On-line version ISSN 0104-6632
Braz. J. Chem. Eng. vol.28 no.4 São Paulo Oct./Dec. 2011
H. GhojavandI, II; B. BonakdarpourI, III, *; S. M. HeydarianIV; J. HamediV
IDepartment of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Phone: + (98) (21) 64543146, Fax: + (98) (21) 640 5847, Tehran, Iran. E-mail: firstname.lastname@example.org
IIIOR Research Institute, Research and Technology Directorate, National Iranian Oil Company, Tehran, Iran
IIIFood Process Engineering and Biotechnology Research Centre, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
IVDepartment of Biotechnology, Institute of Advanced Technology (IAT), Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
VMicrobial Biotechnology Lab., Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
Submerged cultivation of Saccharopolyspora erythraea, at different initial spore concentrations, was carried out to study the inter-relationship between inoculum concentration, morphology, rheology and erythromycin production. Pellet morphology was dominant in runs at 103 and 104 spore/ml initial spore concentrations, whereas there was a significant presence of clump morphology in runs at initial spore concentrations of 105-107 spore/ml. The S. erythraea cultivation broths exhibited Newtonian rheology in runs at initial spore concentrations of 103 and 104 spore/ml, whereas at higher initial spore concentrations the rheological data could be fitted with the power law model. Runs in which clump morphology was predominant resulted in the highest erythromycin productivities. The findings of the present work suggest that the predominance of clump morphology, smaller sized clumps and, in the case of non-Newtonian S. erythraea cultivation broths, a decrease in viscosity enhance erythromycin production.
Keywords: Erythromycin; Saccharopolyspora erythraea; Morphology; Inoculum spore concentration.
The information available about the morphology of actinomycetes grown in submerged culture is restricted, even though this group of microorganisms is used to produce many of the antibiotics in current use (Whitaker, 1992). The morphology of these filamentous microorganisms is an important factor when they are employed in the industrial production of secondary metabolites, such as antibiotics. Control of mycelial morphology is often regarded as a prerequisite to ensure increased productivities in industrial applications (Papagianni and Mattey, 2006). For example, a mycelial form of Streptomyces griseus is required for streptomycin production, but pelleted and fragmented forms are undesirable (Schatz and Waksman, 1945). On the other hand, pellets of S. nigrificans have been shown to produce more glucose isomerase than the mycelial form (Whitaker, 1992).
One of the factors that has been reported to affect the morphology of filamentous microorganisms is the inoculum size (Whitaker, 1992; (Glazebrook and Vining, 1992; Lawton et al., 1989; Smith and Calam, 1980; Tucker and Thomas, 1992; Tucker and Thomas, 1994). An inoculum with high concentration of viable spores tends to produce a disperse form of growth, whereas the use of an inoculum with low spore concentration normally results in pellet formation (Whitaker, 1992). The inoculum spore concentration, in which the transition from predominantly pellet to a predominantly clump or dispersed mycelial form occurs, depends on the strain and can be affected by nutrient concentrations and other fermentation conditions (Braun and Vecht-Lifshitz, 1991).
The effect of spore concentration on the morphology of filamentous fungi has been extensively investigated (Smith and Calam, 1980; Tucker and Thomas, 1992; Du et al., 2003), whereas there is less information for actinomycetes. For these types of bacteria the inoculum size has been reported to affect the degree of fragmentation of hyphae (Tresner et al., 1967), the mycelial aggregate size (Glazebrook and Vining, 1992) and the average pellet size (Vecht-Lifshitz et al., 1990), although in some cases the morphology has been reported not to be strongly affected by the inoculum size (Glazebrook and Vining, 1992).
Saccharopolyspora erythraea is an industrially important actinomycete used for the production of erythromycin. The effect of various parameters such as shear, biomass concentration and medium components (Bushell et al., 1997a; Hamedi et al., 2004; Heydarian et al., 1999; Rostamza et al., 2008; Sarra et al., 1996) on the morphology of this actinomycete has been previously reported. To the knowledge of the authors, there is no report in the literature about the effect of inoculum size on the morphology, rheology and erythromycin production by this microorganism; furthermore, the relative erythromycin productivities of pellet and clump forms of this actinomycete have not been considered in any previous study.
