Abstract
Prosopis juliflora is a drought-resistant evergreen spiny tree that grows in semi-arid and arid tracts of tropical and sub-tropical regions of the world. Dry pods of P. juliflora are a rich source of carbon (40% total sugar) and nitrogen (15% of total nitrogen) and so can be considered as a good substrate for the microbial growth. The present study was mainly focused on the utilization of these pods for the production and statistical optimization of oxytetracycline (OTC) from Streptomyces rimosus NCIM 2213 under SSF. The spectral characterization and chemical color reactions of purified OTC by UV, FTIR, 1H NMR, 13C NMR, and HPLC revealed that the structure was homologous to a standard sample. A central composite design with 26 trails yielded the following critical values of supplements to be added to the dry pods: maltose (0.125 g/gds), Inoculum size (0.617 mL/gds), CaCO3 (0.0026 g/gds), and moisture content (74.87%) with the maximum OTC yield 5.02 mg/gds.
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Production and Statistical Optimization of Oxytetracycline from Streptomyces rimosus NCIM 2213 using a New Cellulosic Substrate, Prosopis juliflora
Surjith Ramasamy, Harish S. Balakrishna, Uthra Selvaraj, and Kiran Babu Uppuluri *
Prosopis juliflora is a drought-resistant evergreen spiny tree that grows in semi-arid and arid tracts of tropical and sub-tropical regions of the world. Dry pods of P. juliflora are a rich source of carbon (40% total sugar) and nitrogen (15% of total nitrogen) and so can be considered as a good substrate for the microbial growth. The present study was mainly focused on the utilization of these pods for the production and statistical optimization of oxytetracycline (OTC) from Streptomyces rimosus NCIM 2213 under SSF. The spectral characterization and chemical color reactions of purified OTC by UV, FTIR, 1H NMR, 13C NMR, and HPLC revealed that the structure was homologous to a standard sample. A central composite design with 26 trails yielded the following critical values of supplements to be added to the dry pods: maltose (0.125 g/gds), Inoculum size (0.617 mL/gds), CaCO3 (0.0026 g/gds), and moisture content (74.87%) with the maximum OTC yield 5.02 mg/gds.
Keywords: Oxytetracycline (OTC); Prosopis juliflora; Solid state fermentation (SSF); Central composite design (CCD); Purification; Characterization
Contact information: Bioprospecting Laboratory, Department of Biotechnology, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, India;
* Corresponding author: kinnubio@gmail.com
INTRODUCTION
Prosopis juliflora is a shrub of semi-arid and tropical part of the world and is spreading due to its non-utilization by both humans and animals. An average plant yields annually 10 to 50 kg of pods/ tree within 3 years of its seeding. In general, these can be collected from May-June and September-October. The total productivity of pods has been estimated to be about 2 to 4 million metric tons worldwide. On a dry matter basis, these ripened pods consist of 12% crude protein, 15% free sugar, and a moderate level of digestible crude protein (7% DCP). The pods contain tannins below the toxic concentration of animals (Sawal et al. 2004). Indian pods consist of 16.5 to 7.6% crude proteins (Talpada et al. 1987), 28 to 19% fiber content (Anon 1943; Talpada et al. 1988), and 46.3 to 61.6% nitrogen-free extract (Anon 1943; Talpada et al. 1987). Lack of the knowledge about nutritional value of pods and neuro-toxic side effects on pods consuming cattle has restricted their utilization as a fodder (Tabosa et al. 2006) (Saykhedkar and Singhal 2004).
Tetracyclines are a family of antibiotics that share the same four cyclic ring and include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and dameclocycline (DC) (Sevilla-Santos et al. 1987; Pickens and Tang 2010). It acts against Gram positive bacteria, Gram negative bacteria, parasites, and some fungal pathogens (Asagbra et al. 2005). Bacteriostatic and bactericidal activity of tetracycline’s family has extended its application in veterinary science for animal disease, agriculture to control plant pathogens (McManus and Stockwell 2001; McManus et al. 2002), and medical fields for treating infections, etc. (Nikolakopoulou et al. 2005).
