**Actinobacteria as Plant Growth-Promoting Rhizobacteria**

Marcela Franco-Correa and Vanessa Chavarro-Anzola

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61291

### **Abstract**

Actinobacteria commonly inhabit the rhizosphere, being an essential part of this environment due to their interactions with plants. Such interactions have made possible to characterize them as plant growth-promoting rhizobacteria (PGPR). As PGPR, they possess direct or indirect mechanisms that favor plant growth. Actinobacteria improve the availability of nutrients and minerals, synthesized plant growth regulators, and specially, they are capable of inhibiting phytopathogens. Different activities that are performed by actinobacteria have been studied, such as phosphate solubilization, siderophores production, and nitrogen fixation. Furthermore, actinobacteria do not contaminate the environment;instead,theyhelptomaintainthe biotic equilibriumof soil bycooperatingwithnutrientscycling.Theaforementionedisdirectlyrelatedtothequality andproductivityofcrops.Moreover,differentaspectsofthesemicroorganismshavebeen studied, such as production of metabolites that improve plant growth, resilience against unfavorable environmental conditions, and beneficial and synergic interactions with arbuscular mycorrhizal fungi. Taking into account the above-mentioned activities, actinobacteria can be considered as possible plant fertilizers.

**Keywords:** Actinobacteria, PGPR, nutrients, solubilization, growth

### **1. Introduction**

Actinobacteria are one of the major components of microbial populations present in soil. They belong to an extensive and diverse group of Gram-positive, aerobic, mycelial bacteria that play an important ecological role in soil nutrient cycling [1-4]. These bacteria are known for their economic importance as producers of biologically active substances, such as antibiotics,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

vitamins, and enzymes [5]. Actinobacteria are also an important source of diverse antimicro‐ bial metabolites [6-8].

Historically, the most commonly described actinobacterial genus has been *Streptomyces* and *Micromonospora*. In fact, the genus *Streptomyces* is known as one of the largest sources of bioactive natural products [7-9]. Particularly, it has been estimated that approximately twothirds of natural antibiotics have been isolated from actinobacteria, and about 75% of them are produced by members of the *Streptomyces* genus [10, 11].

In the past decade, research has focused on minor groups of actinobacteria, including species that are difficult to isolate and cultivate, and those that grow under extreme conditions, i.e., alkaline and acidic conditions [6, 12]. However, the vast majority of soil actinobacte‐ ria show their optimum growth in neutral and slightly alkaline conditions; thus, the methods of isolation have been traditionally based on this neutrophilic character. Actinobac‐ teria have attracted special interest because these filamentous sporulating bacteria are able to thrive in extremely different soil conditions and they play an ecological role of impor‐ tance in nutrient cycling. Moreover, they were recently considered as plant growthpromoting rhizobacteria [13-15].

Plant growth-promoting rhizobacteria (PGPR) are free-living beneficial bacteria of agricultural importance. The presence of PGPR produces beneficial effects on plant health and growth by suppressing disease-causing microbes and accelerating nutrient availability and assimilation. Hence, in the quest to improve soil fertility and crop yield and reducing the negative impact of chemical fertilizers on the environment, there is a need to exploit PGPR for beneficial agricultural uses. In that regard, we propose to characterize actinobacteria as PGPR.

### **2. Mechanisms involved in the PGPR activity**

### **2.1. Production of plant growth regulators**

Plant organ formation and their subsequent development are mediated by internal factors of vital importance. Growth regulators in plants (PGR "Plant Growth Regulators") are known as plants hormones. PGR are small molecules that affect plant growth and development at very low concentrations [16].

One of the parameters used to determine the effectiveness of certain rhizosphere bacteria is the ability to promote the development of characteristic root system of this type of plant growth regulators [17, 18]. The rapid development of roots, either by primary root elongation or secondary lateral root emergence, allows young seeds to have easy access to nutrients and water from their environment [19].

Different mechanisms are involved such as production of siderophores and indole acetic acid, nutrient solubilization, antagonistic or beneficial synergistic effects. These mechanisms have been studied in our collection of actinobacteria and they will be further explained as follows.

### **2.2. Siderophores production**

vitamins, and enzymes [5]. Actinobacteria are also an important source of diverse antimicro‐

Historically, the most commonly described actinobacterial genus has been *Streptomyces* and *Micromonospora*. In fact, the genus *Streptomyces* is known as one of the largest sources of bioactive natural products [7-9]. Particularly, it has been estimated that approximately twothirds of natural antibiotics have been isolated from actinobacteria, and about 75% of them are

In the past decade, research has focused on minor groups of actinobacteria, including species that are difficult to isolate and cultivate, and those that grow under extreme conditions, i.e., alkaline and acidic conditions [6, 12]. However, the vast majority of soil actinobacte‐ ria show their optimum growth in neutral and slightly alkaline conditions; thus, the methods of isolation have been traditionally based on this neutrophilic character. Actinobac‐ teria have attracted special interest because these filamentous sporulating bacteria are able to thrive in extremely different soil conditions and they play an ecological role of impor‐ tance in nutrient cycling. Moreover, they were recently considered as plant growth-

Plant growth-promoting rhizobacteria (PGPR) are free-living beneficial bacteria of agricultural importance. The presence of PGPR produces beneficial effects on plant health and growth by suppressing disease-causing microbes and accelerating nutrient availability and assimilation. Hence, in the quest to improve soil fertility and crop yield and reducing the negative impact of chemical fertilizers on the environment, there is a need to exploit PGPR for beneficial

Plant organ formation and their subsequent development are mediated by internal factors of vital importance. Growth regulators in plants (PGR "Plant Growth Regulators") are known as plants hormones. PGR are small molecules that affect plant growth and development at very

One of the parameters used to determine the effectiveness of certain rhizosphere bacteria is the ability to promote the development of characteristic root system of this type of plant growth regulators [17, 18]. The rapid development of roots, either by primary root elongation or secondary lateral root emergence, allows young seeds to have easy access to nutrients and

Different mechanisms are involved such as production of siderophores and indole acetic acid, nutrient solubilization, antagonistic or beneficial synergistic effects. These mechanisms have been studied in our collection of actinobacteria and they will be further explained as follows.

agricultural uses. In that regard, we propose to characterize actinobacteria as PGPR.

produced by members of the *Streptomyces* genus [10, 11].

**2. Mechanisms involved in the PGPR activity**

**2.1. Production of plant growth regulators**

low concentrations [16].

water from their environment [19].

bial metabolites [6-8].

250 Actinobacteria - Basics and Biotechnological Applications

promoting rhizobacteria [13-15].

Microorganisms have been forced by environmental restrictions and biologic imperatives to produce specific molecules that can compete efficiently with hydroxyl ions for ferric state of iron. Siderophores are compounds produced by various microorganisms in soil. These organisms rely on chelation phenomena to support their biological activity. Siderophores are extracellular fluorescent pigments that possess affinity to iron (III), they are water-soluble and have low molecular weight (500-1,000 Da) [20]. Furthermore, siderophores are produced by a great variety of microorganisms that grow in scarce iron conditions [21, 22]. These compounds act as specific chelate agents of ferric ion, leaving available the ionic form (Fe+2), which is easily absorbed by microorganisms [23].

Chelation is a usual phenomenon of the biologic systems. This refers to formation of chelates that can be described as a polydentate ligand in coordination with a central ion by two or more atoms [24]. When siderophores form a complex with Fe+3, these are recognized by cell membrane receptors [25, 26]. This facilitates the inclusion of the formed complex to cell. Once in the cell, Fe+3 ions are reduced to a Fe+2 becoming available to be used in different biological processes. Finally, the siderophore [27] is released again. Siderophores are classified into three groups based on their chemical nature of the bounds created with metals. They are known as catecholates, hydroxamates, and hydroxide-carboxylates [24]. Actinobacteria and Enterobac‐ teria are among the microorganisms able to produce siderophores. Actinobacteria is one the most important groups in terms of siderophores production.

The vast majority of nitrogen-fixing microorganisms produce siderophores so as to obtain iron. This is necessary to perform the enzyme nitrogenase. The enzyme is composed of several protein units; a total of 36 iron atoms are required for operating properly [28]. According to determinations made by our research group, the highest levels of siderophores production were obtained by *Streptomyces* MCR24 that was maintained over the time; conversely, the lowest recorded levels were observed in *Streptomyces* MCR30. Despite the fact that in its great majority, the analyzed siderophores produced by actinobacteria contained high concentrations of hydroxamates, it can be assumed that some strains show a possible advantage as PGPR mechanism. The strains tested for production of siderophores hydroxamate were capable of growing in culture media without addition of any source of nitrogen. These results are similar to those found by Carson *et al.* [29] and indicate that the selected strains had such capacity and in one-way or another (high or low) produced siderophores.

Studies performed by Díaz [30] evidenced a higher performance of siderophores production when a monosaccharide like glucose is used as a source of carbon. Studies with actinobacteria have shown that the metabolic rate of this group of bacteria is higher when there is an excess of carbon. This favored the production of some organic compounds related to the physiology of the species [31-34]. In the presence of an easily assimilable source of carbon, some species of *Streptomyces* are capable of synthesizing organic acids. Those acids return to metabolic routes to produce energy and result in various secondary metabolites that in such case can be considered as siderophores [34-36].

Authors like Diaz [30] proved that strains of actinobacteria such as *Streptomyces* MCR3 and *Thermobifida* showed a great synthesis of siderophores of the hydroxamate-type using glucose as the only source of carbon, in contrast to what occurred when sodium succinate was utilized to the same purposed. Concerning *Streptomyces* sp., the production of hydroxamate-Desfer‐ rioxamine [37, 38] B by this microorganism is well documented. On the other hand, *Thermo‐ bifida* has been recently reported as the producer of a siderophore known as fuscachelin A [39].

#### **2.3. Indole acetic acid production**

Indole acetic acid (IAA) is a plant growth regulator and active form of auxins. It plays an important role in plant development through its life cycle [40, 41]. IAA stimulates the growth of the radicular system [28, 42-44], thanks to the development of lateral roots and divisions of the apical meristem that derives in roots elongation [43, 44]. This increases the access of soil nutrients to the plant [40, 46]. IAA has proved to be the main one responsible for plant growth promotion over the nitrogen fixation related to diazotrophic bacteria activity [45].

