct SOD activity was performed according the method used by Nebot et al. [20]. 2.4. Analytical methods Cell growth was monitored by measuring the optical density of the culture broth at 540 nm. The cell concentration was also determined by measuring the dry cell weight (DCW). Residual biomass (RB), the non-PHB biomass, was calculated as biomass total dry weight minus PHB dry weight. The PHB content was determined by gas chromatography (Agilent Technologies 6890N, USA) [6,21]. The glucose concentration was determined using a DNS reagent [22]. The total iron content in the fermentation medium was determined by a,a-bipyridyl method [23]. The
In order to study the effect of a different iron concentration and aeration level on the growth and PHB production for A. chroococcum 23 strain, aerobic batch cultivation in a Burk’s medium with 20 g/l glucose was performed. The fermentation parameters and the results obtained are summarised in Table 1. Glucose was consumed completely after 24 h of cultivation in all variants of cultivation. A supply of iron to the growth medium was necessary for enhanced biomass production in all studied variants of cultivation. Biomass yields and carbon source conversion coefficients increased with the iron presence in the growth medium and under decreased aeration. The highest biomass production (4.4 g/l) was observed for the culture grown in a medium with 36 mM of initial iron concentration and moderate aeration level (CVF 50/750). Fe-limited growth resulted in reduced biomass production and the lowest biomass yield was shown at a high aeration level (CVF 25/750). The PHB intracellular content also was dependent on both aeration level and iron concentration in the growth medium. Under low iron concentration, the PHB content in cells was low compared to higher iron concentration. Decrease of aeration level (CVF 100/750) under Fe-replete conditions resulted in the highest PHB production – 70–72.5% from CDW. These results support the observation that oxygen limitation is one of the factors that promote PHB formation [2,5] and at sufficient oxygen supply, biomass and PHB production increased with the presence of iron in growth medium. A positive influence of iron on biomass production and PHB yield was also observed under conditions of fed-batch fermentation permitting the constant airflow rate and agitation speed (unpublished data). Almost complete consumption of the iron initially present in the growth medium was detected by determination of the total iron content in the fermentation medium after 12 h of incubation. This was independent of the initial iron concentration in the growth medium. The same result was shown when A. chroococcum 23 was grown in media with 900 mM of iron [7]. This may be explained by intensive
Table 1 Growth characteristics of A. chroococcum 23 cells after 24 h growth in Burk’s medium with 20 g/l glucose under incubation conditions differed in initial iron content and aeration level. Aeration (ml medium/ml flask)
Initial iron content (mM)
CDW (g/l)
Yx/s (g/g)a
PHB (%CDW)
RB (g/l)
Iron accumulation (mg/g RB)
Iron intracellular (mg/g RB)
25/750
0.2 36 180
1.6 0.23 2.8 0.22 2.5 0.17
0.08 0.02 0.13 0.02 0.12 0.02
50.6 1.65 54.6 5.11 44.2 3.70
0.8 0.21 1.3 0.17 1.4 0.16
0.01 0.93 0.12 4.70 0.12
0.01 0.88 0.03 2.80 0.02
50/750
0.2 36 180
2.8 0.35 4.4 0.64 4.1 0.57
0.14 0.04 0.20 0.03 0.20 0.03
47.2 2.86 52.0 7.32 51.9 5.51
1.5 0.25 2.1 0.17 1.8 0.12
0.01 1.10 0.04 5.20 0.10
0.01 0.70 0.19 3.50 0.45
100/750
0.2 36 180
2.2 0.27 4.1 0.44 4.0 0.32
0.12 0.04 0.21 0.02 0.21 0.05
49.5 4.01 70.2 7.28 72.5 3.88
1.1 0.09 1.2 0.33 1.1 0.10
0.01 1.95 0.28 8.40 0.61
0.01 1.86 0.28 7.20 1.1
a
Yx/s biomass yield (x) per consumed glucose (s) units.