In this study, the effect of the morphology of a S. erythraea culture on the production of erythromycin in shake flask cultivation was investigated. A change in morphology was achieved by varying the initial spore concentration in the range of 103-107 spore/mL. Morphological and rheological parameters were characterized in order to gain a better insight into the inter-relationship between S. erythraea morphology, rheology of the cultivation broth and erythromycin productivity in shake flask cultivation.
MATERIALS AND METHODS
Microorganism and Inoculation
The culture used throughout this study was a mutant strain of Saccharopolyspora erythraea (NUR001) provided by the Shafa-e-Sari Pharmaceutical Co., Tehran, Iran (Hamedi et al, 2002). The strain was stored as a spore suspension at -20ºC. Spore suspensions were prepared by initially incubating agar slants of S. erythraea at 30ºC for 10-14 day. The surface of the slants were then scoured with a solution containing 20% (v/v) glycerol and 0.1% (v/v) Tween 80 and the resulting spore suspension was subsequently passed through sterilized cotton. The harvested spores were pooled and stored at -20ºC. The concentration of spores in the suspension was determined by the spread plate method. This was done by plating the spore suspension for 48 hours at 30ºC in nutrient agar medium and determining the spore concentration using the plate count method.
500 mL Erlenmeyer flasks containing 200 mL of fermentation medium were inoculated with appropriate volumes of spore suspensions to obtain initial spore concentrations in the range of 103-107 spore/mL and incubated at 30ºC with shaking at 200 rpm. All experiments were performed in duplicate.
The stock culture was maintained on agar slants containing the following nutrient (in g/L): 10 corn steep liquor, 10 starch, 2.5 CaCO3, 3 (NH3)2SO4, 3 NaCl, 20 agar and 2 mL microelement solution. Microelement solution had the following composition (in g/L): 100 MgSO4·7H2O, 2 FeSO4·7H2O, 2 ZnSO4·7H2O, 0.4 CuSO4·5H2O, 0.1 CoCl2·6H2O, and 0.1 % (v/v) HCl. The fermentation medium composition was as follows (in g/L): 30 glucose, 6 yeast extract, 4 bacteriological peptone, 2 glycine, 0.5 MgSO4·H2O and 0.68 KH2PO4 (Heydarian et al., 1999). The initial medium pH was adjusted to pH=7 ± 0.1. Glucose and KH2PO4 were separately sterilized and aseptically added to the sterile medium. Corn steep liquor and starch were obtained from Shafa-e-Sari Pharmaceutical Co.
A semi-automatic image analysis method was used to characterize the morphology of S. erythraea in samples taken from the shake flasks at the same time each day. In this method, a Bel optical microscope (model BIO2-Video, Bel Photonics, Monza (Milan), Italy) with video output connected to a computer monitor was used to take video clips from slides prepared from appropriately diluted and stained samples. The clips were then processed to obtain between 100-300 picture frames for each slide. The number and dimensions of pellets, clumps and free mycelia were determined manually. The morphological parameters measured in this study for these three growth forms are described in Table 1 (Heydarian et al., 1999; Rodríguez Porcel et al., 2005). For each parameter, the values reported are the mean of between 100-300 measurements.
Microbial dry cell weights (DCW) were determined by filtering 5 mL samples through predried and weighted membrane filters (Millipore, 0.45 µm). The filter was then rinsed with distilled water (2×5 mL) prior to drying in an oven at 100ºC for 24 h. The results presented are the mean of DCW obtained in two separate cultivations.
The total erythromycin concentration in the samples taken from shake flasks was measured as follows (Hamedi et al., 2002): After centrifugation, the cell-free broth was diluted with 0.2 M carbonate/bicarbonate buffer, pH 9.6, and then extracted with an equal volume of chloroform. Extracted erythromycin was mixed with Bromophenol Blue reagent. The absorbance of the separated organic phase was measured at 415 nm. The results presented are the mean of total erythromycin concentrations obtained in two separate cultivations.
The rheological properties of S. erythraea fermentation broths were characterized using a Haake rheometer (Model CV100) employing a concentric cylinder sensor system (type ZB15). The measurements were carried out over a shear rate range of 0.3300 s-1, at room temperature.
