Egyptian Pharmaceutical Journal

ORIGINAL ARTICLE
Year
: 2015  |  Volume : 14  |  Issue : 2  |  Page : 94--102

Semisolid state fermentation: effects of beet sugar root : peptone ratio on erythromycin production by Saccharopolyspora erythraea NCIMB 12462


Mohamed AM Farid1, Hoda MA Shata2, Azza M Noor El-Deen1, Nayera AM Abdelwahed1,  
1 National Research Centre, Dokki, Egypt
2 Department of Microbial Chemistry, National Research Centre, Dokki, Egypt

Correspondence Address:
Mohamed AM Farid
Department of Natural and Microbial Products, National Research Centre, Dokki, Giza, PO Box 12622
Egypt

Abstract

Background and objectives Erythromycin, a prominent member of the macrolide antibiotics, is commercially produced by submerged fermentation. However, this process requires high energy expenditures. The objective of the present work was to improve and optimize the cultural conditions of Saccharopolyspora erythraea NCIMB 12462 grown under semisolid state fermentation, with less energy requirements, less waste water generation, and easier product recovery, for the production of erythromycin using beet sugar root (BSR) and peptone. Materials and methods Chemical analysis of BSR was carried out according to the guidelines of AOAC and using high-performance liquid chromatographic. The concentration of erythromycin was measured using the agar diffusion bioassay method. Evaluation of different nitrogen sources for erythromycin production was carried out and the effect of different BSR : peptone ratio on erythromycin production was determined. The impact of initial moisture content (75-88%), size of inoculum, sodium chloride and calcium carbonate concentrations, and incubation period (1-12 days) on erythromycin production using solid state fermentation by S. erythraea NCIMB 12462 was evaluated. Results and discussion Optimization of environmental and culture parameters, concentration of nitrogen sources (ammonium sulfate, yeast extract, and peptone), BSR/peptone ratio, inoculum size, moisture content, and incubation time exhibited a significant increase in erythromycin production compared with the production before optimization. The concentration of erythromycin in optimized medium was 735.65 ± 8.58 μg/g dry BSR (1.36 times more than that of the control medium). The optimal conditions for erythromycin production using solid state fermentation for BSR were a initial moisture level of 77.78%, inoculum size of 2 × 10 6 -2 × 10 7 spores/10 g dry BSR, incubation period of 10 days, and peptone at a concentration of 0.8 g/100 g BSR.



How to cite this article:
Farid MA, Shata HM, Noor El-Deen AM, Abdelwahed NA. Semisolid state fermentation: effects of beet sugar root : peptone ratio on erythromycin production by Saccharopolyspora erythraea NCIMB 12462.Egypt Pharmaceut J 2015;14:94-102


How to cite this URL:
Farid MA, Shata HM, Noor El-Deen AM, Abdelwahed NA. Semisolid state fermentation: effects of beet sugar root : peptone ratio on erythromycin production by Saccharopolyspora erythraea NCIMB 12462. Egypt Pharmaceut J [serial online] 2015 [cited 2020 Sep 18 ];14:94-102
Available from: http://www.epj.eg.net/text.asp?2015/14/2/94/161280


Full Text

 Introduction



Erythromycin is an important antibiotic, which belongs to the macrolide family, and is produced by fermentation of Streptomyces erythreus or Saccharopolyspora erythraea. It is widely applied in pharmaceutical preparations for local applications and in veterinary practice. Erythromycin is widely used clinically in treating respiratory infectious diseases [1] and in the treatment of many acute infections caused by bacteria belonging to Staphylococci spp. and Neisseria spp. [2]. It acts as an antimalarial drug in combination with other drugs in reducing pathogen resistance [3]. Besides all these medical applications, erythromycin is also used as a prophylactic and curative therapeutic agent for many diseases of poultry and farm animals. It also has many applications in animal feeding and cultivation of marine microalgae [4],[5]. Erythromycin is commercially produced by submerged fermentation (SmF). However, this process requires high energy expenditures. In the search for more economical fermentation processes with high antibiotic activity, solid state fermentation (SSF) has gained interest in recent years due to the advantages that presents over SmF, such as higher product yields, less energy requirements, easier aeration, less waste water generation, reduced bacterial contamination, and easier product recovery [6]. With SSF, it is possible to utilize renewable and low-cost natural resources, such as agricultural and wood remains, energy crops, and byproducts of the food industry [6].