Solid-state fermentation (SSF) processes are of particular interest due to their high product yield, low costs due to the efficient utilization, value-addition of widely available wastes, and eco-friendly behavior in disposal (Subramaniyam and Vimala 2012). The production of OTC by SSF has been studied using sweet potatoes, potato residues (Yang and Swei 1996), corn cobs (Yang and Swei 1996; Okorie and Asagbra 2008), cassava peels, peanut shells (Asagbra et al. 2005), sawdust, and rice hulls (Saykhedkar and Singhal 2004).
Statistical optimization has been used to reduce the number of trials and cost of experiments by proper design and analysis to find out the optimum concentrations of a series of medium ingredients that are contribute for the maximum product formation (Zeng et al. 2006; Mao et al. 2007; Kumar et al.2014). Response surface methodology is used for the study of linear, square, and interaction effect of the variables on the production of bio-chemicals (Lotfy 2007; Wang et al. 2008, 2013; Yu et al. 2008; Singh et al. 2012; Uppuluri et al. 2013b).
In the present study, the use of lignocellulosic weed Prosopis juliflora as a potential substrate for the fermentation (SSF) was exploited. OTC production was carried out using the pods of Prosopis juliflora from Streptomyces rimosus NCIM 2213 by SSF. RSM-based central composite design was used to optimize cultural conditions viz., the concentration of maltose, CaCO3, inoculum size, and moisture content for the maximum OTC production. Further, spectral characterization of purified OTC by UV, FTIR, 1H NMR, and 13C NMR was performed in addition to the HPLC analysis. To the best of our knowledge, this is the first report on the utilization of pods of Prosopis juliflora as a solid state fermentation substrate.
EXPERIMENTAL
Materials
Microorganisms and media
Streptomyces rimosus NCIM 2213 was procured from National Chemical Laboratory, Pune, India. It was cultured and maintained on MGYP medium. All the other reference strains used in this study were procured from Microbial Type Culture Collection and Gene bank, Chandigarh, India. Test strains was cultured and maintained on nutrient medium and Muller Hinton agar medium was used for antimicrobial assay.
Pods collection
Prosopis juliflora pods were collected outside the SASTRA university campus, Thanjavur, Tamil Nadu, India. Substrates were collected from the same region throughout the work to avoid bias due to change in nutritional content. Carbon sources including cellulose (Kulić and Radojičić 2011), total organic carbon, reducing sugar as glucose, lignin, fiber, and moisture content were determined using standard assays.
Production of OTC by SSF
Prosopis juliflora pods were washed first with tap water followed by distilled water to remove the adhered surface dust particles. Then a bleaching operation was carried out by immersing them in hot water (75 to 80 oC) for 20 min followed by oven drying at 45 oC. The dried material was grounded to course powder using a mixer grinder. Initially 10 g of dried ground pods powder was taken in a 250 mL Erlenmeyer flask and to which a predetermined quantity of phosphate buffer, pH 7.4 was added, mixed thoroughly, and then sterilized at 121 oC, 1.06 kg/cm2 for 15 min. The sterilized medium was inoculated with 2 mL of inoculum containing 107 spores per mL of Streptomyces rimosus NCIM 2213 and incubated for 9 days at 28 oC. The media were mixed regularly for uniform distribution of organisms.
Purification and Characterization of OTC
Fermented matter was mixed with phosphate buffer, pH 7.4 (1:4 w/v) by stirring on a magnetic stirrer for 30 min at room temperature. The slurry was then centrifuged at 10,000 × g for 10 min at 4 oC to remove the insoluble matter. The clear supernatant was used for purification and estimation of OTC yield. Liquid–liquid extraction using ethyl acetate along with 0.1% calcium chloride as chelating agent was performed with the supernatant to extract the OTC, at pH 4 to 8. All the ethyl acetate fractions were concentrated in a Rota-vapor at 37 oC and subjected to silica column chromatography (Sevilla-Santos et al. 1987; Singh et al. 2013).
The ultraviolet (UV) spectrum of the purified compound in methanol was recorded with a Thermo Evolution 201 spectrophotometer at 200 to 400 nm. The infrared (IR) spectrum of the purified compound was recorded on an FT-IR SPECTRUM RX-I spectrometer in the range of 400 to 4,000 cm-1 using the KBr pellet technique. 1H and 13C nuclear magnetic resonance (NMR) spectra of the purified compound in deuterated water (D2O) were conducted with a 300 MHz BRUKER NM-4950 AVANCE II instrument (Chen et al. 2012; Singh et al. 2012)
Color reactions of the purified compound with concentrated H2SO4, concentrated HCl, and 2N NaOH were also recorded under both the visible light and UV light to confirm the presence of OTC. The reagent mixture was also boiled for determine the variability in color (Patrocinio et al. 1987; Chen et al.2012).
