**3. Bio-nanocomposites**

a tissue with its functional properties [30]. The microscopic structure must allow nutrient diffusion as well as the efflux of metabolites no longer needed to the cell through the scaffold. Finally, the scaffold must have good mechanical properties, enabling handling during culture

One of the biggest health issues worldwide is organ failure derived from disease or a traumatic event; this has been resolved by transplantation of organs from living or deceased patient donors. The list of donors and recipients has increased in the last years and there are many patients on waiting lists for organ donation [31]. According to Gilpin and Yang [31], tissue engineering consists of three important aspects: the participating cells, the signaling molecules used and the scaffold. Scaffolds can be natural or synthetic. Natural scaffolds are derived from decellularization processes using chemical, enzymatic or physical methods. The resulting decellularized scaffold has to be recellularized either with one or different type of cells, in other cases

For more than 20 years, scientists started developing nano-bio-materials and it is thought that nano bio-composites will be more important than non-nanometric materials at the physiological level. The advancement in biomedical research due to the incorporation in biomaterials to

Decellularized scaffolds have been improved by combining them with biomaterials, not only to provide the extracellular matrix required for the cells to proliferate and differentiate but also to provide structural, biochemical and biomechanical support to the regenerated organ. Cheng et al. [35], developed silk-based scaffolds for bone regeneration, but their therapeutic efficacy was not optimal, therefore they developed a composite material of mesoporous bioactive glass/silk scaffold to improve mesenchymal stem cell regeneration activity in a rodent model for postmenopausal osteoporosis. They proved that the composite material provided the optimal environment for mesenchymal stem cell differentiation, attachment, and proliferation as treatment of osteoporotic defects [35]. Sterling and Guelcher [36] proposed another example of scaffolds to heal fractures derive for osteoporosis. In this research, the authors have argued that bone autografts (bone sample from the same patient), that have been used to improve fractured bone healing, have some pitfalls due to the limited amounts of bone that can be harvested, instead, hybrid scaffolds have been fabricated made with silk and calcium phosphate to stimulate bone formation and to reverse bone loss. The same group has shown that local delivery of recombinant bone morphogenic protein from microspheres made with polylactic glycolic acid has improved the mechanical properties of vertebrae in animal models [36]. We have been addressing some examples of the use of nanomaterials in conjunction with biological models or cells, but we are also going to show how these systems have to be visualized

Regarding tissue engineering, once the bio-engineered tissue is developed, it has to be evaluated in its structure and function. Histological and histochemical techniques have been used. For example, it is important not only to evaluate the 3D structure of scaffolds and its possible interaction with cells prior deciding on a biological or clinical application, but also the functionality of the cells contained the manufactured tissues. Different imaging techniques can be used to assure the efficiency of the biocomposites, such as ultrasound, microscopy, magnetic

resonance imaging (MRI), and other optical imaging techniques [20].

induced pluripotent stem cells are used to recapitulate organ functionality [31–33].

biological models has had a great impact in health sciences [34].

for further biomedical characterization.

in bioreactors and transplantation into the host [30].

30 Tissue Regeneration

Biomaterials research has been concerned with the use of nanomaterials to enhance the tissue regeneration process. In this regard, nanomaterials can be classified into organic and inorganic systems. Diversity in organic materials derived mainly from polymers, such as polysaccharides, collagen, and chitosan have been recently used with different morphologies into the biomedical application and stem cell differentiation [19]. In particular, the use of polymer NPs as carriers or drug delivery systems is promising materials used as neuroprotectors to avoid acute ischemic stroke, which is actually considered one of the most common causes of death worldwide [40]. Nanostructured drug delivery systems offer many advantages, such as the avoidance of drug degradation, the possibility to improve the pharmacokinetic profile and the specificity at nano scale.

NPs from different materials have been functionalized with bioactive molecules in order to describe their effects in cells and tissues. Bio-composites of silica NPs with fluorescent compounds from the tree *Eysenhardtia polystachya* were internalized into MCF-7 breast cancer cells and observed with confocal microscopy to analyze their possible anti-tumor effect [41].

Cells interact with each other through their own synthesized ECM, which provides support and allows proliferation and differentiation processes. In consequence, ECM produces high membrane adherence with specific ligands associated with signaling pathways and possible migration, which can regulate the cell growth [42, 43]. Our body possesses natural ECM, mainly conformed by fibrous proteins and proteoglycans, ranging in size from 50 to 500 nm [44]. In this regard, collagen is an important source of ECM, present in the majority of connective tissues, such as bone, skin, and tendons. It is confirmed by a three-dimensional protein network by nano-sized fibers, with high resistance and adherence [29, 45], and recently, many studies have been focused to mimic this behavior and replace it with functional materials.