Hence, in the present paper the production of erythromycin by S. erythraea NCIMB 12462 grown under semisolid state fermentation conditions was investigated. This type of fermentation is a sort of SSF in which the free liquid content has been increased to facilitate nutrient availability and fermentation control [7]. To the best of our knowledge, the production of erythromycin under semisolid state fermentation conditions has not been reported before this study.

 Materials and methods



Microorganisms

S. erythraea NCIMB 12462 obtained from National Collection of Industrial and Marine Bacteria Limited (Aberdeen, Scotland, UK) was used in the present study. The spores of this strain had been preserved in a dormant state in starch-nitrate agar medium slants composed of 20 g/l of starch, 2 g/l of NaNO 3 , 0.5 g/l of K 2 HPO 4 , 0.5 g/l of MgSO 4·7H 2 O, and 24 g/l of agar. The pH was adjusted to 7.2 before sterilization using 1 N NaOH and 1 N HCl. Subculturing was carried out once in 2 weeks, and the culture slants were stored at 4°C. The spores from a fully sporulated 10-day-old slant of S. erythraea NCIMB 12462 were dispersed in 10 ml of sterile distilled water by dislodging them with a sterile loop under aseptic conditions. The spore suspension was used as inoculum for each 250 ml Erlenmeyer flask containing the solid medium. Unless otherwise stated, for pure culture, each flask containing 10 g fresh beet sugar root (BSR) substrate was inoculated with 2 ml spore suspension (10 6 -10 7 spores/ml). Spore count was measured using the dilution plate count method [7].

Bacillus subtilis NRRL B-543 was obtained from Northern Regional Research Laboratory (NRRL, Peoria, Illinois, USA). This bacterium was used as a test organism for the determination of the erythromycin produced by S. erythraea NCIMB 12462 using the agar diffusion method [9].

Inoculum preparation

The spores from a fully sporulated 10-day-old slant of S. erythraea NCIMB 12462 grown on ISP-2 agar slants at 28°C were dispersed in 10 ml of sterile distilled water by dislodging them with a sterile loop under aseptic conditions. The spore suspension was used as inoculum for each 250 ml Erlenmeyer flask containing the solid medium. Unless otherwise stated, for pure culture, each flask containing 10 g fresh BSR substrate was inoculated with 2 ml spore suspension (10 6 -10 7 spores/ml). Spore count was measured using the dilution plate count method [8].

Substrate

BSR, from local market and a local sugar company in Kaffr El-Sheikh, Egypt, was pretreated by thoroughly washing with tap water and was homogenized in an electric mixer to small pieces and used as basic carbon source for semisolid fermentation media.

Analysis of beet sugar root components

Chemical analysis of BSR was performed. Moisture content, crude protein, crude fiber, fat content, and total ash were determined according to the method described by AOAC [10]. The amount of total carbohydrates was obtained by the difference between the weight of the sample taken and the sum of its moisture, ash, total lipid, protein, and fiber contents [11].

Determination of sugar contents

Extraction and determination of total water-soluble sugar, reducing sugar, and nonreducing were carried out according to the methods described by Miller [11]. The amount of nonreducing sugar was obtained by subtraction of the amount of reducing sugar from the total amount of water-soluble sugar.

High-performance liquid chromatographic determination of fructose, glucose, and sucrose in beet sugar root

The HP1100 system equipped with autosampler, quaternary pump, online degasser, and refractive index detector, controlled with ChemStation software (Hewlett Packard, Waldbronn, Germany), was used for chromatographic determination of fructose, glucose, and sucrose in BSR using ultrapure water as mobile phase (0.6 ml/min) and Shimadzu Shim-Pack SCR - 101N column (Tokyo, Japan).

