Effect of cultural conditions and physical factors on production of antibiotic metabolites by selected actinomycetes Archives of Ecotoxicology

Antibiotic resistance is one of the biggest global problem of our times. The leading producers of antibiotics that can be used to curb this problem are actinomycetes. This study was conceived to isolate antibiotic producing actinomycetes from the soils of Menengai crater and identify the cultural and physical factors that favoured production of antibiotics by selected actinomycetes. Soil samples were collected from 32 randomly selected sampling points within Menengai crater. The actinomycetes were isolated using serial dilution technique. The actinomycetes were tested for antagonism against selected bacterial and fungal pathogens using primary and secondary screening bioassays. Based on broad spectrum of activity and the size of zone of inhibition, four potent actinomycetes were selected for further studies. The effect of growth media, pH, temperature, incubation period, aeration, inoculum concentration, carbon source, nitrogen source and salt concentration on growth and production of antibiotic metabolites was determined. The actinomycetes isolated presented varying morphological characteristics. There was a significant difference in the diameters of zones of inhibition produced by the test pathogens when subjected to the antibiotic metabolites from the selected actinomycetes (F = 6.6046 P = 0.001338). The growth and production of antibiotics by the selected actinomycetes was favoured by use of Luria Bertani as the culture medium, a pH of 6, incubation temperature of 28 o C, incubation period of 7d, aeration rate of 200rpm, inoculum concentration of 1%, glycerol as carbon source, oat meal as nitrogen source and a salt concentration of 1.5%. The growth and production of antibiotics by the selected actinomycetes is affected by culture medium, pH, incubation temperature, incubation period, aeration rate, inoculum concentration, carbon source, nitrogen source and salt concentration. There is need to curry out structure elucidation of the antibiotics from the selected actinomycetes.


Introduction
The increase of antibiotic resistance is attributed to a number of factors such as their overuse in agriculture, misuse by human beings and unnecessary prescription by physicians However, the ability of actinomycetes to form these bioactive products is not a fixed. It can be greatly increased or completely lost under different conditions of nutrition and cultivation (Bundale et al., 2015). This is because antibiotic biosynthesis is a specific property of microorganisms which depends greatly on culture conditions. Improvement in the growth and antibiotic production can be varied by manipulating the nutritional and physical parameters of the culture conditions. As a result, media composition plays an important role in the growth and efficiency of antibiotic production in actinomycetes. Padma et al. (2018) asserts that designing an appropriate fermentation medium is of 68 critical importance in the production of secondary metabolites (Tuli et al., 2014). In addition, changes in the form and type of carbon and nitrogen sources affect antibiotic biosynthesis in actinomycetes. Moreover, several growth factors play a significant role in growth and production of antibiotics by actinomycetes (Anuprita et al.,2015).

The study area
Soil sampling was carried out in Menengai crater. The crater has a height of 2,278m above sea level. It is a product of a volcanic eruption that occurred about 200,000 years ago (Omenda et al.,  2014). The crater is dormant but has a high temperature geothermal resource represented by a steaming ground at a temperature of 88 o C (Omenda et al., 2014). Menengai caldera is located in Rongai and Nakuru North Sub-counties at 35 o 28',

Collection of soil samples
Thirty-two soil sampling points were randomly selected. From each sampling point, 100g of soil was collected using a soil auger from the top 5cm. The soil samples were mixed to make a composite sample. The composite sample was packed in new polythene bags and immediately carried to Egerton University, Department of Biological Sciences laboratories. The sample was placed in sterile khaki bags and stored at 4 o C awaiting further processing and isolation of microorganisms (Wekesa et al., 2016).