I. Krallish et al. / Process Biochemistry 44 (2009) 369–372
accumulation of iron into intracellular space and/or iron adsorption on the cell surface. This speculation is supported by intracellular iron content measured in the residual cellular biomass. The results are presented in Table 1. Almost all iron was accumulated intracellularly from the medium with 36 mM iron and ca. 60–86% from medium with 180 mM iron, independent of the aeration level. Iron is a major trace element for all bacteria and normally represents about 0.02% from bacterial dry weight [9]. In our experiment, the intracellular iron content was dependent on cultivation conditions and reached 0.07–0.72% of the cell residual biomass. The highest intracellular iron content was observed at Fereplete cultivation conditions (180 mM of initial iron content in growth medium) with low aeration level (CVF 100/750). Our results support the observation that A. chroococcum, like A. vinelandii, can be easily grown in aerobic media with no added iron because of its ability to produce siderophores for the accumulation of iron, in contrast to A. salinestris, for example, which will not grow in Fe-depleted culture [26]. 3.2. Iron supply and aeration level influence on activity of antioxidant enzymes Several intracellular components are constitutively present and help to maintain an intracellular reducing environment or to scavenge ROS. Among them are non-enzymatic antioxidants (NADPH and NADH pools, glutathione), specific enzymes (SOD, catalase, glutathione peroxidase, proteolytic and lipolytic enzymes) and DNA-repair systems [10]. An effect of the different iron concentration and aeration intensity on the specific activity of catalase and SOD was determined after 18 h of A. chroococcum 23 culture growth at a time when glucose was not completely consumed and cellular oxidative metabolism was not inhibited by carbon substrate depletion (Fig. 1). The activity of catalase increased almost linearly during growth and attained its highest activity in the stationary phase of growth (data not shown). Such an
Fig. 1. Specific enzyme activities of catalase and SOD expressed as mmol/min/mg protein during A. chroococcum 23 cell growth in Burk’s medium with different initial iron content and aeration level (cells were harvested at the end of batch fermentation after 24 h of growth).
371
increase in catalase activity during the stationary phase of growth was also observed for Pseudomonas aeruginosa [27]. The catalase activity showed positive response to the presence of available iron – the lowest activity (15–23 mmol/min/mg protein) was observed in cells grown in Fe-limited medium, but the presence of iron in growth medium increase the enzyme activity 2–6 times, independent of the aeration level (Fig. 1). The highest catalase activity (103 mmol/min/ mg proteins) was attained at low aeration level (CVF 100/750) and 36 mM iron concentration in growth medium. The activity of SOD exceeds the catalase activity severalfold in all cultivation variants and presents the maximal activity 217–222 mmol/min/mg protein at high aeration level (CVF 25/750). If the catalase activity increased during oxygen limitation at Fe-replete conditions, the SOD activity, in contrast, decreases in parallel to the decrease of aeration level. Taking in account that the bacterial respiratory system is one of the main sources for production of superoxide radicals [10,11], the limitation of oxygen respiration led to reduced superoxide radical generation. The dependence of catalase and SOD activity on the availability of iron differed for members of the genus Azotobacter. For A. chroococcum ATCC 9043 under Fe-limiting conditions no activity of catalase and SOD in media containing 0–1.0 mM Fe3+ was observed; however in medium containing 10 mM Fe3+ the activity of both enzymes was increased. In such cases, respiration by ironlimited cells represents a considerable hazard, since not only are superoxide radicals and hydrogen peroxide generated, but also ironlimited cells have very low SOD activity [16]. In contrast, the A. vinelandii strain demonstrated active catalase and low SOD activity under iron starvation conditions and with increased iron availability catalase appeared to decrease at higher iron content, but SOD activity was notably increased [16]. Since there is only a single ironcontaining SOD in both A. vinelandii and A. chroococcum, the restored SOD activity can be explained by modulation of Fe-SOD expression under iron-sufficient and iron-limited growth conditions [28,29]. This implies that intracellular ‘‘free’’ ferrous ion