Solid state fermentation

Ten grams of homogenized fresh BSR in a 250 ml Erlenmeyer flask was moistened with 10 ml mineral salt solution composed of 5 g/l of CaCO 3 , 3 g/l (NH 4 ) 2 SO 4 , and 2.5 g/l NaCl, at pH 7, thoroughly mixed, and autoclaved at 121°C for 30 min. Each flask was inoculated with 2 ml of spore suspension (containing 1 × 10 6 -1 × 10 7 spores/ml) and incubated at 28-30°C for 10 days. The moisture content of the medium after inoculation was 84.33%, including the moisture content of BSR. Unless otherwise specified, these fermentation conditions were maintained throughout the experiment. All experiments were performed in duplicate.

Optimization of the culture condition for erythromycin production

Factors affecting the production of erythromycin by S. erythraea NCIMB 12462 were optimized in 250-ml Erlenmeyer flasks containing 10 g of fresh BSR. The effect of incorporation of additional nitrogenous compounds (ammonium sulfate, ammonium phosphate dibasic, sodium nitrate, potassium nitrate, meat extract, peptone, and yeast extract) to the production medium was studied. The nitrogen content of each added source was equivalent to the amount of nitrogen content in ammonium sulfate (3 g). Moreover, the different physicochemical parameters required to maximize the yield of erythromycin by S. erythraea NCIMB 12462 under SSF were investigated. The optimized parameter was incorporated at its optimized level in the subsequent optimization experiments. The impact of initial moisture content (75-88%), size of inoculum, sodium chloride and calcium carbonate concentrations, and incubation period (1-12 days) on erythromycin production using SSF of S. erythraea NCIMB 12462 was evaluated. All experiments were conducted in duplicate and the mean values were considered. After incubation of each fermentation sample, the crude extract was prepared.

Beet sugar root/peptone ratio

For optimization of the ratio of BSR and peptone as nitrogen source, S. erythraea NCIMB 12462 was grown in solid media prepared with different fresh BSR concentrations (10, 20, 30, and 40 g BSR/250 conical flask) and with different concentrations of peptone. The concentrations of peptone were proportional to the concentration of BSR. For 10 g BSR, peptone was added at a concentration of 0.2, 0.4, 0.6, 0.8, and 1.2 g/100 g BSR. For 20 g BSR, peptone at a concentration of 0.4, 0.8, 1.2, 1.6, and 2.4 g/100 g BSR was added. The other concentrations of BSR (30 and 40 g BSR) were also supplemented with the equivalent ratios of peptone. At the same time, different inoculum sizes of S. erythraea NCIMB 12462 were used to inoculate the sterilized medium based on BSR weight (2 ml/10 g BSR). In another set of experiments, each flask of the prepared media with different BSR and peptone concentrations (0.2, 0.4, 0.6, and 0.8 g/100 g BSR) was inoculated with 2 ml of spore inoculum of S. erythraea NCIMB 12462. The extraction volume of distilled water was also considered. All experiments were carried out at the optimized moisture content and with the equivalent amounts of other constituents optimized previously in the medium and extraction process. The flasks were incubated in static conditions at 28-30°C.

Erythromycin extraction

At the end of fermentation, the harvested biomass of each flask (10 g BSR/flask) was treated with 20 ml of water and shaken in an orbital shaker at 200 rpm at room temperature for 1 h. The whole content of each flask was centrifuged resulting in clear supernatant, and the final clear supernatant was used as the antibiotic source.

Erythromycin assay

The agar diffusion bioassay method [9] that utilizes the antibacterial property of erythromycin to produce a zone of inhibition against B. subtilis was used. A volume of 100 μl of filtrate was filled in the agar hole (0.9 mm diameter) punched in the nutrient agar plates [the antibiotic assay medium (Difco, Maryland, USA) comprised 10.0 g/l of glucose, 10.0 g/l of peptone, 2.5 g/l of meat extract, 5.0 g/l of yeast extract, 10.0 g/l of NaCl, and 20.0 g/l of agar) freshly seeded with 0.1 ml of B. subtilis NRRL B-543 strain as the test organism. The inhibition zone diameter was measured in mm after incubation of plates at 30°C for 24 h, and the concentration of erythromycin was calculated using standard erythromycin (Sigma Aldrich, St. Louis, Missouri, USA) calibration curve. All experiments were conducted in duplicate, and the mean of the two reading is presented as micrograms of erythromycin produced per gram dried BSR.