The production of IAA has been widely studied in actinobacteria [47-53]. Authors like El-Shanshoury [47] suggest that IAA can act as endogen regulator of spore germination of *Streptomyces atroolivacezlz* and can be involved in the differentiation of actinobacteria [46].

*Streptomyces* genus [47-53], *Frankia* sp*.* [47, 51, 54]*, Nocardia* sp. [47, 54], *Kitasatospora* sp. [52] have been widely studied as IAA producers. Studies performed by our research group confirmed the ability of genus *Streptomyces* sp. and *Thermobifida* sp. to produce IAA. Duque and Quintana [50] affirmed that MCR14 (*Streptomyces sannanensis*) was the most important microorganism for IAA production.

### **2.4. Non-symbiotic nitrogen fixation**

The actinobacteria are heterotrophic organisms that require carbon sources to obtain the energy necessary for nitrogen fixation. Therefore, each of the different bacteria differs in the carbon metabolism and the intrinsic ability of nitrogen fixation, showing different rates of acetylene reduction assay (ARA). This test is based on detecting indirectly the presence nitrogenase enzyme, which is in charge of reducing nitrogen (N2) to ammonium. This essay evaluates the enzymatic reduction of acetylene to ethylene (NH4 + ) [55]. Likewise, ARA indirectly measures the microorganism capacity to fixate nitrogen, given that nitrogenase is an unspecific enzyme that catalyzes the reduction of steric analogs of N2 [56].

Actinobacteria log phase was evaluated in free-nitrogen media. From those experiments, it was established that the best period for ARA measurement was found after 10 days of culturing. Strains MCR14, MCR27, and MCR31 were selected from the 10 evaluated strains as they turn the pH indicator in Nfb culture media. This fact did not occur for the rest of the evaluated strains. High rates of nitrogen fixation were expected in the vials where color change was observed (Figure 1) and then positively confirmed by ARA. De los Santos et al. [57] described a similar behavior when a semi-solid media inoculated with the bacteria *Burkholde‐ ria* did not display changes of pH in all vials; however, some of them showed a slight increase of pH due to microbial growth. In those vials, it was possible to observe a blue-green color. This confirms the need of carrying out ARA test in order to guarantee diazotrophic charac‐ teristics of these microorganisms. In Nfb semi-solid media, actinobacteria grew as a thin white film placed few millimeters below the agar surface. Bacteria were able to move through the media, thanks to Nfb consistency and found the optimum place to balance the respiration rate with diffusion oxygen rate [58].

Certain evidence indicates that diazotrophs organisms are capable of nitrogen biologic fixation in considerable wide ranges of pH. Despite the fact that Nfb has a neutral pH, hydrolysis of carbon derived from the metabolism will cause products that may change the pH. These pH values interfere with nitrogen fixation, making it difficult to alkalinize the culture media, and therefore, changing the color to blue. This behavior was evidenced in most of the isolated actinobacteria (Figure 1).

**Figure 1.** Actinobacteria evaluated in NFB modified media

Authors like Diaz [30] proved that strains of actinobacteria such as *Streptomyces* MCR3 and *Thermobifida* showed a great synthesis of siderophores of the hydroxamate-type using glucose as the only source of carbon, in contrast to what occurred when sodium succinate was utilized to the same purposed. Concerning *Streptomyces* sp., the production of hydroxamate-Desfer‐ rioxamine [37, 38] B by this microorganism is well documented. On the other hand, *Thermo‐ bifida* has been recently reported as the producer of a siderophore known as fuscachelin A [39].

Indole acetic acid (IAA) is a plant growth regulator and active form of auxins. It plays an important role in plant development through its life cycle [40, 41]. IAA stimulates the growth of the radicular system [28, 42-44], thanks to the development of lateral roots and divisions of the apical meristem that derives in roots elongation [43, 44]. This increases the access of soil nutrients to the plant [40, 46]. IAA has proved to be the main one responsible for plant growth

The production of IAA has been widely studied in actinobacteria [47-53]. Authors like El-Shanshoury [47] suggest that IAA can act as endogen regulator of spore germination of *Streptomyces atroolivacezlz* and can be involved in the differentiation of actinobacteria [46].

*Streptomyces* genus [47-53], *Frankia* sp*.* [47, 51, 54]*, Nocardia* sp. [47, 54], *Kitasatospora* sp. [52] have been widely studied as IAA producers. Studies performed by our research group confirmed the ability of genus *Streptomyces* sp. and *Thermobifida* sp. to produce IAA. Duque and Quintana [50] affirmed that MCR14 (*Streptomyces sannanensis*) was the most important

The actinobacteria are heterotrophic organisms that require carbon sources to obtain the energy necessary for nitrogen fixation. Therefore, each of the different bacteria differs in the carbon metabolism and the intrinsic ability of nitrogen fixation, showing different rates of acetylene reduction assay (ARA). This test is based on detecting indirectly the presence nitrogenase enzyme, which is in charge of reducing nitrogen (N2) to ammonium. This essay

indirectly measures the microorganism capacity to fixate nitrogen, given that nitrogenase is

Actinobacteria log phase was evaluated in free-nitrogen media. From those experiments, it was established that the best period for ARA measurement was found after 10 days of culturing. Strains MCR14, MCR27, and MCR31 were selected from the 10 evaluated strains as they turn the pH indicator in Nfb culture media. This fact did not occur for the rest of the evaluated strains. High rates of nitrogen fixation were expected in the vials where color change was observed (Figure 1) and then positively confirmed by ARA. De los Santos et al. [57] described a similar behavior when a semi-solid media inoculated with the bacteria *Burkholde‐ ria* did not display changes of pH in all vials; however, some of them showed a slight increase of pH due to microbial growth. In those vials, it was possible to observe a blue-green color.

+

) [55]. Likewise, ARA

evaluates the enzymatic reduction of acetylene to ethylene (NH4

an unspecific enzyme that catalyzes the reduction of steric analogs of N2 [56].

promotion over the nitrogen fixation related to diazotrophic bacteria activity [45].

**2.3. Indole acetic acid production**

252 Actinobacteria - Basics and Biotechnological Applications

microorganism for IAA production.

**2.4. Non-symbiotic nitrogen fixation**

After ARA evaluation, it was observed that isolated MCR24, MCR26, and MCR31 recorded the highest rates of ethylene production at the three incubation times. However, no statistically significant differences were noted. The highest nitrogenase activities were observed in MCR31 cultures. It is highly important to detect nitrogenase activity among our strains of free-living diazotroph actinobacteria by using ARA. The high microbial diversity present in soils of high Andean forest of Colombia (Cundinamarca and Boyacá states) derives in the ability of nitrogen fixation obtained by the isolates.

*Frankia* sp. have an outstanding feature related to vesicles specialized in nitrogen fixation. These vesicles are in charge of protecting the nitrogenase complex. These actinobacteria have been extensively reported by several authors [59-61], due to its fixation characteristics that have been confirmed by acetylene reduction method [62-64]. The morphology of the bacteria cultured in our laboratory facilities using Nfb media did not display any similarity with *Frankia* sp, since different microscopic descriptions such as long and short filaments, tortuous or spiralshaped filaments were found. The above mentioned can be an evidence of the presence of new species that have the ability to grow in nitrogen-free conditions.

Gen *nifH* detection was performed. This gene codifies for nitrogenase enzyme and is the molecular marker most widely used for detection of nitrogen-fixing bacteria and phylogenetic studies [65]. We decided to use the primers purposed by Diallo et al. [66]. These primers are very similar to those used by Valdés et al. [64] in non-Frankia actinobacteria for amplifying the *nifH* gen. Based on this protocol was possible to amplify the gen in all the isolated bacteria. Figure 2 displays a band that corresponds to a gen fragment with an expected size of 320 bp. These amplified genes were purified by means of QiaGEN Minelute PCR Purification Kit and then sequenced. The size of the observed bands is similar to that reported by Valdés et al. [64] for *nifH* gene. Furthermore, background can be observed. Studies performed by Soares et al. [67] affirmed that the "background" or "smearing" could be related with the use of degenerate primers such as PolR and PolF during nested PCR.

**Figure 2.** PCR amplification of an intern fragment of gen *nifH* of 320 bp, in 10 of the isolated diazotrophic actinobacte‐ ria MP (100 bp Invitrogen).

It is important to emphasize that the research performed by Valdés et al. [64] corresponds to one of the first studies that have recorded the use of degenerate primers to detect the presence of *nifH* gene in free-living actinobacteria different from *Frankia* and isolated from *Casuarina equisetifolia*. As *nifH* gene can act as a molecular marker, there are other genes that belong to nitrogenase complex that are capable of indicating such activity in nitrogen-fixing microor‐ ganisms. Studies performed by Fedorov et al. [68] on a new primer system for detecting and amplifying gene *nifDK* portion have pointed out the possible use of such gene as a molecular marker. The progress in the development of new primer systems that target different sites in *nif* operon can be efficiently used for searching nitrogen-fixing actinobacteria in which no nitrogenase activity has been detected before. However, the presence of *nifH* gene by itself does not indicate active nitrogen fixation since nitrogenase activity is regulated at pre- and post-transcriptional level [69] and its activity depends on environmental conditions. Probably, the most suitable indicator of nitrogenase activity complex is ARNm of *nifH* [70].

BLAST search was carried out using GenBank in order to find the gene *nifH* sequence closely related to that found in our study. It was noted that they were similar to the sequence assigned to *Frankia* sp*.* According to the access produced by GenBank, some of the strains (MCR 3 and MCR24) showed similarity score between 96 and 98%. The other strains recorded *nifH* gene alignments similar to genes found in nitrogen-fixing bacteria such as *Rhizobium* and *Bradyrhi‐ zobium*, and others. This suggests that PCR fragments probably belong to the *nifH* gene. Among the noted sequences, the majority of the strains did not record any similarities with diazotro‐ phic bacteria; this was expected to take into account that *Frankia* sp. represent the only nitrogenfixing bacteria by means of actinorrhizal symbiosis. *Streptomyces thermoautotrophicus* UBT1 is capable of fixing atmospheric nitrogen and grows in media free of nitrogen; however, it is not capable of acetylene reduction. This type of nitrogen reduction is unusual and it is believed to be coupled to carbon monoxide and dependent of oxygen [71]. These types of microorganisms are not associated to plants and they do not have other characteristics of agronomic interest. In contrast, the atmospheric nitrogen fixed by the actinobacteria studied in our laboratory can influence plant growth. Nitrogenase activity was confirmed after analyzing the ability to reduce acetylene to ethylene and the presence of *nifH* genes by PCR amplification [64, 72].

