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

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Associated Data

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

Abstract

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.

1. 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 [1–3]. Among actinomycetes, the majority of the novel antibiotics originated from different Streptomyces strains [4].

Streptomyces species are aerobic, filamentous, nonmotile, Gram-positive bacteria characterized by mycelial growth with more than 70% guanine-cytosine (GC) content in their DNA [5]. This 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 [11–13]. 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]. This 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.

2. Materials and Methods

2.1. 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. The 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. The samples were properly labeled and placed in tightly closed polyethylene bags to avoid spillage while being transported to the laboratory.

Table 1

Location and environmental characteristics of the collection site with corresponding bacterial load from each soil sample.