RESULTS AND DISCUSSION
Figure 1 shows the morphological population balances of S. erythraea in shake flask cultures for various initial spore concentrations (103-107 spore/mL) as a function of cultivation time. The results showed the existence of three morphological growth forms of S. erythraea (NUR001) at different cultivation times and for various initial spore concentrations, namely pellet, clump and free dispersed mycelium. These different growth forms are illustrated in Figure 2. However, the proportion of each growth form in the culture media was found to depend on both the time of cultivation and the initial spore concentrations. The results show that, with an increase in initial spore concentration, the proportion of S. erythraea cells exhibiting clump morphology increases; pellet morphology is dominant at initial spore concentrations of 103 and 104 spore/mL (Figures 1a and 1b), whereas at 106 and 107 spore/mL clumps are the predominant morphology (Figures 1d and 1e). The results at 106 spore/mL initial spore concentration are in line with a previous report by Heydarian et al. (1997) for another strain of S. erythraea. An increase in the proportion of clump/dispersed growth morphology with an increase in initial spore concentration has also been reported for other filamentous actinomycetes (Whitaker, 1992; El-Enshasy et al., 2000).
The rheological analysis indicated that, in the runs at 103 and 104 spore/mL initial spore concentration -in which pellet was the dominant morphology the cultivation broth exhibited Newtonian characteristics (Figure 3a). With the increase in the concentration of clumps/dispersed mycelia with further increases in initial spore concentration, the cultivation broth became non-Newtonian and the rheological data could be fitted to the power law model (τ = Kγn) with R2>0.999 (results not presented). In the case of runs at an initial spore concentration of 105 spore/mL, the value of n was near 1(results not presented). For runs with initial spore concentrations of 106 and 107 spore/mL (Figure 3b), the consistency index (Κ) increased, whereas the flow behavior (n) decreased with the increase in the proportion of clump/dispersed morphology, indicating that S. erythraea cultivation broth becomes more non-Newtonian under these conditions. Also, as the cultivation proceeded, K decreased and n increased, indicating that the cultivation broth becomes less non-Newtonian with time.
The morphological data presented in Figure 4 shows that the mean major axis of the clumps decreased from day 4-5 onwards and was lower for runs at 106 spore/ml initial spore concentration compared to the runs at 105 and 107 spore/ml. Also, the change in the rheological parameters can be roughly correlated with the trend of change in mean major axis of the clump. For example, in the runs at 106 spore/ml initial spore concentration there is a sharp change in K and n at day 6, with the values remaining fairly constant thereafter; this corresponds to a sharp drop in mean major axis of the clump at the same day. On the other hand, for runs at 107 spore/ml initial spore concentration, K and n show a gradual change with time during cultivation (Figure 3b) that correlates with a gradual drop in the mean major axis of the clumps (Figure 4). The trend of change in dimensions of the clumps is also illustrated pictorially in Figure 5 for runs at 107 spore/mL initial spore concentration.
The morphological data for cultivations, in which either pellet was the dominant morphological form or was present in a significant amount, is presented in Figure 6. The results suggest an increase in pellet size with cultivation time and a subsequent decrease as a result of pellet fragmentation when the cultivation is allowed to proceed long enough (Figure 6a). The maximum pellet size attained during S. erythraea cultivation was the highest for runs at 103 spore/mL initial spore concentration; the maximum pellet sizes achieved at the two higher initial spore concentration were fairly similar to each other. A decrease in pellet size with an increase in inoculum level has been previously reported for a Streptomyces sp. for initial spore concentrations in the range 102-106 spore/mL (Dobson et al., 2008). For cultures of Streptomyces griseus, Kim and Hancock (2000) reported a similar trend for inoculum concentration in the range 1.5×104 to 1.5×106 spore/mL.
The pellet mean hairy length initially increased and then sharply decreased as the S. erythraea cultivation advanced (Figure 6b). The maximum mean hairy length attained during S. erythraea cultivation increased with the increase in initial spore concentration. To the knowledge of the authors, there is no previous report on the variation of main hairy length during cultivation of actinomycetes, but this result is consistent with those reported for Aspergillus terreus (Rodríguez Porcel et al., 2005) and Aspergillus awamori (Cui et al., 1997) fermentations.
The pH of the fermentation media for the different runs was measured daily (results not presented) and varied in the range 5.5-7, depending on the cultivation time and initial spore concentration.