#### *2.4.1. Phosphorus solubilization*

shaped filaments were found. The above mentioned can be an evidence of the presence of new

Gen *nifH* detection was performed. This gene codifies for nitrogenase enzyme and is the molecular marker most widely used for detection of nitrogen-fixing bacteria and phylogenetic studies [65]. We decided to use the primers purposed by Diallo et al. [66]. These primers are very similar to those used by Valdés et al. [64] in non-Frankia actinobacteria for amplifying the *nifH* gen. Based on this protocol was possible to amplify the gen in all the isolated bacteria. Figure 2 displays a band that corresponds to a gen fragment with an expected size of 320 bp. These amplified genes were purified by means of QiaGEN Minelute PCR Purification Kit and then sequenced. The size of the observed bands is similar to that reported by Valdés et al. [64] for *nifH* gene. Furthermore, background can be observed. Studies performed by Soares et al. [67] affirmed that the "background" or "smearing" could be related with the use of degenerate

**Figure 2.** PCR amplification of an intern fragment of gen *nifH* of 320 bp, in 10 of the isolated diazotrophic actinobacte‐

It is important to emphasize that the research performed by Valdés et al. [64] corresponds to one of the first studies that have recorded the use of degenerate primers to detect the presence of *nifH* gene in free-living actinobacteria different from *Frankia* and isolated from *Casuarina equisetifolia*. As *nifH* gene can act as a molecular marker, there are other genes that belong to nitrogenase complex that are capable of indicating such activity in nitrogen-fixing microor‐ ganisms. Studies performed by Fedorov et al. [68] on a new primer system for detecting and amplifying gene *nifDK* portion have pointed out the possible use of such gene as a molecular marker. The progress in the development of new primer systems that target different sites in *nif* operon can be efficiently used for searching nitrogen-fixing actinobacteria in which no nitrogenase activity has been detected before. However, the presence of *nifH* gene by itself does not indicate active nitrogen fixation since nitrogenase activity is regulated at pre- and

species that have the ability to grow in nitrogen-free conditions.

254 Actinobacteria - Basics and Biotechnological Applications

primers such as PolR and PolF during nested PCR.

ria MP (100 bp Invitrogen).

Phosphorus (P) is one of the major essential macronutrients for plants, which is applied to the soil in the form of phosphatic manure. However, a large portion of the applied phosphorus is rapidly immobilized, becoming unavailable for plants [73]. The free phosphatic ion in soil plays a crucial role. The orthophosphatic ion form is the only ion that can be assimilated by plants in considerable amounts [74]. Soil microorganisms are involved in a wide range of biological processes, including phosphorus transformation of soil. They solubilize soil phosphorus for plants growth [75].

Although the mechanisms used by actinobacteria to solubilize phosphorus are not fully understood, it is known that in the solubilization process, inorganic phosphorus and some organic acids (oxalic and citric, principally) are involved [76-78]; our group performed solubilization quantifications and determined organic acids associated with solubilizing of P [79]. Similarly, Hamdali et al. in 2010 and 2012 [80, 81] have reported that compounds different from organic acids, more specifically metabolites of the viridomicenas and siderophores families, are capable of solubilizing P from various sources, which lead us to explore more about the process of solubilization of inorganic phosphorus generated by this group of organisms.

### *2.4.2. Organic phosphate solubilization*

The organic P is composed of various fractions, compromising the most labile and most resistant to mineralization. However, the main organic component of phosphorus cycle is the microbial biomass [82]. Organic phosphate is mineralized by the phosphatase enzyme, which is excreted by some microorganisms, and is released [83, 84].

Studies performed by our research group evidenced that *Streptomyces* MCR26 has the capacity of secreting acid phosphatases, and therefore, mineralizing sources of organic phosphorous [15]. Additionally, actinobacteria not previously reported as phosphate-mineralizing micro‐ organisms were identified and were related to genus *Saccharopolyspora, Thermobifida* and *Thermonospora*. Actinobacteria from the genus *Micromonospora* sp.*, Nocardia* sp.*, Actinomadura* sp.*, Rhodococcus* sp.*, Actinoplanes* sp.*, Microbispora* sp. and *Streptosporangium* sp. produce phosphatase enzymes which have been classified according to their alkaline or acid activity, depending on reaction conditions [85].

### *2.4.3. Inorganic phosphate solubilization*

The growth of phosphate-solubilizing bacteria (PSB) growth often causes soil acidification, playing a key role in phosphorus solubilization [86]. Therefore, PSB are considered important solubilizers of insoluble inorganic phosphate. In turn, plants reimburse PSB with carbohy‐ drates [87].

Prada et al. [79] isolated 57 strains of actinobacteria from different sampling areas. Soil characterization showed the following: pH ranges from 4.0 to 5.9, total P from 360 to 2830 mg/ kg, available P from 8.7 to 118.4 mg/kg, and organic matter from 2.95 to 13.52%. The results of two qualitative assessments are not totally consistent. Seven of the tested strains F1A, F1B, F1C, F4C, T1A, T1D, and T3A were the best solubilizing strains, in both solid and liquid evaluation media. We performed a quantitative assessment in order to find the strain that has the highest solubilizing capacity and to evaluate which of the two methods is more reliable. The strains T1C, T1H, T3A, T3C, P3E, F1A, F2A, and V2B are as good as *Streptomyces* sp. MCR24 for Ca3 (PO4)2 and these strains solubilized significantly more phosphorus than the other strains. Strains T1H, T1C, T3A, T3C, and F1A are only present in the selection obtained with the methodology reported by Mehta and Nautiyal [88], suggesting that this test can select more strains with true solubilizing ability, and thus it is more reliable.

Perez et al. [89] claimed that isolates that cause a shift of > 1.5 units could be selected for further studies. In order to confirm the usefulness of this cut-off point proposed by Perez et al. [89] and therefore, to select the best strains, we implemented a quantitative assay by measuring the release of soluble phosphorus using the NBRIP broth [90-91]. Figure 3 shows strains T1C, T1H, T3C, P3E, and V2B. They have significantly higher activity than other isolates. However, this result was not observed in the plate assay, probably because one or more acids involved in the process did not diffuse into the agar and, therefore, there was no presence of a solubi‐ lization halo. On the other hand, the evaluation in NBRIP-BPB broth revealed that isolates capable of decolorizing the broth more than 1.5 units were also more efficient in the quantita‐ tive assay. Additionally, Mehta and Nautiyal [88] assay contribute to reduce costs and efforts in microorganisms with bio-fertilizing potential screening. Studies focusing on actinobacteria physiology in Colombia are scarce, especially those focused on agriculture [15, 90-92].

**Figure 3.** Released soluble phosphate activity with two sources of P. Ca3(PO4) 2 5g L-1 is displayed in Y axis and activity with AlPO4 1g L-1 source is displayed in X axis.

#### **2.5. Chitinases production**

microbial biomass [82]. Organic phosphate is mineralized by the phosphatase enzyme, which

Studies performed by our research group evidenced that *Streptomyces* MCR26 has the capacity of secreting acid phosphatases, and therefore, mineralizing sources of organic phosphorous [15]. Additionally, actinobacteria not previously reported as phosphate-mineralizing micro‐ organisms were identified and were related to genus *Saccharopolyspora, Thermobifida* and *Thermonospora*. Actinobacteria from the genus *Micromonospora* sp.*, Nocardia* sp.*, Actinomadura* sp.*, Rhodococcus* sp.*, Actinoplanes* sp.*, Microbispora* sp. and *Streptosporangium* sp. produce phosphatase enzymes which have been classified according to their alkaline or acid activity,

The growth of phosphate-solubilizing bacteria (PSB) growth often causes soil acidification, playing a key role in phosphorus solubilization [86]. Therefore, PSB are considered important solubilizers of insoluble inorganic phosphate. In turn, plants reimburse PSB with carbohy‐

Prada et al. [79] isolated 57 strains of actinobacteria from different sampling areas. Soil characterization showed the following: pH ranges from 4.0 to 5.9, total P from 360 to 2830 mg/ kg, available P from 8.7 to 118.4 mg/kg, and organic matter from 2.95 to 13.52%. The results of two qualitative assessments are not totally consistent. Seven of the tested strains F1A, F1B, F1C, F4C, T1A, T1D, and T3A were the best solubilizing strains, in both solid and liquid evaluation media. We performed a quantitative assessment in order to find the strain that has the highest solubilizing capacity and to evaluate which of the two methods is more reliable. The strains T1C, T1H, T3A, T3C, P3E, F1A, F2A, and V2B are as good as *Streptomyces* sp. MCR24 for Ca3 (PO4)2 and these strains solubilized significantly more phosphorus than the other strains. Strains T1H, T1C, T3A, T3C, and F1A are only present in the selection obtained with the methodology reported by Mehta and Nautiyal [88], suggesting that this test can select more

Perez et al. [89] claimed that isolates that cause a shift of > 1.5 units could be selected for further studies. In order to confirm the usefulness of this cut-off point proposed by Perez et al. [89] and therefore, to select the best strains, we implemented a quantitative assay by measuring the release of soluble phosphorus using the NBRIP broth [90-91]. Figure 3 shows strains T1C, T1H, T3C, P3E, and V2B. They have significantly higher activity than other isolates. However, this result was not observed in the plate assay, probably because one or more acids involved in the process did not diffuse into the agar and, therefore, there was no presence of a solubi‐ lization halo. On the other hand, the evaluation in NBRIP-BPB broth revealed that isolates capable of decolorizing the broth more than 1.5 units were also more efficient in the quantita‐ tive assay. Additionally, Mehta and Nautiyal [88] assay contribute to reduce costs and efforts in microorganisms with bio-fertilizing potential screening. Studies focusing on actinobacteria physiology in Colombia are scarce, especially those focused on agriculture [15, 90-92].

is excreted by some microorganisms, and is released [83, 84].

strains with true solubilizing ability, and thus it is more reliable.

depending on reaction conditions [85].

