Enhanced Isolation of Streptomyces from Different Soil Habitats in Calamba City, Laguna, Philippines using a Modified Integrated Approach

Streptomyces species are considered to be the most prolific sources of various bioactive secondary metabolites that are important for antibiotic production. Here, we describe a modified integrated approach to isolate Streptomyces species from diverse soil habitats, such as dumpsite, garden, forest, grassland, and riverside in Calamba City, Laguna, Philippines. A total of 25 soil samples were collected from a depth of 0–20 cm using systematic random soil sampling. All soil samples were air-dried, crushed, pretreated with calcium carbonate, and incubated on a rotary shaker. Isolation of Streptomyces in soil samples was then performed using the standard serial dilution plate technique on starch casein agar supplemented with nystatin (50 μg/ml) and ampicillin (5 μg/ml). Identification of the Streptomyces isolates was done using a polyphasic method that includes morphological and biochemical characterization. A total of 103 morphologically and biochemically distinct Streptomyces were isolated from diverse soil habitats. The number of Streptomyces isolates varied in each collection site, with the highest number collected from dumpsite soil and the least from forest soil. Most of the hydrogen sulfide producers were noted to be isolated from dumpsite samples. Moreover, more Streptomyces were isolated in soil habitats at higher altitudes with a slightly acidic to alkaline pH and a temperature ranging from 29 to 33°C. Employing the modified integrated approach, we have isolated up to 10 times more Streptomyces compared to early studies. These Streptomyces isolates can be valuable for future drug discovery and development research.


Introduction
Microorganisms have long been a reputable source of secondary metabolites that have been successfully industrialized as medical drugs [1]. It is hypothesized that new metabolites can be discovered through intensive screening of the most prolific producers of chemically diverse secondary metabolites, such as actinomycetes [1][2][3]. Among actinomycetes, the majority of the novel antibiotics originated from different Streptomyces strains [4].
is genus comprises industrially and economically important microorganisms that can produce numerous novel bioactive secondary metabolites [6,7]. Soil is the natural habitat of Streptomyces [6,8], a physically, biologically, and nutritionally complex ecosystem often composed of complex carbohydrates [9]. Streptomyces species are widely distributed in soil environments [10]. However, studies highlighted the importance of collecting soil samples from unique and unexplored habitats for the isolation of possibly novel strains of Streptomyces [11][12][13]. More Streptomyces can also be isolated from nonagricultural soils [8,14]. Similarly, environmental conditions of the soil habitats should be favorable to support the growth of Streptomyces [8].
In the Philippines, there is a wealth of evidence proving the existence of Streptomyces in soil habitats and the antibiotic-producing ability of the isolated strains. Several antibiotics have been discovered from the different Streptomyces strains isolated from the Philippine soil, such as erythromycin (ilotycin) [15], roseoflavin [16], and lagunamycin [17]. More recent local studies have focused on the isolation of antibiotic-producing Streptomyces from the marine environment [18,19], while others have focused on the agricultural application of the compounds derived from different strains of Streptomyces, such as the promotion of plant growth [20] and crop protection [21]. Only a few local studies are directly involved in the isolation of Streptomyces from soil habitats [22,23]. is study thus aims to investigate the effectiveness of a modified integrated approach for the isolation of Streptomyces from different soil habitats in a selected city (Calamba City, Laguna) in the Philippines.

Collection of Soil Samples.
A total of 25 soil samples were collected from randomly selected diverse habitats (i.e., dumpsite, forest, garden, grassland, and riverside) in Calamba City, Laguna, Philippines. Systematic random soil sampling was used for the collection of soil samples, aided by grid maps generated using the ArcMap feature of ArcGIS 10.5 overlaid with maps from Google Maps for better visualization. Randomization of grid areas for dumpsite, forest, garden, and grassland was conducted using an online randomizer (https://www.randomizer.org/); samples were collected from the center of the randomized grids. Riverside samples were collected 1 m away from the river and 15 m away from each other. e environmental conditions of the collection sites were recorded in terms of geographic coordinates, altitude, soil temperature, and soil pH (Table 1). Five 400 g of soil samples were collected at each location from a depth of 0-20 cm. e samples were properly labeled and placed in tightly closed polyethylene bags to avoid spillage while being transported to the laboratory.

Pretreatment of Soil Samples.
Soil samples were air-dried at room temperature for 7 days to remove moisture and reduce the population of bacteria other than Streptomyces species. Air-dried soil samples were then manually crushed and sieved through the sterilized mesh to get rid of large debris as previously described [24][25][26]. Soil-CaCO 3 mixtures (10:1 w/w) were prepared and incubated for 2 days at 30°C in a closed sterile Petri dish as performed by Oskay [8]. Following the protocol of Njenga et al. [27], soil-CaCO 3 mixtures (20 g) were suspended in 180 ml of sterile distilled water in a flask followed by incubation (28-30°C) on a rotary shaker at 200 rpm for 30 min.

Isolation of Pure Culture of Streptomyces Species.
Isolation of Streptomyces species in soil samples was performed using the standard serial dilution plate technique on starch casein agar (SCA) (M2054, HiMedia Laboratories, India). In brief, all pretreated soil samples were serially diluted up to 10 −6 . To create a uniform suspension, each tube for serial dilution was vortexed. An aliquot of 1 ml of every dilution was plated out in triplicate and was overlaid with approximately 20-25 ml of SCA. After gentle rotation, isolation media were incubated at 28-30°C for 7 days. e culture media were prepared following the manufacturer's protocol and were supplemented with 50 μg/ml of nystatin (Afunginal ® , ACME Laboratories Ltd., Bangladesh) and 5 μg/ml of ampicillin (Ampicin ® , Sandoz International GmbH, Germany) to minimize the growth of contaminating fungi and bacteria, respectively. e total bacterial count (CFU/g of soil) and total Streptomyces count (CFU/g of soil) were also quantified following the procedure of Kizito and Nwankwo [14]. Purification of isolates was carried out by streaking colonies on new plates of SCA using sterile wire loops.
e isolated pure cultures of Streptomyces species were transferred to the SCA slants and stored at 4°C until further use.