needs to be carefully controlled, especially in the case of low activities of antioxidant enzymes. In our experiments under Fe-limited conditions of growth, catalase activity was low independent of aeration level, but SOD activity was markedly higher and positively influenced by aeration level. This suggests that the function and regulation mechanisms of both enzymes as well as their dependence on the presence of iron, which is necessary for both enzyme synthesis, are not similar and clearly defined for different Azotobacter strains. Bearing in mind the importance of iron supply during cell growth and enzymes activity expression, the dependence of the activity of both enzymes on the accumulation level of intracellular iron becomes clear. Enzymatic activity after 18 h of growth was attributed to intracellular iron content (Fig. 2). In contrast to the large differences in intracellular iron content in both cells grown in Fe-replete medium, catalase and SOD activities did not differ significantly. SOD presented quite a high activity at all intracellular iron concentrations and the enzyme activity decreased in parallel to the decrease of aeration level. In Fedepleted media SOD activity also decreased together with the decrease in aeration level, but its activity was higher even at low aeration level (CVF 100/750), compared with the enzyme activity in Fe-replete media under the same aeration level. Contrastingly, the catalase activity showed a strong positive dependence on the intracellular iron concentration in Fe-replete medium and low activity under Fe-limited growth conditions (Fig. 2). The results are in agreement with other reported work showing that sufficient iron supply is required for enzymatic protection against toxic oxygen products [29,26]. A. chroococcum 23 as a member of the genus Azotobacter having extremely high metabolic and respiratory activity typically possesses very active catalase and SOD enzymes to protect the cells from toxic partially reduced oxygen products [10,12,13]. This can explain the high activity of SOD even under Felimiting conditions, as seen in our experiments (Fig. 2). In that case,
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Fig. 2. Effect of the intracellular iron accumulation and aeration level on the specific enzyme activities of catalase and SOD in extracts of cells harvested until the beginning of the stationary phase of growth (cells were harvested after 18 h of growth).
the cells required a small amount of iron, either from medium or as carry-over of intracellular iron in the inoculums, for growth and siderophores production for iron accumulation sufficient for SOD synthesis [8,16]. Therefore, the correct ratio between SOD and catalase activity, is very important for the oxidative metabolism of cells and should be investigated under regulated oxygen and iron supply during fed-batch or continuous cultivation. Further investigations considering the importance of PHB synthesis as a sink for the excess of reducing equivalents under normal and oxidative stress conditions are continuing. Acknowledgement This work was supported by a grant 04.1133 from the Latvian Council of Science. References [1] Steinbu¨chel A, Schlegel HG. Excretion of pyruvate by mutants of Alcaligenes eutrophus, which are impaired in the accumulation of poly(b-hydroxybutyric acid) (PHB), under conditions permitting synthesis of PHB. Appl Microbiol Biotechnol 1989;31:168–75. [2] Anderson A, Dawes E. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Mol Biol Rev 1990;54:450–72. [3] Madison LL. Huisman GW Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 1999;63:21–53.
[4] Page WJ, Knosp O. Hyperproduction of poly-b-hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Biotechnol 1989;55:1334–9. [5] Lee IY, Stegantseva EM, Savenkova L, Park YH. Effects of nitrogen and oxygen supply on production of poly-b-hydroxybutyrate in Azotobacter chroococcum. J Microbiol Biotechnol 1995;5:100–4. [6] Savenkova L, Gercberga Z, Kizhlo Z, Stegantseva E. Effect of phosphate supply and aeration on poly-b-hydroxybutyrate production in Azotobacter chroococcum. Process Biochem 1999;34:109–14. [7] Gonta S, Savenkova L, Dzene A, Tupureina V. Physical and mechanical characteristics of polyhydroxybutyrate produced by Azotobacter chroococcum in media with different iron concentrations. In: Proceeding of Baltic Polymer Symposium; 2003.p. 94–7. [8] Tindale AE, Mehrotra M, Ottem D, Page WP. Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress. Microbiology 2000;146:1617–26. [9] Andrews SC. Iron storage in bacteria. Adv Microb Physiol 1998;40:281–351. [10] Cabiscol E, Tamarit J, Ros J. