**Abstract**

Actinobacteria isolated from less studied sites on our planet represent a huge opportunity for the discovery of novel microorganisms that may produce unique compounds with biological activity. The class actinobacteria encompasses 80% of the microbes that produce the antibacterial compounds used in medicine today. However, the resistance acquired/showed by pathogenic microorganisms opens the opportunity to explore Mexican ecosystems as a source of novel actinobacteria. Air samples have shown to be an excellent site of study, marine ecosystems which include sediments and marine organisms are important sources of novel actinobacteria and soil samples are still a promising source to isolate this microbial group. The isolation of novel actinobacteria is a dynamic strategy that depends on the expertise, patience, and talent of the techniques applied and needs to be fully explored to untap the unknown actinobacterial diversity with potential in biology.

**Keywords:** actinobacteria, air samples, discovery, marine resources, soil samples

#### **1. Introduction**

Megadiverse countries constitute exceptional areas on Earth where most of the planetary biodiversity is present. The complexity of these areas is huge, but in most of the cases, two major points are key: (1) the geographical location, and (2) the abiotic and biotic elements present. Mexico is one of the top five megadiverse countries in the world and its macrodiversity and endemism are well represented by amphibians, mammals, plants, and reptiles [1]. However, the knowledge of microscopic organisms such as archaea, bacteria, protozoa, microscopic algae, and microscopic fungi, that inhabit aquatic, atmospheric, marine, and soil ecosystems is neither poorly known, studied nor understood.

The vision of this chapter is to contribute to the knowledge, research, and study of microscopic life in different Mexican ecosystems, as they are often ignored or poorly mentioned in federal texts or even in biotic inventories. Our examples are

some members of the class actinobacteria [2], and we aim to demonstrate why it is so important to study these bacteria in such detail to fully explore and untap the unknown actinobacterial diversity with potential in biology. Using a dynamic isolation strategy on air, soil, and marine sediments and sponges collected from yet unexplored sites of the Mexican territory, we have been able to cultivate novel actinobacteria. Our findings showed that expertise, patience, and talent of the techniques applied are keys in the hunt for new potential microbes.

The isolation of microorganisms, including actinobacteria, is not new but a dynamic strategy that is continuously changing, and the developed to date is a powerful tool. For more than two centuries, researchers from Japan, the UK, and USA have shown that beneficial microorganisms isolated from the soil are important to Biology. In recent years the isolation of the first genus of actinobacteria from the marine origin [3] and novel marine species [4] have shown the importance of exploring the marine environment. Extreme or unexplored sites have also shown the isolation of actinobacteria including putative novel actinobacteria [5].

Our research studying actinobacteria started in 1999 [6, 7], but until 2009 we properly started the exploration of the Mexican (marine) ecosystems [8] as an independent group. We followed bioprospecting, diversity, and systematic approach but designing a selective isolation strategy was the first step for a complete full project or protocol [9].

Actinobacteria is a complex group of bacteria, they present forms such as rods or bacilli, many differentiate in vegetative mycelium, aerial hyphae, and chain of spores, and in a few genera fragmentation of the hyphae is present. In general, the Gram reaction is positive and the content of guanine plus cytosine is above 69%mol. The morphological characteristics within the class showed how complex this group is. Actinobacteria are considered saprophytes or beneficial microbes, but a small number of species have been shown to be either pathogenic [10] or opportunistic [11]. This microbial group has been isolated or cultivated using classical methods from almost every sample taken on Earth and they are always detected when using molecular methods to study this group in a given environmental sample.

Actinobacteria also have the innate ability to produce secondary metabolites with biological activity, to date, this class encompasses 80% of the microbes that produce the antibacterial compounds used in medicine. Complete Genome Sequencing of some genera of actinobacteria such as *Streptomyces* [12] and *Salinispora* [3, 4] have shown the biotechnological potential that these organisms contain and maybe explored and exploited for human wellbeing.