256 Actinobacteria - Basics and Biotechnological Applications

*2.4.3. Inorganic phosphate solubilization*

drates [87].

The chitin is a homopolymer comprised of N-acetyl-D-Glucosamine residues with α-1, 4 bonds. It is widely spread in nature as a structural component of fungi (22-44%), insects and crustaceans (25-58%), and protozoa [93-96]. The chitin is hydrolyzed by a complex chitinase that comprises three enzymes such as exochitinase, endochitinase and N-acetyl-D-Glucosa‐ mine.

Actinobacteria are considered as the dominant organisms involved in the decomposition of chitin in soil [1] and also promising antagonist agents for biocontrol due to the hydrolysis reaction over the fungi mycelium [97]. The species that belong to *Streptomyces* genus are considered as the principal chitinolytic microbial group in soil, due to its capacity to degrade this polymer [98].

Within a group of 30-isolated actinobacteria, 20 were able to hydrolyze chitin after 3 days of growth on mineral agar supplemented with chitin, as the sole source of carbon. The clearing zones were observed around bacteria following 9 days of growth, suggesting the presence of chitinolytic activity with secreted proteins into the culture medium [99].

### **2.6. Antagonistic activity against phytopathogenic fungi**

Antagonism is defined as a mechanism of action based primarily on the direct inhibitory activity between two microorganisms [100] that have opposite actions within the same system. In order to evaluate the chitinolytic activity of seven strains of actinobacteria against *Fusarium oxysporum*, *Phytophthora infestans*, *Rhizoctonia solani* and *Verticillium dahlie*, a set of experiments were performed. Additionally, its ability as plant growth regulator was also considered.

It was observed that the strains had a high, medium, or low inhibition on tests of antagonism against phytopathogenic fungi, but *F. oxysporum* was the most resistant fungal strain. Diverse actinobacteria may act as antagonistic microorganisms of *F. oxysporum* by producing antibi‐ otics (antibiosis). These compounds diffuse through the medium inhibiting the growth of phytopathogenic fungus. Molano et al. [101] determined *in vitro* inhibition of *Fusarium oxysporum* growth by actinomycin, an antibiotic produced by *Nocardia* sp., strain isolated from rhizosphere soil sample lichen (Mosquera, Colombia). Production of such secondary metab‐ olites was toxic to the phytopathogenic fungus.

Strains MCR26, MCR10, and MCR24 proved to be the best as fungal antagonists (Figure 4). Based on these results, it can be inferred that mycelial growth inhibition is not caused by chitinase production, but rather by antifungal products. No inhibition of mycelial growth was observed by strains with chitinolytic activity. Using these results, we moved to the interaction phase with the mycorrhizal fungi, considering that chitinolytic enzymes did not directly affect fungi.

Actinobacteria that belong to *Streptomyces* genus have been commercially used to control plant damages. This genus have demonstrated antagonistic activity against *Alternaria* sp., *Pythium aphanidermatum, Colletotrichum higginsianum, Acremonium lactucum*, and *Fusarium oxysporum* [102,103]

Experiments performed at Unidad de Investigaciones Agropecuarias (UNIDIA) have proved the ability of *Streptomyces cuspidosporus* to inhibit the phytopathogen fungus *Fusarium oxyspo‐ rum* following 8 days of incubation [50]. Additionally, we evidenced antibacterial activity present in actinobacteria. Complete inhibition was observed when *Streptomyces* MCR26 was tested against *Bacillus cereus* and *Escherichia coli*, conversely, *Thermobifida* MCR24 strain which was completely inhibited by *Bacillus cereus* [104].

Theantagonisticpotentialofthe compoundsproducedbythese strainswaspreviouslyreported by our research group (UNIDIA). We evaluated the antagonistic activity in vitro of no mycor‐ rhizal fungi generally found in soil. It was found that *Streptomyces* MCR26 and *Thermobifida* MCR24 partially inhibited *Rhizoctonia solani* and *Phytophthora infestans* growth [15].

### **2.7. Mycorrhiza (MA) helper bacteria**

zones were observed around bacteria following 9 days of growth, suggesting the presence of

Antagonism is defined as a mechanism of action based primarily on the direct inhibitory activity between two microorganisms [100] that have opposite actions within the same system. In order to evaluate the chitinolytic activity of seven strains of actinobacteria against *Fusarium oxysporum*, *Phytophthora infestans*, *Rhizoctonia solani* and *Verticillium dahlie*, a set of experiments were performed. Additionally, its ability as plant growth regulator was also considered.

It was observed that the strains had a high, medium, or low inhibition on tests of antagonism against phytopathogenic fungi, but *F. oxysporum* was the most resistant fungal strain. Diverse actinobacteria may act as antagonistic microorganisms of *F. oxysporum* by producing antibi‐ otics (antibiosis). These compounds diffuse through the medium inhibiting the growth of phytopathogenic fungus. Molano et al. [101] determined *in vitro* inhibition of *Fusarium oxysporum* growth by actinomycin, an antibiotic produced by *Nocardia* sp., strain isolated from rhizosphere soil sample lichen (Mosquera, Colombia). Production of such secondary metab‐

**Figure 4.** Dual culture confrontation method. (a) MCR26 vs *Verticillium dahliae*. (b) MCR10 vs *Phytophthora infestans*. (c)

Strains MCR26, MCR10, and MCR24 proved to be the best as fungal antagonists (Figure 4). Based on these results, it can be inferred that mycelial growth inhibition is not caused by chitinase production, but rather by antifungal products. No inhibition of mycelial growth was observed by strains with chitinolytic activity. Using these results, we moved to the interaction phase with the mycorrhizal fungi, considering that chitinolytic enzymes did not directly affect

Actinobacteria that belong to *Streptomyces* genus have been commercially used to control plant damages. This genus have demonstrated antagonistic activity against *Alternaria* sp., *Pythium aphanidermatum, Colletotrichum higginsianum, Acremonium lactucum*, and *Fusarium oxysporum*

Experiments performed at Unidad de Investigaciones Agropecuarias (UNIDIA) have proved the ability of *Streptomyces cuspidosporus* to inhibit the phytopathogen fungus *Fusarium oxyspo‐ rum* following 8 days of incubation [50]. Additionally, we evidenced antibacterial activity

chitinolytic activity with secreted proteins into the culture medium [99].

**2.6. Antagonistic activity against phytopathogenic fungi**

258 Actinobacteria - Basics and Biotechnological Applications

olites was toxic to the phytopathogenic fungus.

MCR14 vs *Rhizoctonia solani*

fungi.

[102,103]

In general, the ability of certain microorganisms to influence the formation and functioning of the symbiosis MA through various kinds of activities, such as activation in fungal propagules infective pre-symbiotic stages [93,105], facilitate formation of inputs point into the root [106-108] and they increase the growth rate [109-111].

In our studies, it can be seen that the two strains of *Streptomyces* (MCR9 and MCR26) cause a stimulation of spores germination of the fungus MA, while *Thermobifida* MCR24 reduces significantly the germination of spores. It was also observed that isolated from *Streptomyces* and *Thermobifida* improved the growth of the mycelium of *Glomus* FC8 sp. Actinobacteria behavior evaluated in this study confirmed the results obtained by *Streptomyces globisporus* 1- K-4 [112], which showed that the concentration of the bacteria increases in rhizoplane seedling rice almost immediately after the inoculation.

Following the methodology described by Azcón-Aguilar et al. [113] and Barea et al. [114] we determine in this study, with, whether or not germination of *Glomus* sp spores were inhibited by the three isolated actinobacteria. Each petri dish was individually inoculated with actino‐ bacteria (MCR9, MCR24, or MCR26) and the spores. Spores and the correspondent actinobac‐ teria were placed on the apex of a hypothetical hexagon keeping a distance of approximately 3.5 cm between each other. After being inoculated, the germination of the spores was observed after 32 days of incubation at 25°C in dark conditions. Percentage of germination was calcu‐ lated in each treatment. It was recorded that the two strains of *Streptomyces*(MCR9 and MCR26) improved germination of the spores of fungus MA. In contrast, *Thermobifida* MCR24 notably decreases spores germination. Furthermore, *Streptomyces* and *Thermobifida* improved myceli‐ um development of *Glomus* FC8 sp.

Carpenter-Boggs et al. [111] found that actinobacteria such as *Streptomyces orientalis* have a beneficial effect on spores of *Gigaspora margarita.* They also observed that the amount of volatile compounds produced by the isolated ones have a good correlation with the germination of MA spores. Such research can explain why the actinobacteria that belong to our collection improve spore germination of *Glomus* FC8 sp. Moreover, Mousse [109] and Azcón-Aguilar & Barea [115] described that some mycorrhizosphere bacteria were capable of promoting the MA settle. This improves germination of spores.

Through confocal microscopy was observed that chitinolytic strains and strains that showed antagonistic capability against non-mycorrhiza fungi with chitin wall did not cause degrada‐ tion of the mycelium wall of *Glomus* FC8 sp. or to the commercial witness. These results are consistent with other studies that have observed bacteria inside of MA and colonizing fungal hyphae [116-118]. Different studies have proved that microbial antagonists of fungal patho‐ gens do not cause any inhibitory effect against MA [114,119-121].

### **Acknowledgements**

The financial support of the experimental studies and publication was realized by Pontificia Universidad Javeriana - Vicerrectoría de Investigación. Project No. 6677. Authors thank Jorge Andrés Fernández Gonzalez for the revision of the translation of chapter.

### **Author details**

Marcela Franco-Correa\* and Vanessa Chavarro-Anzola

\*Address all correspondence to: franco@javeriana.edu.co

Department of Microbiology, Pontificia Universidad Javeriana, Bogotá, Colombia

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The financial support of the experimental studies and publication was realized by Pontificia Universidad Javeriana - Vicerrectoría de Investigación. Project No. 6677. Authors thank Jorge

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### **Chapter 11**

## *Frankia* **as a Biodegrading Agent**

Medhat Rehan, Erik Swanson and Louis S. Tisa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61825

#### **Abstract**

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45-61.