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 2000;3:3–8. [11] Gonzalez-Flecha B, Demple B. Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli. J Biol Chem 1995;270:13681–7. [12] Dingler C, Oelze J. Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations. Arch Microbiol 1987;147:291–4. [13] Gardner PR, Fridovich I. NADPH inhibits transcription of the Escerichia coli manganese superoxide dismutase gene (sodA) in vitro. J Biol Chem 1993;268:12958–63. [14] Koo MS, Lee JH, Rah SY, Yeo WS, Lee JW, Lee KL, et al. A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J 2003;22:2614–22. [15] Giro´ M, Carrillo N, Krapp AR. Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escerichia coli. Microbiology 2006;152:1119–28. [16] Page WJ, Jackson L, Shivprasad S. Sodium-dependent Azotobacter chroococcum strains are aeroadaptive, microaerophilic, nitrogen-fixing bacteria. Appl Environ Microbiol 1988;54:2123–8. [17] Cornish AS, Page WJ. The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management. Microbiology 1998;144:1747–54. [18] Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. Weinheim: Verlag Chemie; 1970. p. 636–41. [19] Halliwell B. The superoxide dismutase activity of iron complexes. FEBS Lett 1975;56:34–8. [20] Nebot C, Moutet M, Huet P, Xu JZ, Yadan JC, Chaudiere J. Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol. Anal Biochem 1993;214:442–51. [21] Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas chromatographic method for the determination of poly-b-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol 1978;6:29–37. [22] Miller GL. Use of the dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 1959;31:426–8. [23] Osaki S, Johnson DA, Frieden E. The mobilization of iron from the perfused mammalian liver by a serum copper enzyme, ferooxidase I. J Biol Chem 1971;246:3018–23. [24] Page WJ, Huyer M. Derepression of the Azotobaciter vinelandii siderophore system, using iron-containing minerals to limit iron repletion. J Bacteriol 1984;158:496–502. [25] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [26] Page WJ, Manchak J, Yohemas M. Inhibition of Azotobacter salinestris growth by zinc under iron-limited conditions. Can J Microbiol 1996;42:655–61. [27] Frederick JR, Elkins JG, Bollinger N, Hasset DJ, McDermott TR. Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells. Appl Environ Microbiol 2001;67:1375–9. [28] Caldwell JM, Hassan HM. Azotobacter chroococcum does not contain sodA or its gene product Mn-superoxide dismutase. Can J Microbiol 2002;48:183–7. [29] Qurollo BA, Bishop PE, Hassan HM. Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability. Can J Microbiol 2001;47:63–71.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum Irina Krallish *, Svetlana Gonta, Ludmila Savenkova Laboratory of Microbial Storage Product Research, Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda blvd. 4, Riga LV-1586, Latvia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2008 Received in revised form 9 September 2008 Accepted 1 December 2008
The effect of aeration level and iron concentration on Azotobacter chroococcum 23 growth, PHB accumulation and antioxidative enzyme activities was investigated in shake flask experiments. Biomass yield and carbon source conversation coefficients increased in the presence of iron in the growth medium and under decreased aeration. The highest biomass production was observed for the culture grown in a medium with 36 mM of initial iron concentration and moderate aeration level. The highest PHB accumulation level (70–72% from cell dry weight) under our experimental conditions was observed at decreased aeration in the growth medium with 180 mM of initial iron concentration. Results obtained prove that both aeration level and iron supply have a marked influence on the activity of SOD and catalase. Bearing in mind the necessity of iron for the synthesis of both enzymes, only catalase showed a specific dependence on the intracellular iron accumulation level. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Azotobacter chroococcum 23 Poly-b-hydroxybutyrate (PHB) Iron accumulation Oxygen Catalase Superoxide dismutase (SOD)