The more we study and discover actinobacteria the more important they become in pass, present, and future assignments. Microorganisms and microbial biomass, including actinobacteria, represent the major resource for biotechnology and biological areas. We should continue exploring their role in nature in order to understand their biology, ecology, and bioprospecting potential [13–16].

### **2. Selective isolation of actinobacteria from different Mexican ecosystems**

#### **2.1 The atmosphere as a source of novel actinobacteria**

The Earth's atmosphere is divided into six specific layers with completely different characteristics: (1) Troposphere, (2) Stratosphere, (3) Mesosphere, (4) Thermosphere, (5) Ionosphere, and (6) Exosphere. It has been established that the atmosphere plays an important role to transport microorganisms, place to place, continent to continent. The latter has been established using scientific tools in the

#### *On the Selective Isolation of Actinobacteria from Different Mexican Ecosystems DOI: http://dx.doi.org/10.5772/intechopen.104699*

last 200 years and in the last 15 years, NASA has monitored mineral dust particles from the Sahara desert with a robust precision using spaceborne satellites. These Saharan dust plumes contain microorganisms and enter mainland Mexico by the Yucatan Peninsula [17].

The atmosphere is a hostile environment for microorganisms though there are a significant number of them in the troposphere, with air as their main dispersion pathway. The abundance, diversity, survival, and transport of microorganisms, as passive drivers, and how they get stressed severely by the conditions presented in the atmosphere have fully been reported [18]. Most of the microorganisms in the atmosphere are present as spores, while others have adapted to resist desiccation or high/low temperatures [19]. Recent reports have also shown that some microorganisms (i.e., by using specific proteins) can act as ice nucleating particles [20] and that they may play an important role in cloud formation [21]. In general, bacteria (including actinobacteria) present in the atmosphere are attached to suspended particles [17], and their concentration change notably during the dry or wet seasons of each year [22].

#### *2.1.1 Isolation of a streptomycete from air samples of Merida-Yucatan*

As part of the African Dust and Biomass Burning Over Yucatan (ADABBOY) Project [23] in the city of Merida (N 21°02´75.4´´ W 89°65´44.8´´) a selective isolation strategy was carried out in order to cultivate/recovered putative actinobacteria in May 2017. Air samples were impacted using a Quick Take 30 Sample Pump® and a BioStage® SKC (**Figure 1**) in Petri dishes prepared with a slightly modified Glucose Yeast Malt extract agar (GYM medium; Appendix A; Medium 65: DSMZ; www.dsmz.de) supplemented with Rifampicin (5 μg/mL; Sigma-Aldrich, USA) and Nystatin (50 μg/mL; MICOSTATIN® Bristol Myers Squibb, Mexico).

Plates were incubated in two different laboratories and conditions. The first laboratory was in the city of Merida at the Universidad Autónoma de Yucatán, using an aerobic incubator set at 25°C and the plates were incubated for 24 hours. For the second procedure, the plates were transported to a laboratory in Mexico City where the incubation time continued aerobically at 30°C (IncuMax IC-320, Amerex USA) for 8 weeks with eye observation each week. One microorganism with the production of aerial hyphae, a gray mass of spores, and a very deep purple diffusible pigment (**Figure 2**) was selected from the isolation plates for further studies.

**Figure 1.** *The device used for the air particles.*

**Figure 2.** *Morphology and purple diffusible pigment of an airborne streptomycete.*

The selected isolate was coded C6-CCA-May-1. After a purification process using GYM medium and two different techniques (cross streak and serial dilutions), bacterial biomass and spores of the strain were ultra-preserved in 20% glycerol. Morphological characterization was carried out using a GYM medium (**Figure 3**) and a Gram staining procedure (**Figure 4**) was carried out following well-known universal protocols.

