RESEARCH ARTICLE

Morphological diversity of some actinomycetes isolated from Durg district of Chhattisgarh

Devnarayan Patel*, R. Diwan, Hemshikha Sahu

Department of Botany, Govt. Nagarjuna P.G. College of Science, Raipur (C.G.) India.

Authors Email- devpatel121997@gmail.com, rupinderdiwan@gmail.com, hshemshikha16@gmail.com

*Corresponding Author Email- devpatel121997@gmail.com

Graphical Abstract

Introduction

Actinomycetes are primarily found in soil, where they contribute significantly to nutrient cycling and bioactive compound production (Javed et al., 2021). Their distribution varies by soil type and environmental conditions (Kulkarni & Siddique, 2012). Technosol is a type of soil found in industrial areas. Its properties and pedogenesis are primarily determined by its technical origin (Cimermanova & Piknova, 2021). They contain artifacts and cover a horizontal area (Michaela, 2021; Jindal, 2018). High temperatures in an industrial area promote the growth of thermophilic Actinomycetes (Kikani et al., 2010). Actinomycetes are identified based on morphological characteristics.

The isolates were examined microscopically and macroscopically using morphological observations of culture, which included

Straight, flexible, and spiral forms, as well as observed growth patterns (Deepthi & Rosamma, 2014). Actinomycetes are the most abundant and highly diverse groups among all organisms living today (Tiwari & Gupta, 2013). Actinomycetes are eubacteria that grow as filaments or as mycelium, chains of conidia formed by spore-bearing aerial hyphae (Zainordin, 2011). The genus belongs to the Streptomycetaceae family, which is part of the order Actinomycetales (Madhaiyan et al., 2022). Actinomycetes morphology describes the qualities of colonies, cells, and spores. Actinomycetes were isolated using Actinomycetes Isolation Agar medium, and their morphological characteristics were subsequently described according to methodology given by Sarkar & Suthindhiran (2022) and Vimal (2009).

 Industrial areas have a specific type of soil called technosol (Kulkarni & Siddique, 2012). The technical origin of the soil dominates its properties and pedogenesis (Capra et al., 2015). They contain artifacts and cover a horizontal extent (Michaela, 2021) and (Jindal, 2018). An industrial area has high temperatures, which are ideal for the growth of thermophilic Actinomycetes. (Ravi and Krishnan, 2022). Five soil samples were collected from five different industrial areas in Bhilai, including ACC Jamul, Jaypee Cement, Rai Industries, Goyal Industries, and the Bhilai Steel Plant. Isolation was performed to investigate the distribution of Actinomycetes in an industrial area using morphological characteristics.

Literature review

In Abroad studies were conducted by researchers like Peverill, (1999) who studied on soil analysis an interpretation manual. Kaur et al., (2010) described the Physico-chemical Analysis of the industrial effluents and their impact on the soil microflora. Studies on Soil heavy metal contamination in an industrial area: analysis of the data collected during a decade were conducted by D’Emilio, et al., (2013).  Markus. & Bratney (1996) described an urban soil study: heavy metals in Glebe, Australia.  Bashour,  & Sayegh, (2007) worked on  Methods of analysis for soils of arid and semi-arid regions Tóth et al. (2016) discovered heavy metals in agricultural soils across the European Union, with implications for food safety.

In India Dheeba and Sampathkumar (2012) investigated heavy metal contamination in surface soil around industrial areas in Tamil Nadu. Krishna and Govil (2007) discovered soil contamination caused by heavy metals in an industrial area of Surat, Gujarat, Western India. Govil, P. K et al., (2008). Described Heavy metal contamination of soil in the Katedan industrial development area in Hyderabad. Govil, Reddy, and Krishna (2001) investigated soil contamination caused by heavy metals in the Patancheru industrial development area, Andhra Pradesh, India. Ramesh Kumar and Anbazhagan (2018). Heavy metals were analyzed and assessed in soils near industrial areas in Mettur, Tamil Nadu.

