Optimization of Different Parameters for Antimicrobial Compound Production by Soil Microorganisms

AASCIT Journal of Biology 2016; 2(2): 16-20 http://www.aascit.org/journal/biology ISSN: 2381-1455 (Print); ISSN: 2381-1463 (Online) Optimization of Different Parameters for Antimicrobial Compound Production by Soil Microorganisms Riya Banerjee 1, Lavanya M. 2, Prathisha R. 2, Ravi Theaj Prakash U. 2, Vimala Gandhi 2, * 1 Departmant of Microbiology, Bangalore City College, Bangalore, India 2 PG Department of Bioscience, CMR Institute of Management Studies, Bangalore, India Email address rs.vimala@gmail.com (V. Gandhi) * Corresponding author Keywords Optimization, Antimicrobial Activity, Soil Isolates, Antimicrobial Compound Received: August 14, 2016 Accepted: August 27, 2016 Published: January 23, 2017 Citation Riya Banerjee, Lavanya M., Prathisha R., Ravi Theaj Prakash U., Vimala Gandhi. Optimization of Different Parameters for Antimicrobial Compound Production by Soil Microorganisms. AASCIT Journal of Biology. Vol. 2, No. 2, 2016, pp. 16-20. Abstract The current work was carried out to isolation of soil microorganisms from various soil samples and optimization of the soil isolates for antimicrobial activity. A total of 22 soil microorganisms were isolated from soil samples, out of which 3 isolates were found to be good antimicrobial activity. Screening and identification of isolates by gram staining and biochemical characterization was performed. The isolates S2, S3 and S6 were found to be having better antimicrobial activity which has been investigated. The antimicrobial compound is highly effective against many gram positive and gram negative bacterial pathogens. Thus, it is concluded that the isolates S2, S3 and S6 are potential antibiotic producers. The influence of ph, temperature, NaCl concentration, various carbon sources and activity in static vs. shaking condition was investigated. 1. Introduction Antibiotics are one of the pillars of modern medicine. They are small organic molecules which include a chemically heterogeneous group. Antibiotics are antimicrobial compounds of microbial agents, which in low concentrations inhibit the growth or kill other microorganisms. The nineteenth and twentieth centuries saw the discovery and development of many new antibiotics. The tide began when Alexander Fleming discovered Penicillin. In 1877, experiments in Paris demonstrated that the benefits of using harmless good bacteria to treat pathogenic or harmful bacteria. Louis Pasteur described the beneficial effects of injecting animals with harmless soil bacteria to combat anthrax, but the experiment failed to be efficient when injected on humans resulting in toxicity. Among the several functional microbial communities constituted within the soil habitat, a few communities produce several antimicrobial metabolites as their defense mechanism. Considerable research is being carried out to find new chemotherapeutic agents isolated from soil. Soil microbial communities are among the most complex, diverse and important assemblages of organisms in biosphere; and they participate in various biological activities [1]. The serious problems of antibiotic resistance are developing at an alarming rate. Hence, intensive search for new antibiotics has become imperative worldwide [2], [3]. A few microorganisms have been isolated to study their antimicrobial properties. Some of the organisms studied includes, a Streptomyces strain isolated from soil. On screening for its ability to produce antimicrobial compounds, it

17 Riya Banerjee et al.: Optimization of Different Parameters for Antimicrobial Compound Production by Soil Microorganisms was found to be active only against Gram positive bacteria. The genus Bacillus encompasses a number of bacteriocinogenic species, such as B. subtilis which produce subtilin and subtilocin, B. coagulans which produce coagulan and B. megaterium which produces megasin [4]. For the control of several insect pathogens in agriculture, B. thuringiensisis predominantly employed, producing crystal proteins (d-endotoxins) that posses specific activity against certain insects, nematodes, mites and protozoa [5]. More over number of extra cellular compounds are produced by B. thuringiensis including phospholipases, chitinases, proteases and β-exotoxins secreted vegetative insecticidal proteins and antibiotic compounds with antifungal activity [6]. One of the major challenges posed in therapeutic agents is their resistance to several pathogenic bacteria, mostly due to overprescription of antibiotics and its inadequate use [7], [8]. This challenge led to scientists to explore novel antimicrobial compounds for several natural sources including bacterial strains with low virulence possessing a broad antimicrobial activity against clinically significant microorganisms [9]. The present study was aimed to isolate microorganisms from various soil samples and optimizing the parameters for soil isolates with the ability to produce antimicrobial compounds. 