Comparison of the trends of change of dry cell weight and erythromycin production with cultivation time (Fig. 7a and b respectively) indicates that, irrespective of the type of morphology of the cells or the rheology of the cultivation broth, erythromycin production by S. erythraea is growth-associated. The production of secondary metabolites, such as antibiotics, is usually induced under nutrient limitation or other stress conditions. This usually, but not always, means that secondary metabolite production starts when the growth rate declines (i.e., antibiotic production is growth diossociated). In the case of erythromycin production by S. erythraea, previous research has shown that, depending on the environment of S. erythraea cultivation, erythromycin production can be both growth and non-growth associated. (Potvin and Péringer, 1994a, b; Bushell et al., 1997b; Clark et al., 1995; McDermott et al., 1993). For example, the use of ammonium sulfate (Potvin and Péringer, 1994a, b), chemostat cultivation under phosphate limitation (Trilli et al, 1987) and batch culture under N (nitrate) limitation (McDermott et al., 1993; Bushell et al., 1997b) has been reported to result in growth-associated erythromycin production. Various explanations have been given for this phenomenon: Potvin and Peringer (1994b) maintain that erythromycin production by S. erythraea under nutrient-rich conditions can be induced by local diffusional limitations imposed by early formation of mycelia pellets; on the other hand, Bushell et al. (1997b) claim that growth-associated erythromycin production can occur with growth substrates for which S. erythraea has low affinity. Since, in the present study, a complex media was employed for the cultivation of S. erythraea, no explanation can be given for the reason why erythromycin production was found to be growth-associated.
The data presented in Fig 7c show that specific erythromycin production is higher for runs at 106 and 107 spore/ml initial spore concentration compared to runs at lower initial spore concentrations. This correlates with the predominance of clump morphology in the former runs. Furthermore, in the range 103 to 105 spore/ml initial spore concentration, erythromycin production increases with an increase in the proportion of clump morphology. These two observations suggest that, for S. erythraea, clump morphology is more suitable for erythromycin production compared to pellet morphology. Oxygen limitation in the core of the pellet could be the main reason for lower erythromycin productivities for this type of morphology (Chen and Wilde, 1991). In the case of runs in which the clump morphology was noteworthy, the runs at an initial spore concentration of 106 spore/ml resulted in the highest erythromycin productivities; furthermore, the data show that this parameter consistently increases during S. erythraea cultivation. These two observations, together with the data presented in Figure 4 and Figure 3b, suggest that smaller clumps plus reduced non-Newtonian rheological characteristics of the cultivation broth improve erythromycin production. These observations might be related to oxygen limitations that occur with an increase in both clump dimension and S. erythraea cultivation broth viscosity. This can perhaps also explain why, at earlier cultivation times, the erythromycin productivity data (Figure 7c) were more similar to each other for runs at different initial spore concentrations. The oxygen limitation due to the high viscosity of the cultivation broth can be circumvented to a significant extent by carrying out the fermentation in well aerated, agitated bioreactors. However, the oxygen diffusion resistances due to the large dimension of the clumps consist of internal and external resistances and only the latter will be reduced if the fermentation is carried out in well mixed bioreactors.
The results obtained in the present work show that in submerged cultivation of S. erythraea a change in the initial spore concentration in the range 103 to 107 spore/mL had a determining effect on the morphology of the bacterial cells. The predominance of clump morphology increased and pellet morphology decreased with the increase in initial spore concentration. In runs in which there was sizeable pellet morphology, the rheology of the broth was Newtonian and pellet size was the largest and the mean pellet hairy length the lowest in runs at the lowest initial spore concentration. In the runs in which clump morphology was significant, the rheology of S. erythraea cultivation broths became more non-Newtonian with an increase in initial spore concentration. In these runs, a rough correlation was observed between the trend of change of morphological and rheological parameters. The mean major axis of the clumps was the lowest in runs with 106 initial spore concentration, which were the runs in which the highest erythromycin productivity was attained. Based on the results presented in the current study, it can be concluded that, for Saccharopolyspora erythraea cultivation, clump morphology is more suitable for erythromycin production compared to pellet morphology Furthermore, a decrease in clump dimensions, together with lower non-Newtonian broth viscosities, probably as a result of decrease in mass transfer resistances, also enhances erythromycin productivity.
The authors would like to than the Shafa-e-Sari Pharmaceutical Co., Tehran, Iran, for supplying the microbial culture used in the present work.
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(Submitted: January 27, 2011 ; Revised: May 15, 2011 ; Accepted: July 21, 2011)
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