Chromatographic analysis was performed with PEAK high performance liquid chromatography having LC-P7000 isocratic pump, equipped with PEAK LC-UV7000 variable wavelength detector. UV detection was made at 254 nm with column temperature of 30 oC. The flow rate was set to 0.6 mLmin-1, and injections of 20 μL were made. Acetonitrile and deionised water (80% and 20%) were used as the mobile phase (Singh et al. 2012).
Construction of Calibration Curve for OTC from Antimicrobial Assay
An antimicrobial assay was performed by using the disc diffusion method. A disc containing an antimicrobial compound was placed on the Muller Hinton agar plated with the test organism with a standard set of conditions including medium volume, inoculum size, and incubation temperature. After 18 h of incubation, the diameter of zone of inhibition was measured (mm), and OTC yield was computed form the standard curve (Yang and Swei 1996; Asagbra et al. 2005).
Standard oxytetracycline was used for preparing the standard curve, and a regression equation was developed having the form Y = mX + C, where Y is the log of concentration, and X is the zone of inhibition minus the disc size) (cm). E. coli was used as a test organism for plotting the standard and also determining the unknown concentration (Yang and Ling 1989; Asagbra et al. 2005).
Optimization of Selected Medium Components by Central Composite Design
The central composite design (CCD) known as Box-Behnken is the most accepted and widely used design to study the interaction effect of the medium components (Adinarayana and Ellaiah 2002). The CCD is a statistical experimental design where each numeric factor is varied at 5 levels (-2, -1, 0, +1, +2). Four significant variables (maltose concentration, CaCO3 concentration, initial moisture content, and inoculum size) were chosen for the experiment. The experimental design was performed using coded values to avoid bias in the experiments and the effects of linear, nonlinear interactions were studied using response surface methodology. A sum of 26 experiments was performed in triplicates, and the average value was considered for obtaining the polynomial equation (1) to predict optimum value:
Y = μ0 + ∑μiZi+ ∑μjZi2 +∑μijZiZj (1)
In Eq. 1, Y is the predicted yield, μ0 is the linear interaction coefficient, μi is the quadratic interaction coefficient, μij is the interaction coefficient, and Znterms are variables. The design and analysis of experiments were carried out using “STATISTICA 8.0” software, evaluation version (Mao et al. 2007; Kammoun et al. 2008; Yong et al. 2011; Kumar et al. 2014).
Antimicrobial Assay against Different Pathogens
The potency of OTC produced and purified from S. rimosus NCIM 2213 using P. juliflora pods was tested against various pathogens. Reference strains including Bacillus subtilis (MTCC 441), Escherichia coli (MTCC 723), Salmonella typhi (MTCC 531), Staphylococcus aureus (MTCC 3160), Shigella dysenteriae ATCC 23513, Klebsilla pneumonia (MTCC 1667), and Pseudomonas aeruginosa (MTCC 2488) were employed (Singh et al. 2013). An antimicrobial assay was triplicated according to the above procedure, and the zone of inhibition was reported in mm along with SE.
RESULTS AND DISCUSSION
Chemical Composition of Pods
The detailed chemical composition of dried pods of P. julioflora is given in Table 1. This is the first report on the chemical composition of P. julioflorapods. Cellulose was found to be the major principle ingredient in pods. Cellulosic feed stocks and agricultural waste can be good fermentative substrates for the production of bio-chemicals, especially for secondary metabolites.
Table 1. Composition of Dried Pods of Prosopis juliflora
Total reducing sugar, total organic carbon, and fiber content results were in good agreement with the other species of Prosopis Pods (Alcedo 1988; Pasiecznik and Felker 2001; Sawal et al. 2004; Mabrouk et al. 2008).