2. Materials and Methods 2.1. Isolation of Microorganisms from Soil Soil sample were collected from various ecologically stressed niches in and around Central Institute of Mining and Fuel Research (CIMFR), Dhanbad, India. This sample included technological waste soil, garbage dump, medical waste dump, garden soil, industrial waste soil and was subjected to microbiological analysis. Serial dilution was performed and plated on Nutrient Agar Medium. Twenty two different colonies were picked up and transferred into Nutrient agar slants. These cultures were screened for antimicrobial compounds. Six test microorganisms were selected depending on their pathogenicity to human namely Escherichia coli, Bacillus subtilis, Pseudomonas aeroginosa, Staphylococcus aureus, Micrococcus leuteus, and Proteus vulgaris were obtained from CIMFR, Dhanbad, India. 2.2. Screening of Microorganisms for Its Antimicrobial Activity (Agar Well Diffusion) Based on the results of primary screening seven isolates were selected for antibiotic production. Strains showing moderate to good activity were selected and agar well diffusion method was performed. The isolates included S2, S3, S6, S11, S15, S16, and S22. Test culture of Escherichia coli, Bacillus subtilis, Pseudomonas aeroginosa, Staphylococcus aureus, Micrococcus luteus, Proteus vulgaris were spread on agar plates in the form of lawn. Wells were bored using cork borer and the culture filtrate of the producer isolates was poured in the well and incubated for 24hrs at 37 C. Zone of inhibition was measured and the isolates showing good activity was selected for further study. 2.3. Optimization of Antibiotic Production 2.3.1. Effect of ph on Antibiotic Production 25ml of nutrient broth was prepared in different flasks and the ph was varied in all the flasks. The ph ranged from 6.0-9.0. To these flasks equal amount of inocula was added and incubated for 48hrs at 37 C and 150rpm. After incubation, the growth was determined by spectrophotometer. The broth was centrifuged and the activity of the supernatant was defined by well diffusion method. 2.3.2. Effect of Temperatures on Antibiotic Production 25ml of nutrient broth was prepared in different flasks and equal amount of inocula was added. The flasks were incubated for 48hrs at different temperatures (30 C, 37 C and 55 C). After 48hrs, growth was determined spectrophotometrically. The broth was centrifuged and the activity of the supernatant was defined by well diffusion method. 2.3.3. Effect of Carbon Source on Antibiotic Production To the 25ml of nutrient broth prepared, different carbon sources were taken which included glucose, fructose, sucrose, xylose and inositol and equal amount of inocula was added and incubated for 48hrs at 37 C and 150rpm. After 48hrs, growth was determined by spectrophotometer. The broth was centrifuged and the activity of the supernatant was defined by well diffusion method. 2.3.4. Effect of NaCl Concentration 25ml of nutrient broth was prepared in different flasks. The NaCl concentration ranged from 0.5%, 1%, 3% and 5%, equal amount of inocula was added and incubated for 48hrs at 37 C and 150rpm. After 48hrs the growth was determined by spectrophotometer. The broth was centrifuged and the activity of the supernatant was defined by well diffusion method. 2.3.5. Growth and Activity in Static vs. Shaking Condition The growth and activity of the isolates were studied in static as well as shaking fermentation. Two sets of media were prepared for each isolate and were inoculated with equal amount of isolated inoculum. One set of flasks were kept at static condition at 37 C and another at shaking (150rpm) at 37 C for 48hrs. The growth of the isolate was determined by spectrophotometer. The broth was centrifuged and the activity of the supernatant was defined by well diffusion method and inhibition zone was measured.