 Results and discussion



Proximate composition of beet sugar root

The proximate composition of BSR is given in [Table 1]. The root of the beet contains about 65.5% water (g/100 g fresh weight) and 34.5% dry matter. The dry matter comprises about 1.5% fiber, 2% protein, 0.11% fats, 1.06% ash, and 29.83% total carbohydrates. The total sugars represent 81.16% of the root's dry matter. The sucrose content in sugar beet roots represents about 18.39%, whereas the content of glucose and fructose was 2.31 and 1.099%, respectively, as determined by high-performance liquid chromatographic [Figure 1].{Figure 1}{Table 1}

Effect of supplementary organic and inorganic nitrogen sources

The protein content in BSR is very low and thus the nitrogen levels as well as the commercial value decrease greatly when it is used as carbon source [12]. Hence, the exogenous addition of various nitrogen levels to the solid medium was studied. Supplementation of medium with organic, as well as inorganic nitrogen sources, resulted in the improvement in erythromycin production compared with control medium (1.1 μg/g dry BSR) without nitrogen source. In the present studies, nitrogen sources - namely, ammonium sulfate, ammonium phosphate dibasic, sodium nitrate, potassium nitrate, meat extract, peptone, and yeast extract - were used for growth and erythromycin production by S. erythraea NCIMB 12462. The results are shown in [Figure 2]. All tested nitrogen sources supported erythromycin formation by the culture of S. erythraea NCIMB 12462, whereas ammonium sulfate, peptone, and yeast extract were proved to be superior to other nitrogen sources. A high titer of erythromycin (541.38 ± 74.35 μg/g dry BSR) was obtained in a medium containing peptone alone as organic nitrogen source, followed by yeast extract (457.27 ± 44.60 μg/g dry BSR). Among the different inorganic nitrogen sources tested, ammonium sulfate was the best source for erythromycin (482 μg/g dry BSR) production. The order of nitrogen source suitability was as follows: peptone>yeast extract>meat extract ≥ ammonium sulfate>ammonium phosphate dibasic > potassium nitrate>sodium nitrate. It was reported that ammonium salts did not favor the biosynthesis of novobiocin, actinomycin, neomycin, kanamycin, and others; however, for rapamycin, ammonium sulfate was the best nitrogen source [13],[14]. Ammonium salts (nitrate, sulfate, chloride, acetate, and arginine) stimulate the formation of some components of IM-111-81 and azalomycin B, whereas ammonium succinate increased the productivity of AK-111-81 nonpolyenic macrolide antibiotic [15]. Hence, peptone, yeast extract, and ammonium sulfate were selected and used for subsequent studies.{Figure 2}