1. Introduction Poly-b-hydroxybutyrate (PHB) is an energy and carbon storage material accumulated intracellularly by numerous microorganisms. Conditions for optimal production of PHB usually include an excess of carbon source and exhaustion of a single nutrient such as nitrogen, sulphur, phosphate, iron, magnesium, potassium or oxygen [1–3]. Cultivation of Azotobacter spp. strains under nitrogen fixation and oxygen limited conditions resulted in low cells and PHB yields [4,5]. This is due to the large amount of ATP and reducing power such as from NADPH, required for nitrogen fixation which are also required for the synthesis of cellular components, including PHB. In a strain of Azotobacter chroococcum 23 the addition of ammonium, sufficient air and regulation of glucose and phosphate supply increased both cell growth and PHB accumulation and a 36 h fed-batch fermentation resulted in a biomass yield 110 g/l with a PHB cellular concentration of 75% dry weight [5,6]. Iron limitation did not induce accumulation of PHB in A. chroococcum 23 as was demonstrated [1] for Alcaligenes eutrophus (also known as Ralstonia eutropha or Cupriavidus necator), moreover, A. chroococcum 23 is able to grow under concentrations of iron in the medium as high as 900 mM [7]. Iron is an essential nutrient for Azotobacter species and is required for optimal growth, respiratory protection, DNA synthesis, for nitrogenase activity and for protection against toxic oxygen
* Corresponding author. Tel.: +371 67034889; fax: +371 67034885. E-mail address: [email protected] (I. Krallish). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.12.003
products generated by active respiration [8]. Usually incorporated in different iron-containing enzymes and proteins or iron-storage proteins, ‘‘free’’ iron is also present within the cell forming a mobile, predominantly Fe2+ pool in the range 10 6 to 10 8 M [9]. The Fe2+ is generally considered as the metabolically active form of iron and is able to enhance the oxygen toxicity by interacting with oxygen and oxygen-reducing products to generate highly reactive and toxic free radicals [9]. Electron leaking from the respiratory chain at the levels of NADH dehydrogenase and ubiquinone sites gives rise to unspecific oxygen reduction and such formation of radicals such as superoxide (O2 ) and H2O2, as the superoxide radical’s dismutation product. Therefore, the rate of respiration can have a significant impact on above mentioned reactive oxygen species (ROS) production [10,11]. One of the most reactive oxygen species – hydroxyl radicals – are generated from H2O2 via Fenton reaction in the presence of free Fe2+ which catalyses this reaction and needs a source of reducing equivalents to regenerate the metal. Cells protect themselves against ROS toxicity via ROS-scavenging enzymes, including SOD, catalase, glutathione/thioredoxine peroxidase or reductase, glucose-6-phosphate dehydrogenase and a number of small molecules with antioxidative function. Among the latter having non-enzymatic antioxidative functionality, especially NADPH, plays an important role in maintaining the strong reducing environment in the cells [10,12]. NADPH is generated during the metabolism of glucose and is required for the proper operation of a variety of synthetic pathways, as well as for scavenging ROS generated by internal or external environmental stress conditions [10]. For Escherichia coli it was shown that NADPH accumulation resulted in down regulating the soxRS (superoxide response)
I. Krallish et al. / Process Biochemistry 44 (2009) 369–372
370
system by keeping the SoxR sensor in a reduced state or in stimulation of Fenton reaction by redox cycling the free iron in cell cytoplasm [13–15]. In PHB-producing bacteria under growthlimited conditions the excess of NADPH is redirected to the PHB synthesis as the cofactor of acetoacetyl-CoA reductase reaction. In that case, synthesis of PHB is used as an alternative electron sink to consume excess NADPH [3]. In this work, the effect of aeration level and iron concentration in growth medium on A. chroococcum 23 growth and PHB biosynthesis were examined. Under the same conditions the activities of SOD and catalase, the enzymes responsible for H2O2 production and decomposition, were also studied.
intracellular iron content was detected after removal of adsorbed iron by treatment of 10 ml of cell suspension (vortex; 1 min, maximal speed), followed by cell sedimentation using centrifugation. The sediment was subsequently treated with 2.5 ml of 7% perchloric acid overnight at room temperature and for 4 h at 90 8C [24] before iron determination using the bipyridyl method. Protein concentration in cell-free extracts was determined according to Bradford assay [25] with bovine serum albumin as the standard. 2.5. Reproducibility of results All data were generated in three or four independent experiments giving consistent results for every type of culture conditions. Representative results are expressed as the mean S.D.