In Chhatisgarh studies were conducted by researchers like Tiwari et al., (2015) discovered An analytical study of heavy metal concentration in soil of an industrial region in Chhattisgarh, central India. Anurag & Sahu (2021) studied on Impact of cement industries dust on soil properties in Bhatapara, Chhattisgarh. Verma, et al., (2021) was investigated a Study of Soil Nitrogen Level and Splitting on Nutrient Uptake around the Industries of Chhattisgarh, India. Rajak, et al., (2023) was give description on Urban soil quality of Raipur, Chhattisgarh, India.  Verma, et al., (2020) investigated Soil Contamination in the Industrial Vicinity of Bemetara and Raipur District of Chhattisgarh, India Kamavisdar, et al., (2005) was provided an Analytical studies on lead pollution in roadside soil samples of Raipur and Bhilai, Chhattisgarh State, India

Material and Method

Sample collection and preservation

The surface layer of soil was removed, and samples were collected by scraping off 4-5 cm of surface material with a spatula, as described by Gautam & Avasthi (2019). The central portion of each sample was collected in sterile plastic bags using a trowel. A total of five soil samples, labeled SS-I to SS-V, were collected from different industrial sites in Bhilai, including: ACC Jamul (SS-I),  Jaypee Cement (SS-II), Rai Industries (SS-III),  Goyal Industries (SS-IV), Bhilai Steel Plant (SS-V)

These sites were selected for screening of Actinomycetes.The collected soil samples were incubated at room temperature for 24 hours and then stored at 4°C until further processing.

Analysis of soil sample

Physical properties of soils like Soil texture, structure and soil color with pH was analyzed by the using the methodology of Gupta et al., (2010).

Isolation

For the isolation and screening of pure cultures of Actinomycetes, one gram of each soil sample was suspended in 10 ml of sterile water and thoroughly mixed with a shaker. Stock solutions were prepared by transferring 1 ml of the suspension to a new test tube containing 9 ml of sterile water, resulting in a five-fold serial dilution process, which was repeated five times. 0.1 ml of each dilution was spread onto Actinomycete Isolation Agar (AIA) Media and incubated at 37°C for 7 days. Following the incubation period, the culture plates were examined, and colonies with Actinomycetes characteristics were chosen. These colonies were then purified via the streak plate method.  The purified Actinomycetes colonies were transferred onto fresh AIA media.

Characterization

The identification of the isolated was carried out through both macroscopic and microscopic examinations, following the methodology described by Lechevalier (1968). Macroscopic examinations included a detailed analysis of colony morphology on AIA, focusing on characteristics such as: Size, Shape, Configuration, Elevation, Margin, Pigmentation. Microscopic examinations determined the presence or absence of Aerial mycelium, Substrate mycelium, Overall colony structure using the methodology of Malviya et al. (2013). Each isolate was streaked onto an AIA plate and incubated at 37°C for seven days to examine the morphological characteristics of the colonies. The morphological features were documented as part of the identification process, including:

 Colony morphology, Mycelium presence and structure, Pigmentation and other notable characteristics. These observations were carefully recorded to facilitate accurate identification of the isolates.

Results

A total of 34 isolates were obtained from the five soil samples, which exhibited variations in color: SS-I (ash grey), SS-II (medium brown), SS-III (dark brown), SS-IV (light brown) and SS-V (red blood) showed variation in color. The soil texture was predominantly loam with clay or silt, and the pH ranged from 6.8 to 7.6 (Table 1).

A total of nine Actinomycetes isolates were obtained from SS-I, with colony diameters ranging from 0.1 cm to 1 cm. The aerial mycelium was predominantly powdery white, except for ACC-V and VI (orange) and ACC-VIII (blue spot). The substrate mycelium was mainly creamy white, except for ACC-V (light orange). The texture of the isolates varied, with the following characteristics: Most isolates were powdery nodular or fuzzy, ACC-III and VII were waxy, ACC-IV and IX were cotton-like Filamentous shapes were observed, although ACC-II, V, and IX exhibited irregular shapes. The elevations were primarily crateriform, except for ACC-II, V, and VIII (raised), and ACC-III and IX (umbonate). The margins were mainly filiform, with ACC-I, V, and VIII being undulate, and ACC-III and VI being entire. All isolates were gram-positive and did not produce pigments, except for ACC-V and VI (orange pigment) and ACC-VIII (blue pigment). A detailed summary of these morphological characteristics is presented in Table 2.