Molecular identification of strain C6-CCA-May-1 was carried out following protocols previously published [8, 24]. First, the DNA of strain C6-CCA-May-1 was extracted and used as a template for PCR amplification using the 16S rRNA gene (Appendix B). The sequence of the 16S rRNA gene PCR product confirmed that strain C6-CCA-May-1 belongs to the genus *Streptomyces*. According to the EZbiocloud phylogenetic approach *Streptomyces* sp. C6-CCA-May-1 was related to *Streptomyces viridiviolaceus* (NBRC 133559T ), *Streptomyces werraensis* (NBRC 13404<sup>T</sup> ), *S. asenjonii* (KNN35.1b<sup>T</sup> ), *Streptomyces minutiscleroticus* (NBRC 13000<sup>T</sup> ) and S*. levis* (NBRC 15423<sup>T</sup> ) (**Table 1**).

A Bayesian phylogenetic tree was constructed in order to establish the taxonomic position of *Streptomyces* sp. C6-CCA-May-1 shows that *Streptomyces* sp.

**Figure 3.** *Aerial hyphae and spore mass of isolate C6-CCA-May-1.*

*On the Selective Isolation of Actinobacteria from Different Mexican Ecosystems DOI: http://dx.doi.org/10.5772/intechopen.104699*

#### **Figure 4.**

*Gram staining of the airborne streptomycete.*


#### **Table 1.**

*List of hits from the EZbiocloud 16S database.*

C6-CCA-May-1 is related to *Streptomyces viridiviolaceus* (**Figure 5**). Moreover *Streptomyces* sp. C6-CCA-May-1 belongs to the *S. glaucus* subclade [25] and according with the probability (number 1) showed in the cluster formed in the phylogenetic tree, *Streptomyces* sp. C6-CCA-May-1 may well represent a novel species. The similarity value amongst *Streptomyces* sp. C6-CCA-May-1 and *S. viridiviolaceus* is 98.2%. A full comparison study based on the phenotypic, morphological microscopic characteristics and chemotaxonony amongst *Streptomyces* sp. C6-CCA-May-1 and *S. viridiviolaceus* could further clarify their accurate taxonomic position and status. Furthermore, genomic analyses are required to fully understand the putative unique biotechnological potential of this airborne streptomycete.

Streptomycetes are an ecologically important group capable of producing diverse bioactive compounds. However, their taxonomy and diversity in air samples remain unknown. For almost two centuries the genus *Streptomyces* has been considered a goldmine and the major producer of bioactive natural products (i.e. antibiotics) [13, 26]. The recent discovery of a new member of the actinomycin family of antibiotics shows the potential to explore old streptomycetes [26], but there still is an open door for the discovery of new bioactive molecules through novel species [9], recovered from "unusual" environments.

**Figure 5.** *Phylogenetic tree of the 16S rRNA gene of the airborne streptomycete.*

#### **2.2 Marine Mexican resources home of novel actinobacteria**

Seventy percent of our planet is covered by the ocean but from one marine research project, there are 10 of terrestrial origin. Little is still known about marine biodiversity (including microorganisms) though their potential is extraordinary and needs to be fully studied and exploited. Mexico is surrounded by the Pacific Ocean, the Sea of Cortez (*aka.* Gulf of California), the Gulf of Mexico, and the Caribbean Sea and this is the main reason why the country shows, at least potentially, a high number of species richness, diversity, and endemism in the coastal areas. The study of the Mexican marine ecosystems and their marine resources is still poorly studied. In contrast, the potential of actinobacteria isolated from marine sediments collected in Mexico has been reported [8, 27–29] and showed that they produce novel and potent compounds with biological activity. Major European research marine programs have shown the importance to study and protect the marine ecosystem but in Latin America, the efforts of conservation and protection of unique marine Mexican sites are urgently needed. It has been recognized that the ancient life of planet Earth started in an aquatic system and that the immense microbial diversity present on it plays an important role in the biochemical cycles. In this subsection, two projects are presented: (a) the isolation of marine actinobacteria from sediments and (b) sponges. Marine sediments were collected from the Revillagigedo Archipelago National Park (RANP) in December 2017 and January 2018. The exploration of microbes from marine sediments of this pristine and unique place has never been studied. Species of the *Aplysina* sponge are ubiquitous inhabitants of tropical and subtropical marine locations [30]. In recent years our group described novel marine sponges of the sponge *Aplysina* (order Verongida) [31] and since 2005 the exploration of the microbiota associated with five different species is an undergoing study.