Effect of different concentrations of the best nitrogen sources

Experiments were performed with varying ammonium sulfate, peptone, and yeast extract concentrations (0.1-0.5, 0.13-0.73, and 0.21-0.86 g/100 g BSR, respectively) in basal medium with 10 g BSR/flask to study the effects of nitrogen sources on erythromycin production by S. erythraea NCIMB 12462 under SSF. Out of the concentrations of three nitrogen sources studied (ammonium sulfate, peptone, and yeast extract), the results in [Figure 3] show that there was a significant increase in the antibiotic production when the medium was supplemented with different ammonium sulfate concentrations. Peptone at 0.73% (w/w) and yeast extract at 0.52 (%w/w) recorded the maximum yield (557.54 = 8.03 and 463.25 = 7.74 μg/g dry BSR, respectively) for organic nitrogen sources. For ammonium sulfate, the maximum yield of erythromycin (492.25 = 4.88 μg/g dry BSR) was obtained at concentration 0.4% (w/w). Further concentration-dependent studies suggested that variation in ammonium sulfate, peptone, and yeast extract concentrations showed a negative impact on erythromycin production. Nitrogen sources have long been known to affect the antibiotic production, and suppress the biosynthesis of antibiotics and other secondary metabolites. The most common observation was a decrease in the levels of antibiotic produced in the presence of an excess of nitrogen source. Some researchers reported that antibiotic biosynthesis may be inhibited or repressed by ammonia and other rapidly utilized nitrogen sources [16],[17]. The phenomenon of catabolic nitrogen repression of ammonium sulfate has been reported to affect many catabolic enzymes, which play a significant role in erythromycin biosynthesis [18]. High ammonium ion concentration had a negative effect on macrolide production, and a strong correlation existed between macrolide production and the level of valine dehydrogenase [16],[19],[20],[21]. The high activity of valine dehydrogenase enhanced the catabolism of branched chain amino acid and was in favor of producing important sources of the macrolide building blocks [17],[19],[20]. This regulation mechanism of ammonium ion was also proved in Streptomyces viridochromogenes AS4.126, the producer of avilamycin [22]. In contrast, some studies reported that ammonium impedes aminoglycoside synthesis, whereas others reveal stimulatory effects such as neomycin [23], gentamicin [24], and streptomycin production [14],[25]. High nitrogen concentrations affect the synthesis of sensitive enzymes involved in primary and secondary metabolism and the utilization of different nitrogen sources from the fermentation medium as well. Therefore, several approaches have been reported to avoid the negative effect of the nitrogen on the fermentative production of metabolites [14].{Figure 3}

Effect of different beet sugar root: peptone ratios on erythromycin production

In general, the C : N ratio in the culture medium is a critical factor for cell growth and metabolite production. The effect of different ratios between BSR and peptone in culture medium on erythromycin production was studied with the controlling of BSR and peptone concentrations, inoculum size, and extraction process. As shown in [Figure 4]a, the medium without peptone showed no erythromycin production. At low BSR concentration (10 g/flask), the increase in concentration of peptone resulted in a significant increase in erythromycin production up to 602.13 ± 11.56 μg/g dry BSR in culture containing 0.8 g peptone/l00 g BSR. In contrast, there was a reduction in erythromycin production as the additions of peptone were increased. In 20 g BSR/flask, the addition of peptone increases the erythromycin production up to 0.4 g peptone/l00 g BSR, and the maximal production was 536.61 ± 94.1 μg/g dry BSR. Moreover, the same behavior of reduction was observed with increasing peptone concentrations. Upon increasing the BSR concentration up to 30 g/flask, the maximal erythromycin production of about 418.58 ± 50 μg/g BSR was obtained in all values of peptone-supplemented culture. However, the repression effect was observed at a high BSR concentration of 40 g/flask with all concentrations of peptone. At higher BSR and peptone concentrations, erythromycin production was greatly influenced (108.48 ± 10-42.23 ± 3 μg/g dry BSR). In contrast, [Figure 4]b shows the effect of different BSR and peptone (0.2, 0.4, 0.6, and 0.8 g/100 g BSR) ratios using fixed inoculum size (2 ml/flask). In these series of experiments, the highest production of erythromycin was also achieved in the presence of 10 g BSR/flask and 0.8 peptone/100 g BSR. At higher BSR and peptone concentrations with 2 ml inoculum size, it was noticed that erythromycin production was slightly influenced (391.49 ± 13.07-201.13 ± 57.2 μg/g dry BSR). From the results of the two experiments, the decrease in erythromycin may be due to carbon source concentration, nitrogen source concentration, as mentioned before, and inoculum size as well.{Figure 4}