3. Results and discussion
2. Materials and methods 2.1. Bacterial strain and media
3.1. Carbon source and iron consumption, product formation
A. chroococcum 23 strain was grown in a basic nitrogen Burk’s medium supplemented with 20 g/l glucose as carbon source [6]. The Fe-replete medium contained 36 mM or 5 times higher – 180 mM of iron added as soluble FeSO47H2O salt. The medium, made from reagent grade chemicals without addition of iron salts, contained about 0.2 mM Fe (Fe-limited medium). The total PO43 concentration in growth medium was 0.5 g/l which was shown to be optimal for obtaining both high PHB cellular concentration and high total PHB yield in the flask experiments [6]. The pH of the medium was adjusted to 6.9. Sugar solution was autoclaved separately. 2.2. Growth conditions Cells, maintained on slants of Burk’s medium, were used as inoculum (1% v/v) for liquid cultures (initial optical density at 540 nm, 0.07–0.09). Flasks experiments were carried out in 750 ml capacity Erlenmeyer flasks at 30 8C and 190 rpm on a rotary shaker. Aeration was decreased by increasing the culture volume per flask (CVF) [4,16,17]. Samples for determination of substrate consumption, biomass yields and PHB production were taken after 24 h of cultivation; enzyme activity determinations after 18 h of culture growth. At the end of cultivation the cells were harvested by centrifugation (4000 g at 4 8C for 20 min) and washed twice with ice-cold 0.9% NaCl solution. 2.3. Preparation of cell-free extract and enzyme activity measurement A. chroococcum 23 biomass was suspended in 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM EDTA and the cells were ruptured by sonication (UP 200S, Germany). The cell-free extract was obtained by centrifugation (15000 g at 4 8C for 20 min) and was stored at 30 8C until analysed. Catalase (EC 1.11.1.6) activity was determined as described by Aebi [18]. Superoxide dismutase (EC 1.15.1.1.) was assayed according to the method of Halliwell [19]. Calculation of the direct SOD activity was performed according the method used by Nebot et al. [20]. 2.4. Analytical methods Cell growth was monitored by measuring the optical density of the culture broth at 540 nm. The cell concentration was also determined by measuring the dry cell weight (DCW). Residual biomass (RB), the non-PHB biomass, was calculated as biomass total dry weight minus PHB dry weight. The PHB content was determined by gas chromatography (Agilent Technologies 6890N, USA) [6,21]. The glucose concentration was determined using a DNS reagent [22]. The total iron content in the fermentation medium was determined by a,a-bipyridyl method [23]. The
In order to study the effect of a different iron concentration and aeration level on the growth and PHB production for A. chroococcum 23 strain, aerobic batch cultivation in a Burk’s medium with 20 g/l glucose was performed. The fermentation parameters and the results obtained are summarised in Table 1. Glucose was consumed completely after 24 h of cultivation in all variants of cultivation. A supply of iron to the growth medium was necessary for enhanced biomass production in all studied variants of cultivation. Biomass yields and carbon source conversion coefficients increased with the iron presence in the growth medium and under decreased aeration. The highest biomass production (4.4 g/l) was observed for the culture grown in a medium with 36 mM of initial iron concentration and moderate aeration level (CVF 50/750). Fe-limited growth resulted in reduced biomass production and the lowest biomass yield was shown at a high aeration level (CVF 25/750). The PHB intracellular content also was dependent on both aeration level and iron concentration in the growth medium. Under low iron concentration, the PHB content in cells was low compared to higher iron concentration. Decrease of aeration level (CVF 100/750) under Fe-replete conditions resulted in the highest PHB production – 70–72.5% from CDW. These results support the observation that oxygen limitation is one of the