Usage of agricultural and forestry residues as fermentative substrates for the production of value-added products including antibiotics under SSF is being widely practiced. Oxytetracycline can be easily produced from Streptomyces rimosus grown on a high cellulose containing medium by SSF. In fact, the oxytetracycline produced by SSF using corn cobs was more stable than that produced by Submerged fermentation (SmF) (Yang and Swei 1996). Peanut shells, corn cob, corn pomace, and cassava peels have been used as substrates for SSF to produce tetracyclines from S. rimosus (Asagbra et al. 2005)
Large quantities of P. julioflora waste have been generated and are spreading all over the world due to the unpalatability of leaves and pods, such that animals do not digest its seed. To overcome the environmental pollution problems associated with the conventional disposal methods of handling, this waste can be used as support-substrates in SSF to produce industrially relevant metabolites such as antibiotics with a great economical advantage.
Production of Oxytetracycline by SSF
The inoculated pods were observed for their time-dependent oxytetracycline production in batch flasks from the 24th hour to the 300th hour. OTC production was observed from the 96th hour, 0.2 mg OTC/gds and the maximum yield was found at the 216th hour, 0.44 mg OTC/gds. After that, a sudden fall of OTC yield was observed from 0.44 to 0.28 mg OTC/gds. This may be due to the autolysis of Streptomyces rimosus. A number of factors such as nutrient depletion, sudden change of environmental conditions, or intolerable limits of secondary metabolites may contribute for the autolysis of a microorganism and hence subsequent reduction of the metabolites. The action of produced antibiotic on the well-grown microorganism also may lead to the decrease in the already formed antibiotic (Okorie and Asagbra 2008; Rodríguez-Jasso et al. 2013).
Physicochemical Characterization of Produced and Purified OTC
The UV spectra of purified OTC showed two characteristic peaks at 349 nm and 277 nm, whereas the standard OTC at 349 nm and 274 nm indicated the presence of a carbonyl group and an aromatic group, respectively (results not shown) (Ruiz Medina et al. 2000; Singh et al. 2013). FT-IR spectra of purified and standard OTC showed the characteristic bands at 3465 cm-1(O–H), 3457, 3441 cm-1 (N–H), 1597.36 cm-1 (C = O), and 1403.46 cm−1 (= C–N) (results not shown) (Ruiz Medina et al. 2000; Singh et al. 2013). The wavelength region 1300 to 1,700 cm-1 is reported to be fingerprint of molecule because it allows the identification of major chemical groups in tetracycline.
Further confirmation of OTC was obtained using 13C NMR and 1H NMR analyses. The 1H NMR spectrum showed identical pattern peaks of various resonances, triplets peaks at 7.15 to 7.108 ppm (Standard OTC) and 7.407 to 7.353 (purified OTC) were indicating CH of aromatic group. Peaks at 1.385 (Standard OTC) and 1.2993 (purified OTC) indicate CH3-C-OH of oxytetracycline. CH3 of aliphatic were noted by peaks in the range 2.474 to 2.629 (Standard OTC) and 2.525 to 2.7(purified OTC) (Chen et al. 2012; Singh et al. 2013). 1HNMR spectra for both standard OTC and purified OTC are shown in Fig. 1. 13C NMR spectra in Fig. 2 show resonances for C signals in the range 193 to 16 ppm for both standard and purified OTC. Functional groups can be identified based on peaks 193.90 to 192.58 (carbonyl of aliphatic ring), 160.89 to 186.45(C=C), 72.89 to 96.05 (1-ethylene), 104.67 to 146.02 (phenyl group), and 42.82 to 73.3 (cyclohexane).
Fig. 1. 1H NMR spectra for a) purified OTC b) standard OTC
Fig. 2. 13C NMR spectra for a) purified OTC b) standard OTC
Further, the produced and extracted yellow-colored compound chromatogram (Fig. 3) by HPLC showed a single peak with the retention time of 6.16 min. The same was observed with the standard OTC, confirming that the produced and extracted compound contains only one product and that it resembles the standard OTC.
The results of chemical characterization of OTC based on the color formation are given in Table 2. Tetracyclines (TC, OTC, CTC) will give different color reactions for various chemical treatments (H2SO4, HCl, 2 N NaOH) at conditions such as boiling and with illumination. The purified compound gave the same color appearances as the standard OTC and with literature reports under both illuminations (UV and Visible) with all the tested chemical methods (Sevilla-Santos et al. 1987; Singh et al. 2013). Based on both the physical and chemical characterization techniques, the produced and purified compound was confirmed as oxytetracycline.