#### *2.2.1 Actinobacteria isolated from marine Mexican resources*

In the present project, a total collection of 34 marine sediments or sponges were collected at RANP during two expeditions (December 2017 and January 2018). A selective isolation strategy using 11 of the marine sediments and two different media was developed following a previously reported study [24]. In order to isolate marine obligate and nonobligate actinobacteria, 1 g of wet sediments was transferred to tubes containing 9 mL of saline solution (0.9%; NaCl; Sigma-Aldrich,

#### *On the Selective Isolation of Actinobacteria from Different Mexican Ecosystems DOI: http://dx.doi.org/10.5772/intechopen.104699*

Mexico), four dilutions were prepared (10<sup>1</sup> to 10<sup>4</sup> ) and 100 μL (Gilson, France) of each dilution were spread onto marine GYM medium and 1:10 marine GYM medium (Appendix A); both media supplemented with Rifampicin [15, 25 and 50 μg/mL] and Nystatin (100 μg/mL). Plates were then aerobically incubated at 30° C (IncuMax IC-320, Amerex USA) for up to 16 weeks. Starting at week eight, the isolation plates were checked by eye looking for actinobacterial colonies. Once putative colonies were noticed each was then streaked in new GYM plates without antibiotics or antifungal compounds until an axenic culture was obtained. The conditions of incubation were as mentioned above. Because of the pressure set in the isolation strategy, not many microbes were able to grow but actinobacteria were successfully cultivated.

A preliminary test to quickly select marine obligate actinobacteria was carried out using marine GYM medium (**Figure 6A** and **C**) and GYM medium (**Figure 6B** and **D**). A positive result was considered when nonmicrobial biomass was observed growing on the surface of GYM medium after 4 weeks of incubation (**Figure 6B**). The ones that presented growth only in the marine GYM medium were considered those marine obligate actinobacteria (**Figure 6A**). It should be pointed out, however, that we were also able to isolate nonobligate actinobacteria that showed the typical characteristics of members of the family Micromonosporaceae [32] (**Figure 6C** and **D**). Up to date there is only one genus that is considered halophile within the Phylum Actinobacteria, and this is *Salinispora* [3, 4]. For 15 years there were only three *Salinispora* species, namely, *S. arenicola*, *S. tropica* [3] and *S. pacifica* [33], but last year seven new species were formally described [4]. The isolation of salinisporae has never been reported from sediments taken from RANP.

The molecular identification using the 16S rRNA gene of obligate and nonobligate marine actinobacteria confirmed that they belong to the genera *Micromonospora*, *Salinispora* and *Williamsia* (**Table 2**). In addition, other bacteria such as species of the genus *Erythrobacter* were also identified (**Table 2**).

To isolate obligate and nonobligate marine actinobacteria from the sponge samples, five different species of *Aplysina* (*A. airapii*, *A. clathrata*, *A. encarnacionae*, *A. gerardogrenii*, and *A. sinuscaliforniensis*) were selected. The selective isolation strategy was based on that previously reported by [3, 24]. Ten grams of each sponge were transferred into 90 mL of saline solution that was previously added to a plastic bottle with a wide mouth (**Figure 7**). The sponge was disintegrated for 5 min using an electric mixer at maximum speed (**Figure 7**). One milliliter of each suspension

**Figure 6.** *Screening of obligate and nonobligate marine actinobacteria.*

#### *Actinobacteria - Diversity, Applications and Medical Aspects*


#### **Table 2.**

*Taxonomic identification of some of the obligate and nonobligate marine actinobacteria from RANP.*

#### **Figure 7.**

*Strategy to isolate actinobacteria from six marine sponges.*

was then used to prepare serial dilutions (up to 10<sup>4</sup> ) in tubes containing 9 mL of saline solution. One hundred milliliters were spread in marine GYM medium and 1:10 marine GYM medium (Appendix A), both media were supplemented with Rifampicin [5 and 15 μg/mL] and Nystatin (100 μg/mL). Plates were aerobically incubated at 30°C (IncuMax IC-320, Amerex USA) for up to 16 weeks.