The *Frankia* actinorhizal plant symbiosis plays an important role in colonization of soils contaminated with toxic aromatic hydrocarbons. Our understanding of the bacterial partner, *Frankia*, in the actinorhizal symbiosis has been greatly facilitated by the availability of sequenced genomes. The analysis of these *Frankia* genomes has suggested that these bacteria are metabolically diverse and have potential for toxic aromatic hydrocarbon degradation. In this chapter, we explore what is known about that metabolic potential.

**Keywords:** *Frankias*-triazines, aromatic hydrocarbon degradation, PAH, bioremediation, bioinformatics, actinobacteria

### **1. Introduction**

*Frankia* are filamentous nitrogen-fixing Gram-positive actinobacteria that are found as freeliving microbes in the soil and in symbiotic associations with actinorhizal plants [1-5]. These bacteria fix nitrogen by converting atmospheric N2 into biologically useful ammonia and supply the host plants with a source of reduced nitrogen. *Frankia* are developmentally complex and form three cell types: vegetative hyphae, spores located in sporangia, and vesicles. Hyphae are septate structures and form the growing state of this microbe. Under appropriate condi‐ tions, either terminal or intercalary multilocular sporangia are produced and contain many spores. When mature, the spores are released from the sporangia. The spores are presumed to aid in the survival and dispersal of *Frankia* in the environment. Vesicles are produced under nitrogen-limited conditions and consist of unique lipid-enveloped cellular structures that contain the enzymes responsible for nitrogen fixation. Thus, vesicles act as specialized structures for the nitrogen fixation process. *Frankia* are able to establish symbiotic nitrogen-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

fixing associations with over 220 species of woody dicotyledonous plants, termed actinorhizal plants, that are found in eight families of angiosperms [1, 3-6]. The symbiosis with *Frankia* allows these actinorhizal host plants to colonize nutrient-poor soil and harsh environments. Actinorhizal plants have been used to recolonize and reclaim industrial wastelands and environments contaminated with heavy metals and toxic aromatic hydrocarbon [7-15]. The metabolic potential of these bacteria has only recently been investigated in the context of bioremediation [16-18].

#### **1.1.** *Frankia* **genomics and identification of metabolic potential**

Based on phylogenetic analysis, *Frankia* strains have been classified into four main lineages [19-23]. Members of lineage 1 are found infective on host plants of the Betulaceae *(Alnus*), Myricaceae, and Casuariaraceae families, while lineage 2 represents strains that are infective on Rosaceae (*Dryas*, etc.), Coriariaceae *(Coriaria)*, Datiscaceae (*Datisca),* and the genus *Ceano‐ thus* (Rhamnaceae)*.* Members of lineage 3 are the most promiscuous and are infective on Eleagnaceae, Rhamnaceae, Myricaceae, *Gynmmostoma,* and occasionally *Alnus.* The fourth *Frankia* lineage consists of the "atypical" strains which are unable to reinfect actinorhizal host plants or form ineffective root nodule structures that are unable to fix nitrogen. Our under‐ standing of this genus has been greatly enhanced by the sequencing of several *Frankia* genomes from the different *Frankia* lineages [24-33]. Analysis of *Frankia* genomes has revealed new potential with respect to metabolic diversity, natural product biosynthesis, and stress toler‐ ance, which may help aid the cosmopolitan nature of the actinorhizal symbiosis [31, 34].

In this chapter, we will describe what is known about the degradation properties of these bacteria.

### **2. Rhizodegradation**

Among bacteria with bioremediation potential, *Frankia* are unique in that these bacteria form a symbiosis with actinorhizal plants. The implications of this trait for bioremediation efforts have only recently been explored. In the context of bioremediation, the most extensively studied system is the *Frankia–Alnus* association. Diverse assemblages of free-living *Frankia* strains are present in soils with polyaromatic hydrocarbon (PAH) contamination [8-10, 15, 35-38]. These *Frankia* strains readily form symbioses with alders, resulting in greatly increased alder fitness in harsh environments. The *Frankia–*alder symbiosis also increases the minerali‐ zation of representative organic pollutants in oil-sands reclamation sites. The *Frankia*–alder symbiosis has been used in reclamation projects because of these traits [5, 8, 36-38]. Free-living *Frankia* also appears to be part of natural degradation communities. Specifically, *Frankia* has been found to be one of the most abundant genera in wastewater treatment communities [35]. Based on these findings, *Frankia* appears to be an underutilized tool in holistic remediation approaches.

### **3.** *S***- triazines degradation**

### **3.1. Overview**

fixing associations with over 220 species of woody dicotyledonous plants, termed actinorhizal plants, that are found in eight families of angiosperms [1, 3-6]. The symbiosis with *Frankia* allows these actinorhizal host plants to colonize nutrient-poor soil and harsh environments. Actinorhizal plants have been used to recolonize and reclaim industrial wastelands and environments contaminated with heavy metals and toxic aromatic hydrocarbon [7-15]. The metabolic potential of these bacteria has only recently been investigated in the context of

Based on phylogenetic analysis, *Frankia* strains have been classified into four main lineages [19-23]. Members of lineage 1 are found infective on host plants of the Betulaceae *(Alnus*), Myricaceae, and Casuariaraceae families, while lineage 2 represents strains that are infective on Rosaceae (*Dryas*, etc.), Coriariaceae *(Coriaria)*, Datiscaceae (*Datisca),* and the genus *Ceano‐ thus* (Rhamnaceae)*.* Members of lineage 3 are the most promiscuous and are infective on Eleagnaceae, Rhamnaceae, Myricaceae, *Gynmmostoma,* and occasionally *Alnus.* The fourth *Frankia* lineage consists of the "atypical" strains which are unable to reinfect actinorhizal host plants or form ineffective root nodule structures that are unable to fix nitrogen. Our under‐ standing of this genus has been greatly enhanced by the sequencing of several *Frankia* genomes from the different *Frankia* lineages [24-33]. Analysis of *Frankia* genomes has revealed new potential with respect to metabolic diversity, natural product biosynthesis, and stress toler‐ ance, which may help aid the cosmopolitan nature of the actinorhizal symbiosis [31, 34].

In this chapter, we will describe what is known about the degradation properties of these

Among bacteria with bioremediation potential, *Frankia* are unique in that these bacteria form a symbiosis with actinorhizal plants. The implications of this trait for bioremediation efforts have only recently been explored. In the context of bioremediation, the most extensively studied system is the *Frankia–Alnus* association. Diverse assemblages of free-living *Frankia* strains are present in soils with polyaromatic hydrocarbon (PAH) contamination [8-10, 15, 35-38]. These *Frankia* strains readily form symbioses with alders, resulting in greatly increased alder fitness in harsh environments. The *Frankia–*alder symbiosis also increases the minerali‐ zation of representative organic pollutants in oil-sands reclamation sites. The *Frankia*–alder symbiosis has been used in reclamation projects because of these traits [5, 8, 36-38]. Free-living *Frankia* also appears to be part of natural degradation communities. Specifically, *Frankia* has been found to be one of the most abundant genera in wastewater treatment communities [35]. Based on these findings, *Frankia* appears to be an underutilized tool in holistic remediation

**1.1.** *Frankia* **genomics and identification of metabolic potential**

bioremediation [16-18].

272 Actinobacteria - Basics and Biotechnological Applications

bacteria.

approaches.

**2. Rhizodegradation**

Triazines are a class of herbicides composed of a heterocyclic six-membered ring with alternating carbon and nitrogen atoms joined by double bonds. These herbicides have been used extensively for control of broadleaf and grassy weeds in corn, sorghum, and sugar‐ cane cultivation. Atrazine and simazine are the most ubiquitous members of the s-tria‐ zine family. Biodegradation of atrazine is a complex process and depends on the nature and amount of atrazine in soil or water [39-41]. There are four major steps in atrazine degradation: hydrolysis, dealkylation, deamination, and ring cleavage. For the hydrolysis step, an amidohydrolase enzyme (AtzA) cleaves the carbon-chlorine (C-Cl) bond and thus dechlorinates atrazine to hydroxylatrazine. This intermediate is dealkylated and deaminat‐ ed at the ethyl and isopropyl groups by the amidohydrolase enzymes, AtzB and AtzC, to produce cyanuric acid. This product is converted to ammonia and carbon dioxide by the AtzD, AtzE, and AtzF enzymes [42-44].

### **3.2.** *S***-triazine degradation pathway in** *Frankia*

In *Frankia,* the first two steps in atrazine degradation have been identified as well as the regulation of their gene expression [17]. The mineralization of atrazine to ammonia and carbon dioxide is generally initiated by hydrolytic dechlorination, catalyzed by the enzyme atrazine chlorohydrolase (AtzA). Alternatively, this reaction is catalyzed by another atrazine chloro‐ hydrolase (TrzN), which is also able to use atrazine derivatives including desethyl-desisopro‐ pylatrazine as substrates. Analysis of the *Frankia* genomes identified candidate genes for the atrazine degradation pathway (Figure 1). The *trzN* gene was identified in *Frankia alni* ACN14a (FRAAL1474) and *Frankia* sp EuI1c (FraEuI1c\_5874) genomes and its amidohydrolase gene product is predicted to remove chlorine from s-triazine compounds to produce hydroxyatra‐ zine or ammeline from atrazine and desethyl desisopropyl atrazine, respectively. Furthermore, a putative *atzB* gene was also identified in both *Frankia* genomes (FRAAL1473 and FraEuI1c\_5875) whose predicted gene product, adenosine aminohydrolase 3, is involved in the dealkylation reaction of the *N*-ethyl group from hydroxyatrazine transforming it into *N*isopropylammelide. Physiological studies showed that *Frankia* ACN14a and EuI1c cultures are able to break down atrazine and desethyl-desisopropylatrazine producing the end products hydroxyatrazine and *N*-isopropylammelide. Although the enzymes were not purified, these data clearly showed metabolism of atrazine. Analysis of gene expression in *Frankia* ACN14a found that the two genes, *trzN* (FRAAL1474) and *atzB* (FRAAL1473) are under control of the *atzR* (FRAAL1471) gene, which encodes a predicted LysR-type transcriptional regulator.