Carbon sources such as corn starch, glucose, sucrose, and molasses are commonly used as growth substrates to produce enzymes, antibiotics, and other secondary metabolites by fermentation. More than 30 examples of secondary metabolites are reported to be suppressed by the presence of the carbon source. Glucose and other carbohydrates, such as glycerol, maltose, mannose, sucrose, and xylose, have been reported to interfere with the synthesis of secondary metabolites. For instance, glucose depresses the formation of aminoglycoside antibiotics (streptomycin, kanamycin, istamycin, neomycin, and gentamicin) through repression of biosynthetic enzymes [26-28]. However, production is frequently limited due to a negative effect exerted by the carbon source. This regulatory mechanism, termed carbon catabolite regulation (CCR), is widely distributed among microbial systems and functions primarily to assure an organized and sequential utilization of carbon sources, when more than one carbon source is present in the environment [14]. Actinomycetes (Gram-positive) bacteria are subjected to CCR. This group possessing a high guanine and cytosine (GC) content in DNA includes Streptomyces, a genus characterized by its ability to produce secondary metabolites. The synthesis of these compounds is usually sensitive to CCR. For example, glucose depresses the formation of many aminoglycoside antibiotics produced by actinomycetes (streptomycin, kanamycin, istamycin, neomycin) through repression of biosynthetic enzymes [14],[26]. Polysaccharides (e.g. starch), oligosaccharides (e.g. lactose), and oils (e.g. soybean oil, methyloleate) are often preferable for fermentations yielding secondary metabolites [14]. Irrespective of the type of fermentation, whether it is a SSF or SmF, inoculum level also affects the yield of final product greatly [29],[30].

Effect of inoculum size

The effects of the inoculum on erythromycin production were studied by adding different concentrations (0.5 × 10 6 -0.5 × 10 7 , 1 × 10 6 -1 × 10 7 , 2 × 10 6 -2 × 10 7 , 3 × 10 6 -3 × 10 7 , and 4 × 10 6 -4 × 10 7 spores/10 g BSR) of spores to the solid medium and fermentation was carried out for 10 days. The moisture content, pH of the substrate, and incubation temperature were kept at their optimum levels. The maximum production of erythromycin (616.50 ± 16.8 μg/g dry BSR) was obtained in the fermentation medium that was inoculated with 2 ml of spore suspension (2 × 10 6 -2 × 10 7 spores/10 g BSR) of S. erythraea NCIMB 12462. Lower and higher inocula levels than the optimum level, resulted in decreased erythromycin activities [Figure 5]. In previous reports, authors have reported that adequate inoculum can initiate fast mycelium growth and product formation, thereby reducing the growth of contaminants. A decrease in antibiotic production was observed when the inoculum size was increased beyond the optimum level. Antibiotic production attains its peak when sufficient nutrients are available to the biomass. Conditions with a misbalance between nutrients and proliferating biomass result in decreased antibiotic synthesis [29],[31]. A low inoculum density may give insufficient biomass causing reduced product formation, whereas an inoculum density higher than the optimum density may produce too much biomass and may deplete the nutrients necessary for secondary metabolite production [32].{Figure 5}

Effect of different concentrations of sodium chloride and calcium carbonate

The effect of inorganic salt supplementation on erythromycin production is shown in [Figure 6]. Addition of 0.5% (w/w) CaCO 3 resulted in maximal erythromycin secretion (616.15 μg/g dry BSR) with Streptomyces strains than with controls. CaCO 3 can significantly affect the pH of the medium during the course of fermentation, which in turn may influence antibiotic production. Calcium carbonate has been used as a source of Ca +2 [33]. Moreover, it compensates lowering of the pH by consumption of carbon sources and maintains the pH of broth at optimum level for the production of erythromycin. A comparison of the concentration of the antibiotic in medium I and ISP-2 reveals that addition of this salt in the seeding media is not useful and that less pellet form of hyphae were observed in the media without calcium carbonate. The presence of 0.06% (w/w) NaCl also enhanced erythromycin production; thus, the salt requirements for the production of this antibiotic were apparently provided by the solid substrates.{Figure 6}