factors that promote PHB formation [2,5] and at sufficient oxygen supply, biomass and PHB production increased with the presence of iron in growth medium. A positive influence of iron on biomass production and PHB yield was also observed under conditions of fed-batch fermentation permitting the constant airflow rate and agitation speed (unpublished data). Almost complete consumption of the iron initially present in the growth medium was detected by determination of the total iron content in the fermentation medium after 12 h of incubation. This was independent of the initial iron concentration in the growth medium. The same result was shown when A. chroococcum 23 was grown in media with 900 mM of iron [7]. This may be explained by intensive
Table 1 Growth characteristics of A. chroococcum 23 cells after 24 h growth in Burk’s medium with 20 g/l glucose under incubation conditions differed in initial iron content and aeration level. Aeration (ml medium/ml flask)
Initial iron content (mM)
CDW (g/l)
Yx/s (g/g)a
PHB (%CDW)
RB (g/l)
Iron accumulation (mg/g RB)
Iron intracellular (mg/g RB)
25/750
0.2 36 180
1.6 0.23 2.8 0.22 2.5 0.17
0.08 0.02 0.13 0.02 0.12 0.02
50.6 1.65 54.6 5.11 44.2 3.70
0.8 0.21 1.3 0.17 1.4 0.16
0.01 0.93 0.12 4.70 0.12
0.01 0.88 0.03 2.80 0.02
50/750
0.2 36 180
2.8 0.35 4.4 0.64 4.1 0.57
0.14 0.04 0.20 0.03 0.20 0.03
47.2 2.86 52.0 7.32 51.9 5.51
1.5 0.25 2.1 0.17 1.8 0.12
0.01 1.10 0.04 5.20 0.10
0.01 0.70 0.19 3.50 0.45
100/750
0.2 36 180
2.2 0.27 4.1 0.44 4.0 0.32
0.12 0.04 0.21 0.02 0.21 0.05
49.5 4.01 70.2 7.28 72.5 3.88
1.1 0.09 1.2 0.33 1.1 0.10
0.01 1.95 0.28 8.40 0.61
0.01 1.86 0.28 7.20 1.1
a
Yx/s biomass yield (x) per consumed glucose (s) units.
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accumulation of iron into intracellular space and/or iron adsorption on the cell surface. This speculation is supported by intracellular iron content measured in the residual cellular biomass. The results are presented in Table 1. Almost all iron was accumulated intracellularly from the medium with 36 mM iron and ca. 60–86% from medium with 180 mM iron, independent of the aeration level. Iron is a major trace element for all bacteria and normally represents about 0.02% from bacterial dry weight [9]. In our experiment, the intracellular iron content was dependent on cultivation conditions and reached 0.07–0.72% of the cell residual biomass. The highest intracellular iron content was observed at Fereplete cultivation conditions (180 mM of initial iron content in growth medium) with low aeration level (CVF 100/750). Our results support the observation that A. chroococcum, like A. vinelandii, can be easily grown in aerobic media with no added iron because of its ability to produce siderophores for the accumulation of iron, in contrast to A. salinestris, for example, which will not grow in Fe-depleted culture [26]. 3.2. Iron supply and aeration level influence on activity of antioxidant enzymes Several intracellular components are constitutively present and help to maintain an intracellular reducing environment or to scavenge ROS. Among them are non-enzymatic antioxidants (NADPH and NADH pools, glutathione), specific enzymes (SOD, catalase, glutathione peroxidase, proteolytic and lipolytic enzymes) and DNA-repair systems [10]. An effect of the different iron concentration and aeration intensity on the specific activity of catalase and SOD was determined after 18 h of A. chroococcum 23 culture growth at a time when glucose was not completely consumed and cellular oxidative metabolism was not inhibited by carbon substrate depletion (Fig. 1). The activity of catalase increased almost linearly during growth and attained its highest activity in the stationary phase of growth (data not shown). Such an
Fig. 1. Specific enzyme activities of catalase and SOD expressed as mmol/min/mg protein during A. chroococcum 23 cell growth in Burk’s medium with different initial iron content and aeration level (cells were harvested at the end of batch fermentation after 24 h of growth).