Bioinformatics analysis of the *Frankia* genomes revealed a potential full pathway for atrazine degradation in the *Frankia* sp EuI1c genome (Figure 2). The *atzC* (FraEuI1c\_4724) gene, which encodes a putative amidhydrolase enzyme, was identified and is predicted to be involved in the dealkylation of the *N*-isopropyl group from atrazine to produce cyanuric acid. With other bacterial systems, cyanuric acid is hydrolyzed to ammonium and carbon dioxide via the *atzDEF* operon [43, 45]. In *Frankia* EuI1c, the *atzD* (FraEuI1c\_3137) gene product is predicted

**Figure 1.** Gene cluster organization in *Frankia alni* ACN14a for atrazine degradation. The cluster contains a putative *trzN* (FRAAL1474), putative *atzB* (FRAAL1473), and putative LysR-family transcriptional (*atzR*).

to transform cyanuric acid into carboxybiuret, which spontaneously decarboxylates to biuret. Putative *atzE* (FraEuI1c\_1007 and 1008), and *atzF* (FraEuI1c\_3831) genes were also identified in the *Frankia* EuI1c genome and their gene products expected to complete *s*-triazine mineral‐ ization by converting biuret to allophanate and ammonia plus carbon dioxide. A *trzR* (FraEuI1c\_3136) gene, which encodes a GntR family transcriptional regulator, is found before the *atzD* gene and is involved in the expression of that gene (Rehan unpublished).

**Figure 2.** The atrazine degradation steps in *Frankia* strains EuI1c and ACN14a include atrazine dechlorination and dealkylation and ring cleavage by TrzN, atzB, and atzD enzymes.

### **4. Aromatic compounds degradation**

#### **4.1. Biphenyl and polychlorinated biphenyl**

Biphenyls and polychlorinated biphenyls (PCBs) are some of the most recalcitrant xenobiotics found in the environment. The degree of chlorination differs greatly among the PCBs, ranging from 1 to 10, as does their position on the carbon atoms. Since the mid-1980s, the use of PCBs has been phased out in many countries. However, due to their toxicity, persistence in the environment, and potential carcinogenicity, they are still a major global environmental problem [46-48].

Bacteria degrade biphenyl and PCBs via the *meta*-cleavage pathway, which is encoded by the *bph* operon, and produces tricarboxylic acid and chlorobenzoate (CBA) as intermediates [47-50]. The first enzyme in this pathway is biphenyl dioxygenase, which is a multimeric complex consisting of the large α and small β subunits, and the ferredoxine and ferredoxine reductase subunits. The degradation process is initiated by biphenyl dioxygenase which incorporates two oxygen atoms at the 2 and 3 carbon positions of the aromatic ring (called 2,3 dioxygenation) to generate hydroxyl groups. For PCBs degradation, biphenyl dioxygenase catalyzes the initial 2,3-dioxygenation, and dihydrodiol dehydrogenase converts the product into 2,3-dihydroxybiphenyl. The enzyme, 2,3 dihydroxybiphenyl dioxygenase, cleaves the dihydroxylated ring to produce (chlorinated) 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). A hydrolase enzyme then hydrolyzes HOPDA to (chlorinated) benzoic acid and 2 hydroxypent a-2,4-dienoate.

### *4.1.1. Biphenyl degradation pathway in Frankia*

to transform cyanuric acid into carboxybiuret, which spontaneously decarboxylates to biuret. Putative *atzE* (FraEuI1c\_1007 and 1008), and *atzF* (FraEuI1c\_3831) genes were also identified in the *Frankia* EuI1c genome and their gene products expected to complete *s*-triazine mineral‐ ization by converting biuret to allophanate and ammonia plus carbon dioxide. A *trzR* (FraEuI1c\_3136) gene, which encodes a GntR family transcriptional regulator, is found before

**Figure 1.** Gene cluster organization in *Frankia alni* ACN14a for atrazine degradation. The cluster contains a putative

**Figure 2.** The atrazine degradation steps in *Frankia* strains EuI1c and ACN14a include atrazine dechlorination and

Biphenyls and polychlorinated biphenyls (PCBs) are some of the most recalcitrant xenobiotics found in the environment. The degree of chlorination differs greatly among the PCBs, ranging

dealkylation and ring cleavage by TrzN, atzB, and atzD enzymes.

**4. Aromatic compounds degradation**

**4.1. Biphenyl and polychlorinated biphenyl**

the *atzD* gene and is involved in the expression of that gene (Rehan unpublished).

*trzN* (FRAAL1474), putative *atzB* (FRAAL1473), and putative LysR-family transcriptional (*atzR*).

274 Actinobacteria - Basics and Biotechnological Applications

At least four *Frankia* strains (ACN14a, CcI3, EUN1f, and EuI1c) are resistant to biphenyl and polychlorinated biphenyl (PCB) at concentrations up to 5mM [51, Swanson unpublished results]. Data mining for known organisms capable of biphenyl degradation [46, 52] and the availability of a *Frankia* genome database enabled the identification of genes potentially involved in biphenyl degradation in several of the *Frankia* strains listed above. Five genes were identified that encode enzymes involved in biphenyl degradation: the alpha and beta subunits of the aromatic-ring-hydroxylating dioxygenase, a Rieske (2Fe-2S) iron–sulfur domain protein, an alpha/beta hydrolase fold protein, and a short-chain dehydrogenase/reductase (SDR). These enzymes are putatively capable of oxidizing and hydroxylating benzene rings, and are also known as the upper meta-cleavage pathway. A lower pathway of aromatic ring degradation consisting of three genes (encoding the 2-hydroxypenta-2,4-dienoate hydratase; acylating acetaldehyde dehydrogenase; and 4-hydroxy-2-oxovalerate aldolase) is located downstream of this operon [53, Swanson and Tisa unpublished data]. Figure [3] shows the gene neighbor‐ hood of the Biphenyl degradation genes. These genes were also found in *Frankia* strain EUN1f and Dg1 genomes (Swanson and Tisa unpublished). Both the meta-cleavage upper and the lower pathways are commonly referred to as the *bph* operon in several other PCB-degrading bacteria. *Rhodococcus* RAH1, a species closely related to *Frankia*, utilizes *bph* genes homologous to those found in *Frankia* to metabolize PCBs as a sole carbon and energy source [54]. Since at least two genes (Aromatic-ring-hydroxylating dioxygenase, subunit alpha-like protein (FraEuI1c\_4097) and short-chain dehydrogenase/reductase (FraEuI1c\_4101) in the *bph* operon in *Frankia* are upregulated in the presence of biphenyl, it is likely that *Frankia* also uses the *bph* operon to metabolize biphenyl and PCBs (Rehan and Tisa unpublished)

**Figure 3.** The gene neighborhood of *bph* operon in *Frankia* EuI1c in comparison to *Rhodococcus equi 103S* and *Photorhab‐ dus luminescens laumondii* TTO1 operon. (1) Aromatic-ring-hydroxylating dioxygenase, subunit alpha. (2) Rieske (2Fe-2S) iron–sulfur domain protein. (3) Aromatic-ring-hydroxylating dioxygenase, subunit beta. (4) Alpha/beta hy‐ drolase fold protein. (5) Short-chain dehydrogenase/reductase SDR.

#### **4.2. Phenol degradation**

#### *4.2.1. Overview*

Phenol (or hydroxybenzene) consists of a benzene ring substituted with a hydroxyl group. Derivatives of this molecule are colloquially known as phenolic compounds. Phenolic compounds are ubiquitous chemicals with diverse properties and uses. The simplest phenolic compound, phenol, is widely used in oil and coal processing, tinctorial and metallurgic industries, and many other industrial applications. Phenol also enters the environment via vehicle exhaust and as the product of natural metabolic processes, and chlorophenols are widely used as biocides in agricultural applications [for a review see 55]. While anthropogenic phenolics are often hazardous, natural phenolic compounds are mostly harmless in the concentrations that are found in foods such as coffee and tea, and some are used as antibiotics [56, 57]. However, the toxicity of some phenolics, particularly phenol and chlorinated phenols, has prompted considerable research activity devoted to phenol remediation. Acute and chronic exposure to phenol and chlorophenol has serious health effects. Phenol and chloro‐ phenol cause lipid peroxidation which ultimately leads to tissue necrosis, and liver and kidney damage [58]. Additionally, chlorophenol exposure is associated with elevated risks of cancer, immune deficiencies, and teratogenic effects [59-61].

#### *4.2.2. General phenol degradation pathway*

One of the most promising techniques for removing anthropogenic phenolics from the environment is bioremediation. As was the case for many compounds, the degradation pathway for phenol was first elucidated in a *Pseudomonas* strain [62]. Most bacteria degrade phenolics using catechol catabolic enzymes, most importantly catechol-2,3-dioxygenase. Phenols are first hydroxylated to form catechol, and then catechol-2,3-dioxygenase cleaves the benzene ring at the meta position [62]. Therefore, the degradation pathway that begins with catechol-2,3-dioxygenase is called the meta pathway (Figure 4). While the meta pathway is most prevalent, degradation can also begin with cleavage at the para or ortho position using catechol-1,2-oxygenase [63-65]. After ring cleavage, 2-hydroxymuconic semialdehyde hydro‐ lase catalyzes a decarboxylation reaction yielding 4-oxalocrotonate. 4-oxalocrotonate is hydrated by 2-oxopent-4-enoate hydratase to form 4-hydroxy-2-oxovalerate. 4-hydroxy-2 oxovalerate aldolase then splits 4-hydroxy-2-oxovalerate into pyruvate and acetaldehyde, which can then be incorporated into the central metabolic pathways [62].