Effect of initial moisture content

The influence of initial moisture content of the solid substrate was investigated at various moisture levels (75, 77.77, 80, 82.85, 85, 86.36, and 88%) of BSR using distilled water before autoclaving, and the initial moisture percent was calculated. All experiments were carried out at initial pH 7, and excess distilled water was added to get the desired moisture percent. [Figure 7] shows the yields of erythromycin under seven different moisture contents. The moisture content of 77.77% (w/w) provides the best environment for erythromycin production (715.8 μg/g dry BSR). A decrease in erythromycin yield was observed when the moisture contents were much higher or lower than the optimum level. However, any further increase in moisture level in SSF causes free water in the fermentation medium, which may lead to limit gas exchange and higher vulnerability to bacterial contamination, whereas low moisture leads to reduced solubility of nutrients and substrate swelling. For SSF, moisture is a key parameter to control the growth of microorganism and metabolite production [6]. The importance of substrate moisture level in SSF for the production and secretion of secondary metabolites has been well established [6]. In SSF, the intensity of microbial growth generally depends on the initial moisture level and it indirectly affects the production titer. This result was similar to the findings of Mahalaxmi et al. [29], who reported that an initial substrate moisture content less than 40% gave less rifamycin B production, but that of 50-56% could give the highest rifamycin B production. The highest tylosin production was obtained at 70% initial moisture contents [33]. Moreover, the maximum yield of neomycin production was obtained from 70% moisture at day 8. The results from the previous study stated that the ideal moisture content was 80% and the reduction in antibiotic yield could occur with low and with higher moisture levels [14]. Higher initial moisture in SSF leads to suboptimal product formation due to reduced mass transfer process, and decrease in initial moisture level results in reduced solubility, minimized heat exchange and oxygen transfer, and low availability of nutrients to the culture [34]. Low moisture levels decrease the solubility and availability of nutrients, minimize heat exchange and oxygen transfer rates, thus lowering the activity of microbial cultures and resulting in reduced productivity [34]. Higher substrate moisture in SSF resulted in less productivity because of reduced mass transfer process, such as diffusion of solutes and gases to the cells during fermentation process.{Figure 7}

Effect of incubation period

The culture of S. erythraea NCIMB 12462 was grown in time course studies to determine the optimum time for production of erythromycin under SSF using BSR as the main carbon source, peptone as nitrogen source, and at optimized moisture level, inoculum size, and BSR/peptone ratio. Erythromycin production started earlier (i.e. during the lag phase) and antibiotic production was observed from second day of fermentation and reached maximum (735.65 ± 8.577 μg/g dry BSR) on the 10th day [Figure 8]. Further incubation after this time did not show any increment in the level of erythromycin production. The highest erythromycin produced after medium optimization represents 1.36-fold increase over the activities attained before optimization. It has been reported that antibiotic and secondary metabolite production are related to morphological characters of actinomycetes during the course of fermentation. It was reported that antibiotic production in liquid culture is correlated with mycelial fragment diameter in actinomycete cultures [35]. Smaller fragments appear to grow at the same rate as larger particles, but are incapable of significant antibiotic production. This phenomenon appears to account for loss of biosynthesis in liquid culture in species able to produce antibiotic on agar [36]. It was concluded also that, for S. erythraea cultivation in SmF, clump morphology is more suitable for erythromycin production compared with 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 [37].{Figure 8}

Erythromycin production by S. erythraea has been reported to take place in pellets 80-90 μm in diameter or larger, supporting the idea that the antibiotic is produced at a fixed distance from the hyphal end. Consequently, mycelia that are too small and have not developed to this length would be incapable of producing antibiotics [38].

 Conclusion



Overall, the present study revealed that S. erythraea NCIMB 12462 is effective in erythromycin production using BSR as carbon source under SSF.

It is interesting to notice that organic nitrogen (peptone and yeast extract) and the inorganic (ammonium sulfate) source had significant effect at individual level. The optimum production of erythromycin (735.65 ± 8.57694 μg/g dry BSR) was achieved by using BSR and peptone with optimized process parameters such as moisture level, inoculum level of 2 ml/10 g BSR v/w, incubation period of 10 days, 100 : 0.8 (w/w) ratio of BSR to peptone, and incubation temperature at 30°C. An overall 1.36-fold improvement in erythromycin production was achieved due to optimization.

 Acknowledgements



Conflicts of interest

None declared.

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