Starting at week eight, the isolation plates were checked by eye looking for actinobacterial colonies (**Figure 8**). Once putative colonies were selected they were streaked in new GYM plates until axenic culture were obtained. The conditions of incubation were the same as mentioned before.

The preliminary test to select marine obligate actinobacteria was carried out as mentioned previously. Obligate marine actinobacteria (**Figure 9A**) were isolated from *A. clathrata* and *A. gerardogrenii* and non-obligate marine actinobacteria from *A. gerardogrenii* and *A. encarnacionae* (**Figure 9B** and **C**). One isolate presented a red-wine color diffusible pigment (**Figure 9B**) and another was a symbiont (**Figure 9C**).

We isolated marine obligate actinobacteria that were preliminarily assigned to the genus *Salinispora*, one nonobligate marine actinobacteria with typical characteristics of members of the family Micromonosporaceae [32] and one symbiont actinobacteria. The isolation of salinisporae has never been reported from sponges collected or studied in Mexico and there are also non-reports about symbionts marine actinobacteria.

*On the Selective Isolation of Actinobacteria from Different Mexican Ecosystems DOI: http://dx.doi.org/10.5772/intechopen.104699*

**Figure 8.** *Morphology of the marine obligate actinobacteria.*

**Figure 9.** *Morphology of obligate and nonobligate marine actinobacteria.*

The microbial communities of marine obligate and nonobligate actinobacteria associated with marine sediments remain poorly characterized [34] and we must continue searching for these gifted microorganisms [35]. Culture-dependent methods captured approximately 3% of the total count of the microbes and in some reports around 39 genera have been only detected in culture. The latter shows the importance to carry out/improve, innovative and original selective isolation techniques since these may be more effective than previously recognized.

### **2.3 Soil is still an extraordinary resource to isolate important actinobacteria**

Soil is one of the most complex ecosystems on Earth and its amount of organic matter, mineral composition, and diversity of microorganisms will determine its ecology. There are different kinds of soils but in general, those with less anthropogenic impact will be richer in microorganisms. Mexico encompasses 26 types of soil out of the 32 recognized in the world [36, 37] and this is due to several causes, namely: (1) the complexity of the topography originated from the volcanic activity in the Cenozoic Era, (2) the wide altitudinal gradient (from 0 to 5, 600 m.a.s.l.), (3) by the five main climates present according to the Köppen classification [38], (4) the enormous diversity of landscapes present and, finally (5) the different kind of rocks that the Mexican territory enclose. It is well recognized that actinobacteria are abundant in soils and that they play an important role in the degradation and recycling of organic matter. Soil microorganisms have a remarkable ability to produce compounds with biological activity such as antibiotics, and historically this has been exemplified by streptomycin which is produced by a streptomycete named

*Streptomyces griseus* subsp. *griseus* recovered from soil. Since 1940 soil has been a major resource to selective isolate important actinobacteria, not only streptomycetes but also several other biotechnological genera such as *Amycolatopsis* and *Saccharopolyspora*, the producers of the glucopeptide -vancomycin-, and the macrolide -erythromycin-, respectively.

Mexico encompasses nearly 4, 000 insular regions of outstanding natural beauty, their biodiversity is remarked by high number of endemism (plants and animals) and most of these regions are federal protected. Revillagigedo Archipelago National Park (RANP) [39] encompasses four tropical volcanic Islands: (1) Socorro, (2) Clarion, (3) San Benedicto, and (4) Roca Partida. The RANP is considered as one of the best-preserved areas in the world.