**Figure 4.** General phenol degradation pathway.

**Figure 3.** The gene neighborhood of *bph* operon in *Frankia* EuI1c in comparison to *Rhodococcus equi 103S* and *Photorhab‐ dus luminescens laumondii* TTO1 operon. (1) Aromatic-ring-hydroxylating dioxygenase, subunit alpha. (2) Rieske (2Fe-2S) iron–sulfur domain protein. (3) Aromatic-ring-hydroxylating dioxygenase, subunit beta. (4) Alpha/beta hy‐

Phenol (or hydroxybenzene) consists of a benzene ring substituted with a hydroxyl group. Derivatives of this molecule are colloquially known as phenolic compounds. Phenolic compounds are ubiquitous chemicals with diverse properties and uses. The simplest phenolic compound, phenol, is widely used in oil and coal processing, tinctorial and metallurgic industries, and many other industrial applications. Phenol also enters the environment via vehicle exhaust and as the product of natural metabolic processes, and chlorophenols are widely used as biocides in agricultural applications [for a review see 55]. While anthropogenic phenolics are often hazardous, natural phenolic compounds are mostly harmless in the concentrations that are found in foods such as coffee and tea, and some are used as antibiotics [56, 57]. However, the toxicity of some phenolics, particularly phenol and chlorinated phenols, has prompted considerable research activity devoted to phenol remediation. Acute and chronic exposure to phenol and chlorophenol has serious health effects. Phenol and chloro‐ phenol cause lipid peroxidation which ultimately leads to tissue necrosis, and liver and kidney damage [58]. Additionally, chlorophenol exposure is associated with elevated risks of cancer,

One of the most promising techniques for removing anthropogenic phenolics from the environment is bioremediation. As was the case for many compounds, the degradation pathway for phenol was first elucidated in a *Pseudomonas* strain [62]. Most bacteria degrade phenolics using catechol catabolic enzymes, most importantly catechol-2,3-dioxygenase. Phenols are first hydroxylated to form catechol, and then catechol-2,3-dioxygenase cleaves the

drolase fold protein. (5) Short-chain dehydrogenase/reductase SDR.

276 Actinobacteria - Basics and Biotechnological Applications

immune deficiencies, and teratogenic effects [59-61].

*4.2.2. General phenol degradation pathway*

**4.2. Phenol degradation**

*4.2.1. Overview*

### *4.2.3. Phenolic compounds and Frankia*

*Frankia* spp. both produce and are affected by phenolic compounds. However, it is unclear whether *Frankia* may degrade phenol and other phenolic compounds. The response of *Frankia* to phenolics was first studied in the context of plant–microbe interactions. Despite apparent functional and morphological similarities between *Frankia* nodules and leguminous nodules, the molecular and physiological mechanisms that control nodulation are distinct. Therefore, the unique process of nodulation by *Frankia* is still an area of intense research. *Alnus* spp. (Alders) plants are a major host plant for *Frankia,* and also have unusually high levels of phenolics in their root exudates, which affect the growth of *Frankia*. Most *Alnus* phenolics tested inhibit *Frankia* growth to varying degrees [66, 67]. Specifically, benzoic acids are less inhibitory than cinnamic acids such as caffeinic acid. However, one plant phenolic, o-hydroxyphenyl‐ acetic acid, promoted *Frankia* growth, and both benzoic and cinnamic acids caused increased branching of *Frankia* hyphae. Low concentration plant phenolics also mediate a global shift in *Frankia* gene expression, while higher concentrations (above 30 mg L-1) simply inhibit biosyn‐ thesis [33]. Interestingly, *Frankia* also increases phenolic expression of their host plant, causing them to produce more phenol, flavonoids, and hydroxycinnamic acid [68].

*Frankia* may promote excretion of phenolics as a way to increase available nutrients. However, this explanation depends on *Frankia* having the ability to degrade phenolic compounds. While no study has demonstrated that *Frankia* degrades phenolic compounds, there is genetic evidence that this bacterium may have the ability to degrade phenolics. First, some *Frankia* strains have genes coding for the production of catechol and other phenolic compounds [34]. Because bacteria often salvage the biomolecules they produce, the presence of an anabolic pathway suggests that a catabolic pathway is also present [69]. Furthermore, multiple *Frankia* strains contain catechol-2, 3-dioxygenase, the most important enzyme in the phenol degrada‐ tion pathway (Swanson and Tisa unpublished data) [64]. A closely related bacterium, *Rhodococcus* spp., uses the catechol-2,3-dioxygenase pathway to grow with phenol as its sole carbon source [70]. The same species is also able to break down the more recalcitrant penta‐ chlorophenol via the para pathway [71]. This suggests that *Frankia* may break down phenol, a trait that could be applied in bioremediation efforts. Several *Frankia* strains are able to grow on phenol, quercetin, catechol, and other phenolic compounds (Furnholm, Greenleaf, and Tisa unpublished data), but the metabolism of their breakdown has not been studied.

#### **4.3. Naphthalene degradation**

#### *4.3.1. Overview*

Naphthalene is a ubiquitous polyaromatic hydrocarbon composed of two benzene rings joined at the 9 and 10 carbons (Figure 5). Naphthalene is produced by distilling and crystallizing coal tar, and also as by-product of fossil fuel combustion and cigarette smoke [72]. Naphthalene is used in a number of industrial applications including as feed stock for the production of plastics and resins, and as a component of creosote-based wood preservatives. Naphthalene is also used in tincture and leather tanning industries [72]. Unlike many organic pollutants, naphthalene does not bioaccumulate. Instead, naphthalene is metabolized and excreted in the urine of rats and humans [72, 73]. Nonetheless, naphthalene is a problematic pollutant with numerous toxic effects. Acute exposure to naphthalene causes hemolytic anemia, and liver and neurological damage [74]. Chronic naphthalene exposure is associated with elevated cancer risk [75, 76]. The toxicity of naphthalene and its prevalence as a pollutant has spurred research on remediation techniques, including bioremediation and biodegradation.

**Figure 5.** Structure of naphthalene.

#### *4.3.2. Degradation pathway*

The naphthalene biodegradation pathway was first studied in a strain of *Pseudomonas* which has two related naphthalene degradation pathways. The upper pathway catabolizes naph‐ thalene to produce salicylate and a molecule of pyruvate [77]. The lower pathway breaks salicylate down into acetyl Co-A and pyruvate [78]. The first step of the upper pathway is catalyzed by four proteins: naphthalene dioxygenase reductase, naphthalene dioxygenase ferredoxin, and naphthalene dioxygenase Fe-S protein small and large subunits. This collection of enzymes oxidizes naphthalene to produce *cis*-naphthalene dihydrodiol, which is subse‐ quently dehydrogenated by naphthalene *cis*-dihyrdodiol dehydrogenase to form 1,2-dihy‐ droxynaphthalene. 1,2-dihydronaphthalene dioxygenase then produces 2 hydroxychromene-2-carboxylate which is then cleaved by 2-hydroxychromene-2-carboxylate dehydrogenase to form *cis*-*o*-hydroxybenzylpyruvate. 1,2-dihydroxybenzylpyruvate aldolase then splits *cis*-*o*-hydroxybenzylpyruvate producing pyruvate and salicylaldehyde. Finally, salicylaldehyde dehydrogenase carboxylates salicylaldehyde to form salicylate [77, 78].

In the lower pathway, salicylate hydroxylase hydroxylates salicylate to produce catechol. The remaining benzene ring is then cleaved by catechol-2,3-dioxygenase to produce 2-hydroxy‐ muconic semialdehyde [78]. Hydroxymuconic semialdehyde dehydrogenase then produces 2-hydroxyhexa-2,4-diene-1,6-dioate which is subsequently isomerized by 4-oxalocrotmate isomerase to produce 2-oxohexa-3-ene-1,6-dioate. This is then transformed into 2-oxopent-4 enoate by 4-oxalocrotomate decarboxylase. 2-oxopent-4-enole hydratase produces 4-hy‐ droxy-2-oxovalerate, which is subsequently split into acetaldehyde and pyruvate by 2-oxo-4 hydroxypentanoate aldolase. Finally, acetaldehyde dehydrogenase converts acetaldehyde into acetyl Co-A [78]. Both of these pathways are also found in *Rhodococcus* spp, a close relative of *Frankia* [79].

### *4.3.3. Naphthalene degradation in Frankia*

Not surprisingly, *Frankia* also metabolizes naphthalene as a sole carbon and energy source via a related pathway [18]. Specifically, *Frankia* uses the protocatechuate pathway to convert naphthalene or a naphthalene derivative into acetyl Co-A and succinyl Co-A (Figure 6) [18]. This finding confirms the role of *Frankia* in naphthalene degradation, which was suggested by earlier field studies [8-10, 37, 38]. In symbiosis with alders, *Frankia* increases polyaromatic hydrocarbon degradation in oil-sand tailings for the first 1.5 years [8, 10, 37]. However, after 2.5 years, alders without *Frankia* symbionts demonstrated naphthalene degradation equal to the degradation or *Frankia*-inoculated alders [8]. The *Frankia*-alder symbiosis thrives in PAHcontaminated areas [15]. Interestingly, alder plants found in these PAH-contaminated areas maintained a symbiosis with *Frankia* lineage III as opposed to the normal lineage I, suggesting that this pollutant affected nodulation and/or survival of the actinorhizal plants. Taken together, these findings indicate that *Frankia* could be a useful tool in naphthalene remediation.

#### **4.4. Protocatechuate**

#### *4.4.1. Overview*

no study has demonstrated that *Frankia* degrades phenolic compounds, there is genetic evidence that this bacterium may have the ability to degrade phenolics. First, some *Frankia* strains have genes coding for the production of catechol and other phenolic compounds [34]. Because bacteria often salvage the biomolecules they produce, the presence of an anabolic pathway suggests that a catabolic pathway is also present [69]. Furthermore, multiple *Frankia* strains contain catechol-2, 3-dioxygenase, the most important enzyme in the phenol degrada‐ tion pathway (Swanson and Tisa unpublished data) [64]. A closely related bacterium, *Rhodococcus* spp., uses the catechol-2,3-dioxygenase pathway to grow with phenol as its sole carbon source [70]. The same species is also able to break down the more recalcitrant penta‐ chlorophenol via the para pathway [71]. This suggests that *Frankia* may break down phenol, a trait that could be applied in bioremediation efforts. Several *Frankia* strains are able to grow on phenol, quercetin, catechol, and other phenolic compounds (Furnholm, Greenleaf, and Tisa

unpublished data), but the metabolism of their breakdown has not been studied.