Pretreatment of soil for isolation of actinomycetes
The soil sample was air dried on the laboratory benches for one week to reduce the population of Gram negative bacteria

Isolation of actinomycetes
Actinomycetes were isolated using serial dilution technique. From the composite soil sample, 1g of soil was added to a test tube containing 9mL distilled water and shaken vigorously at room temperature (23±2 o C), using a vortex mixer (PV-1, V-32, rpm 200) for 10 min. Aseptically, 1mL aliquot from the stock solution was transferred to a test tube containing 9mL of sterile physiological saline and vortexed. From these test tube, 1mL of aliquot was again transferred and mixed with another 9mL of distilled water to make 10 -2 dilution factor. Similarly, dilutions up to 10 -6 were made using serial dilution technique (Sujatha and Swethalatha, 2017). After serial dilution, 0.1mL aliquots of the soil suspensions were separately placed on sterile media plates using a micropipette. The samples were spread on the surface of culture media Luria Bertani (M1) using an L-shaped glass rod. The plates were incubated at 28±2°C and observed after 7d. Actinomycete isolates were distinguished from other microbial colonies by characteristics such as tough, leathery colonies partially submerged in the agar medium (Yang et al., 2018). Colonies with suspected actinomycetes morphology were sub-cultured on M1 agar medium and incubated at 28±2 o C for seven days. The pure cultures were maintained as slant culture on M1 agar medium as well as in glycerol at -4°C for further studies. Characterization of the actinomycetes was carried out using morphological and biochemical characteristics (Orooba et al., 2017).

Primary screening of actinomycetes for antagonism to test microorganisms
Antagonistic activity of the isolates to the test pathogenic microorganisms was determined by cross streak method (Mohammadipanah and Wink, 2015). As positive control, vancomycin (30µg/mL) was steaked at the centre of Mueller-Hinton agar for bacterial pathogens and clotimazole (1% topical solution) for fungi. The pathogens were streaked perpendicularly to the positive control. Plane Petri dishes were used as negative controls. Antagonism was measured by determination of the size of the inhibition zone in millimeters following incubation of bacteria cultures at 37 ± 2 o C for 24h and fungal pathogens at 28 ± 2 o C for seven days (Elbendary et al., 2018).

Secondary screening of actinomycete isolates for antibiotic production
About 250mL of M1 broth in conical flasks was prepared and separately inoculated with isolated actinomycete cultures using a sterile wire loop. The flasks were incubated at 28±2 o C for 7d in an orbital shaker. Ethyl acetate was added in 1:1 ration and centrifuged for 10 minutes at 600rpm. Antibiotic activity against the selected test pathogens was determined using Kirby Bauer disk diffusion technique  Unless where otherwise stated, incubation was carried out at 28±2°C for 7 d in an orbital shaker (Gallenkamp, Model 10X 400) at 100rpm. The metabolites were assayed for antibiotic activity using S. aureus (ATCC 25923) using Karby Bauer disk diffusion technique.

Data analysis
Data was analyzed using Statistical Package for Social Sciences (SPSS) Version 17.0 software. Diameter of zones of inhibition were compared by performing One-way ANOVA. Statistical values with p ≤ 0.05 were statistically significant.

Morphological characteristics of actinomycete isolates
A total of 138 actinomycetes were isolated from the soils of Menengai crater. The actinomycetes isolates gave varying colours of the aerial and substrate mycelia (Figure 1).

Figure 1
Cultural characteristics of actinomycetes isolated from soils of Menengai crater.

Antibiotic properties of the metabolites from the selected actinomycetes
There was a significant difference in the diameters of zones of inhibition produced by the test pathogens when subjected to the antibiotic metabolites from the selected actinomycetes (F = 6.6046 P = 0.001338). Among the bacteria, the mean diameter of zone of inhibition varied from 24

Culture media
There was no significant difference in the zones of inhibition produced by antibiotic metabolites extracted from isolates grown on starch casein, Luria Bertani and starch nitrate broth when tested against S. aureus (ATCC 25923) (F = 2.5618, P = 0.128). Antibiotic metabolites from cultures grown in Luria Bertani broth (M1) produced the largest zones of inhibition in metabolites from isolates PAN 75 (30±0.1mm) and the smallest from PAN 25 (23±0.2mm) (Figure 2). However, in starch casein broth, zones of inhibition ranged from PAN 25 (18 ± 0.1mm) to PAN 41 (25± 0.2mm). In starch nitrate broth, the zones of inhibition varied from metabolites from isolate PAN 25 (19±0.3mm) to PAN 75 (24±0.2mm). Among the three culture media, Luria Bartani medium was the most ideal for growth and production of antibiotics by the four actinomycetes isolates.