AASCIT Journal of Biology 2016; 2(2): 16-20 18 2.4. Morphological and Biochemical Characterization Test The producer strains selected from secondary screening were characterized by morphological and biochemical tests according to the methods described by Bergey s manual of determinative bacteriology. Biochemical characterization of isolates was done by indole test, methyl red test voges- Proskauer test, citrate utilization test, catalase test, amylase test, and litmus test and protease test. 2.5. Solvent Extraction 10ml broth from production media was taken and centrifuged at 10,000 rpm for 15mins. The supernatant was collected in separate tubes and mixed with 10ml of ethyl acetate. The mixture was left for 30mins and then centrifuged at 10,000 rpm for 15mins. The supernatant was collected and kept to evaporate at room temperature for 2-3days. The crude obtained was weighed and used for further analysis. 3. Results and Discussion Different strains of bacteria were isolated from different soils and locations as shown in Table 1. A total of 22 soil organisms have been isolated from various ecologically stressed niches in and around Central Institute of Mining and Fuel Research (CIMFR), Dhanbad, India. Such us industrial waste soil (5), garbage dump (4), technological waste soil (3), agricultural land soil (3), garden soil (2), geo-environmental lab area (3) and medical waste dumps near central hospital (2). Table 1. Isolation of microorganisms from different locations showing the number of isolates and the isolate code. SAMPLE LOCATION No. OF ISOLATES ISOLATE CODE 1 Technological waste soil 3 S1, S2, S3 2 Garbage dump 4 S4, S5, S6, S7 3 Agricultural land soil 3 S8, S9, S10 4 Garden soil 2 S11, S12 5 Industrial waste soil 5 S13, S14, S15, S16, S17 6 Geo-environmental lab area 3 S18, S19, S20 7 Medical waste dumps near central hospital 2 S21, S22 All the test organisms were coded as EC (Escherichi coli), BS (Bacillius subtilis), ML (Micrococcus luteus), PA (Pseudomonas aeroginosa), SA (Staphylococcus aureus) and PV (Proteus vulgaris). On the basis of secondary screening it was seen that only isolates S2, S3 and S6 showed good antimicrobial activity against most of the test strains. It was found that S2 showed maximum activity after 48hrs of production while S3 and S6 showed maximum activity after 72hrs of production (Table 2). Thus only S2, S3 and S6 were selected for further study. Table 2. Screening of microorganisms: Activity profile of inhibition of the producer strains at 24hrs, 48hrs and 72hrs. Zone of inhibition (in mm). Isolates Test strains EC BS ML KP SA PV 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 S2 08 11 07 07 18 18 - - - 11 07 07 06 06 06 06 - - S3 09 - - 06 07 13 06 06 16 06 07-05 06 04 04-04 S6 08 06-08 - 21 10 10 12 09 08-07 - 04 - - - S11 04 06 06 - - - 05 05 04 - - - - 04 - - - - S15 - - 03 - - - 04 06 06 - - 04 - - 04 - - 04 S16-04 - - - - - 02 - - - 04 05 05 04 - - 04 S22 - - - - - - - - - - - - - - - - - - Optimization of antibiotic production was carried out, where growth and activity of the isolates was studied on various physical and chemical parameters such as ph, temperature, NaCl concentration and carbon sources. When the isolates S2, S3 and S6 were inoculated on media with varying ph and growth was studied by spectrophotometer, it was found that optimal ph for the growth of S2 was 8.0 while that for S3 and S6 was 6.0 and 8.0 respectively (Figure 1). In case of S2 and S6, it was observed that as the ph of the medium increased, there was an increase in the optical density till ph 8.0, after which the OD was observed to decrease. But in case of S3, the OD values gradually decreased on increasing the ph of the medium While maximum antimicrobial activity in the production medium was observed at neutral ph 7, there was none at ph 6 and 8 was studied by Rajendran et al., 2014 [10]. Figure 1. Effect of ph on growth isolated (S2, S3 and S6).

19 Riya Banerjee et al.: Optimization of Different Parameters for Antimicrobial Compound Production by Soil Microorganisms When the media with S2, S3 and S6 was incubated at different temperatures and growth studied by spectrophotometer, it was found that optimum temperature for the growth of S2 was 23 C, while that for S3 and S6 was 37 C and 50 C respectively (Figure 2). Raytapadar and Paul, 2001, reported that maximum activity at 37 C which is the optimum temperature for the antimicrobial compound production [11]. Figure 4. Effect of NaCl concentration on the growth of isolates. It was observed that when the isolates were grown in static condition the growth of all the 3 isolates was increased, while the activity of S2 was highly increased (Figure 5). Figure 2. Effect of temperature on the growth of isolates. When the isolates S2, S3 and S6 were grown in media substituted with different carbohydrate substrate and when studied it was found that sucrose and inositol amplified the growth of S2 while for S3 xylose and S6 sucrose proved to be better substrates (Figure 3). Figure 5. Growth and activity in static vs. shaking conditions. Figure 3. Effect of various carbohydrate substrates on the growth of isolates. When the isolate S2, S3 and S6 were inoculated in media with different NaCl concentrations and when the growth was studied by spectrophotometer it was found that optimum NaCl concentration for the growth of S6 was 3% while that for S3 and S2 was 0.5% (Figure 4). It was observed that as in S2 and S3, as the concentration of NaCl in the medium increased, a decrease in the OD values were observed, while that of S6, there was an increase in the OD values at 3% NaCl concentration, which further decreased on increasing the concentration. The producer strains selected from screening was characterized by morphological, biochemical tests according to the methods described by Bergey s Manual of Determinative Bacteriology. Morphological characterization is as mentioned below. Gram staining showed that S2 and S6 were positive rods where as S3 strain was positive cocci. Assimilation of carbon source showed the best results in fructose. Strain S2 and S6 showed the best results at ph where as S3 showed optimum results at ph 6. All three strains showed mild growth in 0.5% NaCl concentration. Moderate growth was seen at 31 C and optimum growth was seen at 27 C. The crude obtained after solvent evaporation was weighed and was observed to be 1.251496 mg/ml for S2, 0.43912 mg/ml for S3 and 1.42516mg/ml for S6. The maximum yield was observed for isolate S6, followed by S2 and S3 respectively. This shows that the strains S2, S3 and S6 could produce potent antibiotics and maximum yield of the antibiotic producing strains could be achieved by optimizing different growth parameters.