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increase in catalase activity during the stationary phase of growth was also observed for Pseudomonas aeruginosa [27]. The catalase activity showed positive response to the presence of available iron – the lowest activity (15–23 mmol/min/mg protein) was observed in cells grown in Fe-limited medium, but the presence of iron in growth medium increase the enzyme activity 2–6 times, independent of the aeration level (Fig. 1). The highest catalase activity (103 mmol/min/ mg proteins) was attained at low aeration level (CVF 100/750) and 36 mM iron concentration in growth medium. The activity of SOD exceeds the catalase activity severalfold in all cultivation variants and presents the maximal activity 217–222 mmol/min/mg protein at high aeration level (CVF 25/750). If the catalase activity increased during oxygen limitation at Fe-replete conditions, the SOD activity, in contrast, decreases in parallel to the decrease of aeration level. Taking in account that the bacterial respiratory system is one of the main sources for production of superoxide radicals [10,11], the limitation of oxygen respiration led to reduced superoxide radical generation. The dependence of catalase and SOD activity on the availability of iron differed for members of the genus Azotobacter. For A. chroococcum ATCC 9043 under Fe-limiting conditions no activity of catalase and SOD in media containing 0–1.0 mM Fe3+ was observed; however in medium containing 10 mM Fe3+ the activity of both enzymes was increased. In such cases, respiration by ironlimited cells represents a considerable hazard, since not only are superoxide radicals and hydrogen peroxide generated, but also ironlimited cells have very low SOD activity [16]. In contrast, the A. vinelandii strain demonstrated active catalase and low SOD activity under iron starvation conditions and with increased iron availability catalase appeared to decrease at higher iron content, but SOD activity was notably increased [16]. Since there is only a single ironcontaining SOD in both A. vinelandii and A. chroococcum, the restored SOD activity can be explained by modulation of Fe-SOD expression under iron-sufficient and iron-limited growth conditions [28,29]. This implies that intracellular ‘‘free’’ ferrous ion needs to be carefully controlled, especially in the case of low activities of antioxidant enzymes. In our experiments under Fe-limited conditions of growth, catalase activity was low independent of aeration level, but SOD activity was markedly higher and positively influenced by aeration level. This suggests that the function and regulation mechanisms of both enzymes as well as their dependence on the presence of iron, which is necessary for both enzyme synthesis, are not similar and clearly defined for different Azotobacter strains. Bearing in mind the importance of iron supply during cell growth and enzymes activity expression, the dependence of the activity of both enzymes on the accumulation level of intracellular iron becomes clear. Enzymatic activity after 18 h of growth was attributed to intracellular iron content (Fig. 2). In contrast to the large differences in intracellular iron content in both cells grown in Fe-replete medium, catalase and SOD activities did not differ significantly. SOD presented quite a high activity at all intracellular iron concentrations and the enzyme activity decreased in parallel to the decrease of aeration level. In Fedepleted media SOD activity also decreased together with the decrease in aeration level, but its activity was higher even at low aeration level (CVF 100/750), compared with the enzyme activity in Fe-replete media under the same aeration level. Contrastingly, the catalase activity showed a strong positive dependence on the intracellular iron concentration in Fe-replete medium and low activity under Fe-limited growth conditions (Fig. 2). The results are in agreement with other reported work showing that sufficient iron supply is required for enzymatic protection against toxic oxygen products [29,26]. A. chroococcum 23 as a member of the genus Azotobacter having extremely high metabolic and respiratory activity typically possesses very active catalase and SOD enzymes to protect the cells from toxic partially reduced oxygen products [10,12,13]. This can explain the high activity of SOD even under Felimiting conditions, as seen in our experiments (Fig. 2). In that case,
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Fig. 2. Effect of the intracellular iron accumulation and aeration level on the specific enzyme activities of catalase and SOD in extracts of cells harvested until the beginning of the stationary phase of growth (cells were harvested after 18 h of growth).
the cells required a small amount of iron, either from medium or as carry-over of intracellular iron in the inoculums, for growth and siderophores production for iron accumulation sufficient for SOD synthesis [8,16]. Therefore, the correct ratio between SOD and catalase activity, is very important for the oxidative metabolism of cells and should be investigated under regulated oxygen and iron supply during fed-batch or continuous cultivation. Further investigations considering the importance of PHB synthesis as a sink for the excess of reducing equivalents under normal and oxidative stress conditions are continuing. Acknowledgement This work was supported by a grant 04.1133 from the Latvian Council of Science. References [1] Steinbu¨chel A, Schlegel HG. Excretion of pyruvate by mutants of Alcaligenes eutrophus, which are impaired in the accumulation of poly(b-hydroxybutyric acid) (PHB), under conditions permitting synthesis of PHB. Appl Microbiol Biotechnol 1989;31:168–75. [2] Anderson A, Dawes E. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Mol Biol Rev 1990;54:450–72. [3] Madison LL. Huisman GW Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 1999;63:21–53.
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