pH
The zones of inhibition produced by antibiotic metabolites from cultures of isolates PAN

Inoculum concentration
Inoculum size affected production of antibiotic metabolites from isolate PAN 25, 41, and 110 significantly (F = 4.31, P = 0.02). The zones of inhibition produced by antibiotic metabolites from cultures inoculated with 0.5% starter inoculum ranged from 23 ± 0.1mm in metabolites from isolate PAN 25 to 33 ± 0.2mm in PAN 75 (Figure 7). At a concentration of 1.0% of starter inoculum, the zones of inhibition varied from 27 ± 0.2mm in metabolites from isolate PAN 25 to 36 ± 0.1mm in PAN 75. However, at 1.5% starter inoculum concentration, the zone of inhibition ranged from 25 ± 0.2mm (isolate PAN 25) to 32 ± 0.3mm (isolate PAN 75). At 2.0% starter inoculum concentration, the biggest zone of inhibition was given by metabolites from PAN 75 (30 ± 0.2mm) while the smallest was presented by PAN 25 (23 ± 0.1mm). However, at 2.5% starter inoculum, the zones of inhibition varied from 20 ± 0.3mm in metabolites from isolate PAN 25 to 27 ± 0.3mm in PAN 75. Antibiotic production by the actinomycete isolates grown on Luria Bertani medium and incubated at 28 o C reached its peak at a concentration of 1% and declined with increase in inoculum concentration.

Carbon source
The carbon source affected production of antibiotic metabolites significantly (F = 5.65, P = 0.0078). The zones of inhibition by the metabolites when glucose was used as the main carbon source in the culture medium varied from isolate PAN 25 (19 ± 0.1mm) to PAN 75 (30 ± 0.3mm) ( Among the carbon sources, glycerol was the most ideal for growth and production of antibiotics by the four actinomycetes isolates.

Nitrogen source
Nitrogen source affected production of antibiotic metabolites by the actinomycete cultures significantly (F = 8.18, P = 0.0016).
When malt extract was used as the main nitrogen source, the zones of inhibition varied from 24 ± 0.1mm in metabolites from isolate PAN 25 to 30 ± 0.1mm in PAN 41 (

Salt concentration
The levels of salt concentration affected antibiotic metabolite production significantly (F = 13.41, P = 0.00012). At a salt concentration of 0.5%, the zones of inhibition produced by the metabolites varied from (20 ± 0.2mm) in metabolites from isolate PAN 25 to PAN 75 (31 ± 0.3mm) (Figure 8). However, at a concentration of 1.0%, the zone of inhibition was highest in metabolites from isolate PAN 75 (35 ± 0.2mm) and lowest in PAN 25 (22 ± 0.1mm). At a concentration of 1.5%, the zones of inhibition varied from metabolites from isolate PAN (25 ± 0.2mm) to PAN 75 (37 ± 0.1mm). In addition, at 2.0%, the zones of inhibition ranged from metabolites from isolate PAN 25 (23 ± 0.3mm) to PAN 75 (35 ± 0.1mm). At a salt concentration of 2.5% the biggest zone of inhibition was observed in metabolites from isolate PAN 75 (30 ± 0.2mm) and the smallest in PAN 25 (21 ± 0.2mm). Antibiotic production by the actinomycete isolates grown on Luria Bertani medium and incubated at 28 o C reached its peak at a salt concentration of 1.5% and declined with increase in inoculum concentration.

Figure 8
Effect of salt concentration on the production of antibiotic metabolites by the selected actinomycetes.