Morphological Characterization of Actinomycetes was done for Isolates from SS-II a total of 7 Actinomycetes isolates (JP-I to JP-VII) were recovered from SS-II, exhibiting colony diameters ranging from 0.1 cm to 1.3 cm. The aerial mycelium displayed a predominantly white coloration, with notable exceptions in JP-IV (bluish) and JP-VI (brown). Conversely, the substrate mycelium exhibited a range of colors, including white, yellow, blue, and creamy.Textural analysis revealed a predominantly powdery-fuzzy morphology, although JP-III, JP-IV, and JP-VI exhibited a cotton-like texture. Morphological examination further revealed filamentous shapes, with JP-III and JP-V displaying irregular and circular shapes, respectively. Elevation patterns varied, with convex and crateriform shapes observed, except for JP-III and JP-VI (raised) and JP-IV (umbonate). Margin characteristics were primarily filiform, with JP-I and JP-IV exhibiting entire margins, and JP-II and JP-VI displaying undulate margins. Gram staining confirmed that all 7 isolates were gram-positive. Notably, JP-II and JP-V exhibited yellow diffusible pigments. A comprehensive summary of these morphological characteristics is presented in Table 2.

Ten Actinomycetes isolates were obtained from SS-III, with colony diameters ranging from 0.1 to 0.7 cm. The aerial mycelium exhibited a range of colors, including white, creamy white, and transparent white, with the exception of RAI-VI (yellow) and RAI-VII (brown). The substrate mycelium also displayed a range of colors, including white, creamy white, transparent white, and chalky white. Notably, RAI-III exhibited bluish pigmentation, while RAI-X displayed yellowish-white coloration. Textural analysis revealed a predominantly powdery to fuzzy morphology, with the exception of RAI-IV and RAI-VII, which exhibited a waxy texture. Morphological examination further revealed filamentous and circular shapes, with RAI-VII displaying an irregular shape. Elevation patterns varied, with umbonate, raised, and convex shapes observed. However, RAI-VI exhibited a crateriform elevation. Margin characteristics were primarily entire and undulate, with the exception of RAI-V and RAI-IX, which displayed filiform margins. All isolates were gram-positive. Additionally, RAI-IV and RAI-VIII exhibited diffusible pigments, with yellow and reddish-brown pigmentation observed, respectively (Table 2)

Three Actinomycetes isolates were isolated from S.S. IV, with colony diameters ranging from 0.2 to 0.3 cm. The aerial mycelium displayed a range of colors, including white, creamy, and yellow, while the substrate mycelium was predominantly white, except for GYL-III, which exhibited yellow coloration. The texture of the isolates was cotton-like and fuzzy, with shapes varying from filamentous to irregular. The elevations were characterized as crateriform, umbonate, and raised, while the margins were primarily entire, except for GYL-II, which displayed undulate margins. All isolates were gram-positive, indicating a thick peptidoglycan layer in their cell walls. Notably, GYL-III exhibited brown diffusible pigments, suggesting the production of secondary metabolites (Table 2)

In soil sample V, a total of 5 isolates were found. The colony sizes of the respective samples ranged from 0.2 to 0.8 cm. The color of the aerial mycelium was white, except for BSP-I and BSP-IV, which were grey. In contrast, the color of the substrate mycelium was grey, except for BSP-III and BSP-V, which were white. The texture of the samples was observed to be powdery and fuzzy, except for BSP-II, which was waxy, and BSP-V, which was nodular. The shapes of the isolates were primarily filamentous, except for BSP-I and BSP-V, which were circular. The elevations were raised, umbonate, and convex, except for BSP-IV, which was crateriform. The margins were filamentous to undulate; however, BSP-III and BSP-V had entire margins. All samples were reported as gram-positive. Notably, BSP-IV exhibited grey diffusible pigments (Table 2).

Table 1.  Physico-Chemical properties of Soil Samples

Table 2.  Morphological Characteristics of Actinomycetes

Graph 1 Total Number of Isolates from 05 Soil Samples

Graph 2 Diffusible Pigments in Isolates

Figure 1 Pure culture of isolates from S.S. I (ACC I to ACC IX)

Figure 2 Pure cultures of isolates from S.S. II (JP I to JP VII)

Figure 3 Pure culture of isolates from S.S. III (RAI I to RAI X)

Figure 4 Pure culture of isolates from S.S. IV (GYL I to GYL III)

Figure 5 Pure culture of isolates from S.S. V (BSP I to BSP V)