### *2.3.1 Actinobacteria isolated from unexplored soils of Mexico*

In the present project, soil samples were collected at five different sites of Socorro Island (SI) (**Figure 10**) in 2016 and 2017, respectively (**Table 3**). A selective isolation strategy using five soil samples and three different media was developed in order to isolate actinobacteria. One gram of each soil was transferred to tubes containing 9 mL of saline solution (0.9%; NaCl; Sigma-Aldrich, Mexico). Modified Pikovskaya agar (Appendix A), GYM without antibiotics or antifungal compounds, and marine GYM supplemented with Rifampicin [5 μg/mL] and Nystatin (100 μg/mL) were used as isolation media. The dilutions used for modified Pikovskaya and marine GYM were 10<sup>1</sup> to 10<sup>4</sup> and for GYM 10<sup>6</sup> to 10<sup>8</sup> . One hundred microliters (Gilson, France) of each dilution were spread in the media and then aerobically incubated at 30°C (IncuMax IC-320, Amerex USA) for up to 4 weeks.

**Figure 10.** *Sites of sampling in Socorro Island.*


*On the Selective Isolation of Actinobacteria from Different Mexican Ecosystems DOI: http://dx.doi.org/10.5772/intechopen.104699*

#### **Table 3.**

*General information of the sampling sites.*

**Figure 11.** *Isolation plate (a), selected actinobacteria (b), and axenic culture (c).*

The isolation plates were checked by eye looking for actinobacteria and selected colonies were streaked in new GYM media plates until axenic cultures were obtained (**Figure 11**). The conditions of incubation were the same as mentioned above. The cultivable actinobacteria diversity was remarkable but only 215 isolates were selected from the isolation plates. Eighty-six isolates presented morphological differentiation based on aerial hyphae and spore mass so that they were considered as streptomycetes (**Figure 12**).

Molecular identification using the 16S rRNA gene of 10 selected strains morphologically resembling streptomycetes confirmed that they indeed belong to this extensive and important genus (**Table 4**). The phylogenetic tree constructed using five of sequences of the selected strains showed that they are different amongst them and from those more related *Streptomyces* type species selected (**Figure 13**).

**Figure 12.** *Morphological diversity of actinobacteria from Socorro Island soils.*

#### *Actinobacteria - Diversity, Applications and Medical Aspects*


#### **Table 4.**

*Preliminary identification of selected streptomycetes from the different sites.*

#### **Figure 13.**

*Phylogenetic tree of the 16S rRNA gene selected streptomycetes isolates.*

The strains from the soil samples collected at RANP may well represent novel species, but more studies to delineate its novelty are certainly needed. According to our results site 5 (S5) presented the higher percentage of actinobacteria like-strains (26%), S1 23.7%, S2 20.5%, S3 18.1% and S4 11.2%, respectively. Soil samples from unexplored sites with outstanding natural beauty represent a significant resource to isolate potential streptomycetes and use their genetic reservoir. Soil is one of the most complex, dynamic, and in constant evolution ecosystem and so it surely is its actinobacteria portion.

Soil actinobacteria, particularly *Streptomyces* is still a never-ending source for the discovery of secondary metabolites with diverse biological activities [13, 14, 26] and due to its ubiquity, survival capabilities, and metabolic versatility, it is the most studied genus within the class Actinobacteria [2, 15]. The diversity of actinobacteria isolated from SI at RANP shows the potential of our study [40] and represents the entree to explore its secondary or specialized metabolite potential [13–16, 41, 42] and diversity. Actinobacteria are amongst the most abundant organisms in soil but their potential role in this environment is still unknown. Socorro Island harbors

diverse actinobacteria and according to the selected streptomycetes (**Figure 13**) they are unique and may well represent candidates of novel taxa. Improved isolation strategies are needed to recover culturable and unculturable yet soil actinobacteria to fully explore their diversity and biotechnological applications for human and environmental welfare.