Naphthalene is a ubiquitous polyaromatic hydrocarbon composed of two benzene rings joined at the 9 and 10 carbons (Figure 5). Naphthalene is produced by distilling and crystallizing coal tar, and also as by-product of fossil fuel combustion and cigarette smoke [72]. Naphthalene is used in a number of industrial applications including as feed stock for the production of plastics and resins, and as a component of creosote-based wood preservatives. Naphthalene is also used in tincture and leather tanning industries [72]. Unlike many organic pollutants, naphthalene does not bioaccumulate. Instead, naphthalene is metabolized and excreted in the urine of rats and humans [72, 73]. Nonetheless, naphthalene is a problematic pollutant with numerous toxic effects. Acute exposure to naphthalene causes hemolytic anemia, and liver and neurological damage [74]. Chronic naphthalene exposure is associated with elevated cancer risk [75, 76]. The toxicity of naphthalene and its prevalence as a pollutant has spurred

research on remediation techniques, including bioremediation and biodegradation.

The naphthalene biodegradation pathway was first studied in a strain of *Pseudomonas* which has two related naphthalene degradation pathways. The upper pathway catabolizes naph‐

**4.3. Naphthalene degradation**

278 Actinobacteria - Basics and Biotechnological Applications

**Figure 5.** Structure of naphthalene.

*4.3.2. Degradation pathway*

*4.3.1. Overview*

Under oxic conditions, microbial degradation of many aromatic compounds occurs through the catechol or protocatechuate branch of the ß-ketoadipate pathway via either *ortho* cleavage

**Figure 6.** Putative naphthalene degradation pathway in *Frankia* [18]. (Figure is recopied with permission from *Canadian Journal of Microbiology*.)

by catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase or *meta*-cleavage by catechol-2,3-dioxygenase and protocatechuate-4,5-dioxygenase.

#### *4.4.2. Potential protocatechuate degradation pathway in Frankia*

Besides the protochatechuate pathway found in *Frankia* QA3 [18], several other potential protocatechuate pathways have been identified from bioinformatics analysis of the available *Frankia* genomes. In *Frankia* EuI1c, a potential operon (*FraEuI1c\_2560* -to- *FraEuI1c\_2564*) for a putative protocatechuate pathway was identified (Figure 7). This operon encodes the predicted gene products involved in the putative pathway including protocatechuate 3,4-dioxygenase alpha and beta subunits, fumarate lyase, 3-oxoadipate enol-lactonase, and 4-hydroxybenzoate 3-monooxygenase. These gene products are similar to the protocatechuate degradation pathway found in *Rhodococcus opacus* 1CP [80, 81]. These results suggest that *Frankia* may use the protocatechuate degradation pathway to degrade many aromatic ring compounds after their conversion to protocatechuate.

**Figure 7.** The proposed protocatechuate degradation pathway in *Frankia* strains EuI1c and EUN1f.

### **5. Hydrocarbons**

#### **5.1. Overview**

by catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase or *meta*-cleavage by

**Figure 6.** Putative naphthalene degradation pathway in *Frankia* [18]. (Figure is recopied with permission from *Canadian*

Besides the protochatechuate pathway found in *Frankia* QA3 [18], several other potential protocatechuate pathways have been identified from bioinformatics analysis of the available *Frankia* genomes. In *Frankia* EuI1c, a potential operon (*FraEuI1c\_2560* -to- *FraEuI1c\_2564*) for a putative protocatechuate pathway was identified (Figure 7). This operon encodes the predicted gene products involved in the putative pathway including protocatechuate 3,4-dioxygenase alpha and beta subunits, fumarate lyase, 3-oxoadipate enol-lactonase, and 4-hydroxybenzoate 3-monooxygenase. These gene products are similar to the protocatechuate degradation pathway found in *Rhodococcus opacus* 1CP [80, 81]. These results suggest that *Frankia* may use the protocatechuate degradation pathway to degrade many aromatic ring compounds after

catechol-2,3-dioxygenase and protocatechuate-4,5-dioxygenase.

*4.4.2. Potential protocatechuate degradation pathway in Frankia*

their conversion to protocatechuate.

*Journal of Microbiology*.)

280 Actinobacteria - Basics and Biotechnological Applications

Petroleum-based energy and products are used extensively around the world. The pervasive‐ ness of petroleum inevitably leads to serious environmental pollution. Petroleum is a complex mixture of hydrocarbons, cycloalkanes, aromatic hydrocarbons, and more complex chemicals like asphaltenes. These chemicals and their derivatives, which are termed petrogenic com‐ pounds, are released into the environment as a result of oil spills and combustion of petroleumbased products [82]. Oil spills are one of the most serious sources of petroleum pollution and devastate aquatic and marine environments. Ongoing research to identify new methods for petroleum remediation is important because oil spills and other types of petroleum-derived pollution continue to pose environmental health risks.

Hydrocarbon-degrading bacteria and fungi are widely distributed in marine and freshwater environments, as well as soil habitats [83, 84]. In *Pseudomonas*, the alkane hydroxylase (mon‐ ooxygenase) system consists of three components: alkane hydroxylase (AlkB), rubredoxin, and rubredoxin reductase. This system is responsible for the first oxidation step in the utilization of n-alkanes [85]. Similar alkane hydroxylase systems have been found in a variety of alkanedegrading bacteria [86, 87]. *Alcanivorax* sp. strain 2B5 will degrade C13–C30 n-alkanes and branched alkanes (pristine and phytane) from crude oil as the sole carbon source via a novel alkane hydroxylase gene (alkB). Other *Acinetobacter* are able to use n-alkanes with chain length C10–C40 as a sole source of carbon. In addition, the presence of multiple alkane hydroxylases in two *Rhodococcus* strains were characterized and both organisms contained at least four alkane monooxygenase gene homologs (*alkB1*, *alkB2*, *alkB3*, and *alkB4*) [76, 88].

A bioinformatics approach was used to identify these potential hydrocarbon degradation pathways among the sequenced *Frankia* strains. Functionally analyzed genes for the known hydrocarbon degradation pathways [84, 88] were used to probe the *Frankia* genome database and identify potential pathways. Our preliminary results (Rehan unpublished data) revealed that the *F*. *alni* ACN14a genome possesses a putative alkane-1 monooxygenase (Alkane omegahydroxylase) gene (*FRAAL1986*), which is one of the known enzymes involved in the break‐ down of n-alkanes (Figure 8). Furthermore, a similar gene (*Franean1\_2192*) was also found in the *Frankia* sp. EAN1pec genome. These bioinformatics results support the hypothesis that *Frankia* may be able to degrade oil-spill-derived hydrocarbons. However, these preliminary results need further study.

**Figure 8.** Potential alkane-1 monooxygenase identified in *F. alni* ACN14a.

### **6. Future aspects**

Clearly, we have only begun to scratch the surface of the metabolism of *Frankia* and its biodegradative potential. These initial studies correlating metabolic capacity to gene function are the first step in exploiting the bacteria for their bioremediation ability. Further bioinfor‐ matics data mining are necessary to elucidate the unique metabolic potential of *Frankia*. However, these *in silico* studies require "wet lab" experiments to confirm these capabilities.

From limited field studies, actinorhizal nodule occupancy seems to be under control by environmental conditions. The presence of *Frankia* lineage III strains inside alder nodules found under PAH-stressed soils suggests that this lineage may have a greater metabolic potential. The larger genome size of this lineage compared to the other infective strains also supports this hypothesis. However, further experiments are required to confirm this postulate.

### **Acknowledgements**

We thank Michele Greenleaf, Teal Furnholm, and Kaci B. Kus for their efforts on our degra‐ dation studies. Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is scientific Contribution Number 2613. This work was supported by USDA National Institute of Food and Agriculture Hatch Project NH585. MR was supported by an Egyptian Channel Fellowship from The Egyptian Cultural Affairs and Missions Sectors.

### **Author details**

and identify potential pathways. Our preliminary results (Rehan unpublished data) revealed that the *F*. *alni* ACN14a genome possesses a putative alkane-1 monooxygenase (Alkane omegahydroxylase) gene (*FRAAL1986*), which is one of the known enzymes involved in the break‐ down of n-alkanes (Figure 8). Furthermore, a similar gene (*Franean1\_2192*) was also found in the *Frankia* sp. EAN1pec genome. These bioinformatics results support the hypothesis that *Frankia* may be able to degrade oil-spill-derived hydrocarbons. However, these preliminary

Clearly, we have only begun to scratch the surface of the metabolism of *Frankia* and its biodegradative potential. These initial studies correlating metabolic capacity to gene function are the first step in exploiting the bacteria for their bioremediation ability. Further bioinfor‐ matics data mining are necessary to elucidate the unique metabolic potential of *Frankia*. However, these *in silico* studies require "wet lab" experiments to confirm these capabilities. From limited field studies, actinorhizal nodule occupancy seems to be under control by environmental conditions. The presence of *Frankia* lineage III strains inside alder nodules found under PAH-stressed soils suggests that this lineage may have a greater metabolic potential. The larger genome size of this lineage compared to the other infective strains also supports this hypothesis. However, further experiments are required to confirm this postulate.

We thank Michele Greenleaf, Teal Furnholm, and Kaci B. Kus for their efforts on our degra‐ dation studies. Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is scientific Contribution Number 2613. This work was supported by USDA National Institute of Food and Agriculture Hatch Project NH585. MR was supported by an Egyptian Channel Fellowship from The Egyptian Cultural Affairs and Missions Sectors.

results need further study.

282 Actinobacteria - Basics and Biotechnological Applications

**6. Future aspects**

**Acknowledgements**

**Figure 8.** Potential alkane-1 monooxygenase identified in *F. alni* ACN14a.

Medhat Rehan1 , Erik Swanson2 and Louis S. Tisa2\*

\*Address all correspondence to: louis.tisa@unh.edu

1 Department of Genetics, Kafrelsheikh University, Kafr El-Sheikh, Egypt

2 Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, USA

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