AASCIT Journal of Biology 2016; 2(2): 16-20 20 Table 3. Morphological and biochemical characterization f soil microorganisms. Characterization test S2 S3 S6 Morphological characterization Dirty yellow, abundant, thick Opaque, abundant, thin, transparent growth White, abundant, thick Gram staining Gram + Rod Gram + Cocci Gram + Rod Catalase - + + Amylase + - + Protease - + - Litmus test + + + Indole test + - + Citrate utilization test + - + Methyl red test + - + Voges-proskaur + - - 4. Conclusion Three different isolates showed significant and potent antibiotic activity. The morphological, biochemical studies of all the strains showed the characteristic features of the family bacteria. The strain S2, S3 and S6 isolates was found to be better antimicrobial activity in the comparison with other soil isolates of bacteria. The study showed that ph, temperature, NaCl concentration and various carbon sources positively influenced the production of the antimicrobial compounds. The three isolates tested in our study i.e., S2, S3 and S6 had different properties, each requiring specific conditions for maximum growth along with their antimicrobial compound production. References [1] Hackl, E., Boltenstern, S., Bodrossy, L. and Sessitsch, A. 2004. Comparison of diversities and compositions of bacterial populations inhabiting natural forest soil. Appl. Environ. Microbiol. 7: 5057-5065. [2] Haque, S. F. K., Sen, S. K. and Pal, S. C. 1996. Antimicrobial spectra and toxicity of antibiotics from Streptomycetes antibiotic user 15-4. Indian Journal of Microbiology. 36: 113-114. [3] Oskay, M., Tamer, A. U. and Azeri, C. 2004. Antibacterial activity of some Actinomycetes isolated from farming soils of Turkey. African Journal of Biotechnology. 3 (9): 441-446. [4] Von Tersch, M. A. and Carlton, B. C. 1983. Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216. Journal of Bacteriology. 155: 872-877. [5] Feitelson, J. S., Payne, J. and Kim, L. 1992. Bacillus thuringiensis: insects and beyond. Journal of Biotechnology. 10: 271-275. [6] Stabb, E. V., Jacobsen, L. M. and Handelsman, J. 1994. Zwittermycin A producing strains of Bacillus cereus from diverse soils. Appl. Environ. Microbiol. 60: 4404 4412. [7] Akbar, A. and Anal, K. A. 2013. Prevalence and antibiogram study of Salmonella and Staphylococcus aureus in poultry meat. Asian Pac. J. Trop. Biomed. 3 (2): 163-168. [8] Talpur A. D., Memon, A. J., Khan, M. I., Ikhwanuddin, M., Danish, M. M., Daniel and Abol-Munafi, A. B. 2012. Inhibition of pathogens by lactic acid bacteria and application as water additive multi isolates in early stages larviculture of P. pelagicus (LINNAEUS, 1758). The J. Anim. Plant Sci. 22 (1): 54-64. [9] Ananou, S., Garriga, M., Jofré, A., Aymerich, T., Gálvez, A., Maqueda, M., Martínez-Bueno, M. and Valdivia, E. 2010. Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages. Meat Science. 84: 594 600. [10] Rajendran, R., Abirami, M., Jagadeeswari, S. and Prabhavathi, P. 2014. Production, optimization and partial purification of antimicrobial compound from Streptomyces exfoliates. A Journal of Science and Technology. 2 (1): 50-55. [11] Raytapadar, S. and Paul, AK. 2001. Production of antifungal antibiotics by Streptomyces aburaviensis IDA -28. Microbiol. Res. 155 (4): 315-323.