2.4.
Characterization of Streptomyces Isolates. Streptomyces isolates were identified using a polyphasic method that includes cultural, morphological, and biochemical characteristics based on the guidelines recommended by Bergey's Manual of Systematic Bacteriology and the International Streptomyces Project [8,24,28]. All colonial morphological characteristics were observed on SCA and were used for classification and differentiation. e morphological characteristics consist of the color of aerial and substrate mycelia, melanoid pigment production of the colony, Gram stain appearance (i.e., viewed under 1000x magnification), and spore chain morphology. Streptomyces isolates were categorized into six color series (i.e., gray, white, red, yellow, green, and variable (brown, pink, orange, violet)), which were determined through comparison with the Inter-Society Color Council-National Bureau of Standards (ISCC-NBS) color charts [29]. Isolates were also either classified as melanoid producers (i.e., brown-black colonies) or melanoid nonproducers (i.e., colonies other than brown and black) [30]. Isolated strains were also grouped according to the shape of spore chains observed under light microcopy, such as rectiflexibiles (straight to flexuous), retinaculiaperti (open loops, hooks, or spirals consisting of one or two turns), and spirales (tight spirals) [24,28]. Lastly, biochemical tests including catalase, motility, nitrate reduction, urea hydrolysis, hydrogen sulfide (H 2 S) production, indole production, methyl red, Voges-Proskauer, citrate utilization, and utilization of carbon sources (e.g., glucose, lactose, and sucrose) and gas production observed on triple sugar iron slant were conducted [6]. Table 1 shows the soil characteristics and the frequencies of bacterial and Streptomyces isolates from each soil habitat examined in this study. We observed that more Streptomyces species were isolated from soil habitats (e.g., dumpsite and garden) at higher altitudes, with slightly acidic to alkaline pH and temperatures ranging from 29-33°C, which is consistent with the reported optimum conditions that support Streptomyces growth [24,30].

Results and Discussion
Bacterial load varied in each collection site, where the highest bacterial count of 4.6 × 10 7 CFU/g of soil was from grassland, while the lowest count was from the forest with 6.2 × 10 6 CFU/g of soil. ree hundred sixty-seven distinct colonial growths were seen on SCA post-incubation; 103 (28%) of these isolates were morphologically and biochemically distinct Streptomyces species (Figure 1), with the highest number isolated from dumpsite soil (35 out of 103). e same observation was noted in previous studies of Oskay [8] and Kizito and Nwankwo [14] where higher densities of Streptomyces have been isolated in nonagricultural soils like dumpsites. e color of the isolated Streptomyces colonies varied as depicted in Figure 2, although more than half are gray and white. Melanin production was seen on 14 Streptomyces colonies, notably from dumpsite, garden, and riverside soils. Barka et al. [30] suggest that melanin production has a vital role in enhancing Streptomyces' capability to adapt and survive in challenging and harsh environmental conditions.
Microscopic examination revealed all 103 isolates are Gram-positive, filamentous with branching mycelia (Figure 3) and the majority possesses rectiflexibiles (74%) spore chains. Table 2 displays biochemical test results consistent with the usual metabolic profile of Streptomyces species-catalasepositive, indole-negative, and can utilize glucose as their carbon source. Interestingly, most of the hydrogen sulfide producers (21 out of 37) came from the dumpsite, which is known to contain alternative sources of nutrients (e.g., food waste, paper, plastic) for microbial use with hydrogen sulfide as an end product [31,32]. In addition, Long et al. [33] reported that the accumulation of hydrogen sulfide may cause an increase in soil pH, which favors the growth of Streptomyces. is could likewise explain the abundance of Streptomyces isolated from dumpsites as seen in this study.
Our approach to Streptomyces isolation combined pretreatment, enrichment, and selective inhibition methods. e pretreatment employed in this study consisted of desiccation, crushing, calcium carbonate supplementation, and incubation on a rotary shaker, as opposed to published studies that used these techniques singly or in combination [8,[24][25][26][27]. Starch casein agar as an enrichment culture medium [8,34] and antibiotic supplementation (e.g., nystatin and ampicillin) for selective inhibition [25,35] have been shown to be most effective in isolating Streptomyces.     Performing this modified integrated approach, a substantially higher number of Streptomyces were isolated compared to similar studies (Table 3). Up to 10X more Streptomyces were isolated in this study compared to published literature using one pretreatment method alone and no antibiotic supplementation. Approximately 2-7X the number of Streptomyces isolates were isolated in our study employing the modified integrated approach, in contrast to early studies that applied antibiotic supplementation and one to two pretreatment methods only.

Conclusion
We have demonstrated in this study the utilization of a modified integrated approach for enhanced isolation of Streptomyces from soil habitats. We isolated 103 soil Streptomyces from different soil habitats in Calamba City, Laguna, Philippines, with the highest number collected from dumpsite soil. ese isolates can be valuable for novel antibiotic discoveries. Further studies are now being conducted to investigate the potential antibiotic activity and to identify the isolates at the species level through molecular methods.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Ethical approval was obtained from the University of the Philippines Manila Research Ethics Board (UPMREB) (UPMREB code: 2020-145-EX).    International Journal of Microbiology 5