Fig. 3. HPLC chromatogram for purified OTC
Table 2. Chemical Tests for Standard and Purified OTC
*UB: Unboiled; B: Boiled
Calibration Curve from Antimicrobial Assay
A calibration curve was plotted between the log of concentration and the zone of inhibition (against E. coli) for the range of 2 μg to 30 µg under the standard set of conditions (Fig. 4). A linear equation was developed and used for the computing OTC yield in mg/mL throughout the study; Y = 0.1427X – 0.647, with R2= 0.9891 (Yang et al. 1996; Yang and Ling 1989; Okorie and Asagbra 2008; Okorie et al. 2008).
Statistical Optimization
Preliminary studies involving factors such as the influence of environmental conditions (initial pH, incubation time, and temperature), nutritional conditions (carbon sources such as sucrose, maltose, lactose, glucose, and fructose, nitrogen sources (both organic and inorganic), and inoculum size were conducted. The four most significant variables were chosen for further optimization by CCD for enhanced production of OTC from S. rimosus using dry pods.
Fig. 4. Calibration curve for OTC from antimicrobial assay
The selected significant variables, maltose concentration, CaCO3 concentration, inoculum size, and moisture (%) were optimized for maximum production of oxytetracycline by RSM (Kumar et al. 2014). The CCD experimental model was applied, leading to a second order polynomial equation for determining the optimized value (Zeng et al. 2006). Table 3 shows the Central Composite Design of the variables in real units for
Table 3. Central Composite Design of the Variables in Real Units for the Response of OTC Yield with Its Predicted and Observed Values
the response of OTC yield (average value) along with its predicted and observed values. Regression analysis of the central composite design is given in Table 4.
Equation 2 was framed by considering the only significant variables (p<0.005) on the OTC production; hence insignificant variables, p>0.005, were omitted in the equation (Gharibzahedi et al. 2012).
In Eq. 2, Y is the OTC yield with respect Zn independent variables, for the equation based on linear, quadratic, and interaction coefficients of the variables.
Accuracy and fitness of the model depend on the coefficient of determination (R2) and adjusted R2 value. The smaller the difference between 1 and the R2value, the stronger the model (El Enshasy et al. 2008; Uppuluri et al. 2013b). The obtained values for R2 (0.95714) and R adj (0.906) from the analysis of CCD experimental values show its accuracy and fitness. The strength of the model was exposed by response surface plots with respect to OTC yield and two other variables at a time (Uppuluri et al. 2013a,b). The correlation was plotted between observed and predicted values, showing values close to the diagonal line.
Table 4. Regression Analysis of the Central Composite Design
Oxytetracycline production ranged from 5.01 to 0.45 mg /gds based on the experimental values. Critical values of the selected variables are given in Table 5.
Table 5. Critical Value Obtained through RSM (where the predicted OTC value is 5.02 mg/gds)
Real experiments were performed at critical values of selected variables and yield was found to be closer to predicted yield 5.02 mg /gds.
Antimicrobial Activity of Purified OTC
Purified OTC showed relatively good antimicrobial activity against various selected Gram positive and Gram negative organisms compared to the standard OTC (Table 6). The tested pathogens were sensitive to purified compound and further no growth was observed. This clearly demonstrated the bactericidal activity of purified OTC.
Table 6. Antimicrobial Spectrum (Zone of Inhibition) by Standard and Purified OTC
CONCLUSIONS
- The present study exploited the use of Prosopis juliflora pods as a novel and cheap cellulosic substrate for the fermentative production of oxytetracycline (OTC).
- High cellulosic content of these pods provides a very good platform for the growth of microorganisms, especially actinomycetes and fungi.
- So, the cultivation of micro-organisms on these pods surely will be a value-added process capable of converting these materials into various biochemicals, especially biopharmaceuticals.
- A universally accepted statistical optimization process (RSM), was used to further increase the OTC production from 0.4 to 5 mg/gds.
- But still, scale-up needs to be done to develop a commercial process with techno-economical feasibility.
ACKNOWLEDGMENTS
The authors are grateful to the management, SASTRA University for providing the necessary facilities to carry out the research work.
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Article submitted: July 14, 2014; Peer review completed: September 14, 2014; Revised version received: October 11, 2014; Accepted: October 12, 2014; Published: October 15, 2014.