Discussion
In early stages of studying actinomycetes, morphological characteristics were considered adequate in characterizing actinomycetes (Messaoudi et al., 2015). Generally, actinomycetes have leathery or chalky appearance which is usually folded, branching, filamentous and bearing aerial mycelia (Cockell et al., 2016). In this study, a total of 138 actinomycete cultures were isolated from the soils of Menengai crater. The isolates had different morphological characteristics which differed with a study carried by asserted that the environment in which actinomycetes are growing in to a great extent, influences the metabolic activities of the organisms thus influencing the types of antibiotics produced.
In the current study, several culture media were compared for their suitability in the production of antibiotic metabolites by the selected actinomycetes. Luria Bertani (M1) was the best medium for production of antibiotic metabolites by the selected actinomycetes. reported that an agitation of 150 rpm was most suitable for production of antibiotic metabolites when studying the effects of aeration by agitation on antibiotics production from actinomycetes isolated from South West Indian ridge. Differences in antibiotic metabolites production based on agitation are attributed to variations in oxygen and nutrient requirements among actinomycetes (Trabelsi et al., 2016).
The effect of inoculum concentration on production of antibiotic metabolites was studied by inoculating M1 broth with varying concentration of the selected actinomycetes at 0.5%, 1.0%, 1.5%, 2.0% and 2.5%. An inoculum concentration of 1% was the best for optimum production of antibiotic metabolites. Jami et al.
(2015) obtained an inoculum concentration of 2% for optimum production of antibiotic metabolites which differed with the results of this study. The concentration of the inoculum determines the duration of the actinomycetes get to lag phase before the cultures enters the log phase and thus in turn affects production of antibiotic (Wurch et al., 2016). In addition, an inoculum concentration exceeding the optimum concentration level leads to reduced antibiotic production due to nutrients been directed towards formation of biomass rather than production of metabolites (Qais et al., 2016).
In testing the effect of carbon source on production of antibiotic metabolites, different carbon sources such as glucose, sucrose, maltose, glycerol and starch were used in this study. The results demonstrated that glycerol was the best carbon source for production of antibiotics in the selected actinomycetes. These findings differed with those of a previous study carried out by Priyambada and Roymon (2017) where starch was found to be the most suitable carbon source. A possible reason for the difference may be variations in carbon source requirements between the isolates in the present and the previous study (Waheeda and Shyam 2017). The preference of glycerol by the actinomycetes isolates studied may be explained by their ability to break the building blocks in glycerol (Semra and Mustafa, 2017).
In this study, malt extract, yeast extract, oat meal, corn meal and and peptone were used to study nitrogen source requirements of the selected actinomycetes. The results showed that oat meal was the best nitrogen source for production of antibiotic metabolites. In a similar study carried out by Ser et al. (2017), soybean was found to be the best nitrogen source for production of antibiotic metabolites while yeast and corn steep solids encouraged cellular growth of actinomycetes but not production of antibiotics. Further, Sengupta et al. (2015) showed that peptone was the best nitrogen source for production of antibiotic metabolites. This was contrary to the findings of this study. The possible reason for the difference could be variations in metabolic pathways followed by the actinomycetes when metabolizing the various nitrogen sources (Singh et al., 2016).
In actinomycetes, nitrogen affects their rate of growth and therefore indirectly determines production of antibiotic metabolites (Tao et al., 2015).
In the current study, a salt concentration of 1.5% favoured optimum production of antibiotic metabolites by the selected actinomycetes. According to Waheeda and Shyam (2017), salt concentration affects production of antibiotic metabolites due to its effects on osmotic pressure of the medium and the actinomycetes inoculum. Usman et al. (2016) found optimum concentration of NaCl to be 1%, which differed with the results of the present study. The observed variations in the amount of NaCl required for optimal production of antibiotic metabolites between the previous and the current study may be attributed to differences in growth requirements in actinomycetes especially the ability to survive in saline environments (Waithaka et al., 2017).

Conclusion
The soils of Menengai crater have actinomycetes some of which produce antibiotics. The growth and production of antibiotics by the selected actinomycetes is affected by culture medium, pH, incubation temperature, incubation period, aeration rate, inoculum concentration, carbon source, nitrogen source and a salt concentration. There is need to curry out structure elucidation of the antibiotics from the selected actinomycetes. In addition, similar studies from other harsh environments need to be carried out.

Conflict of Interest
The authors declare that they had no competing interests.