Several studies have been directed to design and understanding the composition and structure of new hybrid polymers. These hybrid materials are made of Au and Ag NPs supported on a polymer grill; the matrix prevents NPs aggregation, provides mechanical backing and keeps biocompatibility. In this area, CTS appears as a unique material with polycationic, chelating, and film forming properties. Additionally, through NPs incorporation its antibacterial

Trends in Tissue Regeneration: Bio-Nanomaterials http://dx.doi.org/10.5772/intechopen.75401 33

The AgNPs synthesis allows the production of stable metal NPs. When these NPs are incorporated in CTS electrospun fibers, it is possible to obtain high antimicrobial nanomaterials [62]. This behavior is generated due to the polycationic characteristics of CTS matrix and their interaction with the embedded AgNPs, linked by electrostatic attractions [63]. It has been reported that amine/hydroxyl groups presented in CTS matrix can interact with metal ions, in order to form stable complexes, and it is possible to in situ synthesize metal NPs in CTS solution, with high morphology control [64]. Moreover, AuNPs are also used due to their excellent biocompatibility and especially because it was found that CTS-AuNPs nanocomposites enhance the proliferation of human fibroblasts. This significant enhancement of biocompatibility may be due to the altered surface morphology. The size of the nanometric surface domains could have

The mechanical properties of CTS (e.g. swelling), are not good enough for medical applications; to solve this it was inserted into the structure, a natural synthetic polymer CTS-based, grafted with glycidyl methacrylate (CTS-g-GMA) [66]. This arrangement of polymers pro-

Ag and Au NPs show a collective oscillation of their electrons from the conduction band when they interact with a specific electromagnetic field; this property is called surface plasmon resonance (SPR). After all the evidence collected from the interaction between noble metals and natural polymers, the results are the evident success in the aggregation of AuNPs and AgNPs. This behavior was confirmed by UV-vis analyses, where the SPR bands were used to identify the metallic elements. As result, characteristic SPR for the AgNPs was located at 427 nm, while

Fibers obtained by electrospinning have been synthesized modifying the film method, the viscosity did not allow the correct stretched from the solutions, then it was necessary to add to the mixture polyethylene oxide and a surfactant to enhance the viscosity in order to obtain

These results are promising, the combined UV-vis spectra from the materials show SPR in 432 nm for AgNPs and 532 nm for AuNPs. Transmission electron microscopy (TEM) allows to observe fibers with several particles inserted in the surface, as presented in **Figure 2**. In this case, is not possible to determine if observed NPs are Ag or Au using only TEM imaging (elemental analyses show the presence of both elements), but the presence of them indicates that the NPs synthesis was successful and the electrospinning method is an option to perform

The major contribution of this research is that normally both metals, Au and Ag, are reduced chemically by separated and joined after these chemical reductions, nevertheless both nanostructured materials shown above as, films and fibers, underwent the chemical reduction *in situ*.

the SPR peak of the AuNPs was located around 530 nm [58], as shown in **Figure 1**.

materials with the characteristics to be used in biomedical applications.

effect increases and it can either stimulate or inhibit human cells activity [61].

an impact on cellular responses [65].

nanometric fibers of the polymer with NPs.

vided a new material with better biomedical applications.

The challenge in the research of materials able to replace the ECM is the recreation of a functional nanostructured network which allows cellular growth and differentiation. In fact, there are a lot of techniques for this task but there is one in particular that has been used more frequently in the recent years by researchers because it actually generates a fibrous structure like the ECM [46]. Electro spinning technique can produce nano-sized fibers from different sources, such as polymers, biocompatible systems, sol-gel, and nanocomposite materials. This technique generates three-dimensional porous fibers with high electrostatic attraction, associated with their high surface area/aspect ratio [47]. In this regard, this technique works from a solution (polymer, nanocomposite, and others) passed through a syringe, ending from a Taylor cone to control the efflux. A voltage source creates a drop and is collected at different distances to create variable morphology fibers. The surface tension produced between the collector and the needle is created by the electrostatic forces of the fibers [47].

Chitosan (CTS) has been defined as one of the most common biopolymers and chemically is a linear polymer derived from the deacetylated process of chitin, which is obtained from crustaceans [48]. The main characteristics of CTS are their biocompatibility and degradability [49], and can be easily processed in many different structures such as films, scaffolds, and fibers. CTS has been studied as antibacterial, biocompatible material, as a carrier for specific drug delivery and wound healing dressings [50, 51]. Some of its chemical properties are its solubility in organic acids [52] and low solubility in water. In order to improve the biological behavior of CTS material, different authors propose the addition of nano-sized structures to increase the physic-chemical and antibacterial properties, such as silver nanoparticles (AgNPs) [53] and gold nanoparticles (AuNPs) [54].

NPs of noble metals are some of the most promising materials, owing to their high surface area and their facility of functionalization or coordination with organic molecules. For example, AuNPs are easily prepared in colloidal solutions. Novel research has been done exploring the potential use of AuNPs as phototherapeutic agents, in the detection and treatment of cancer, in gene therapy and in the transport and selective vectorization of drugs and macromolecules [17, 54]. Otherwise, the AgNPs are widely applied to produce artificial skin, sterilized materials, functional contraceptive devices, antibacterial surgical instruments, bone prostheses, bone coating, surface cleaners, antimicrobial paints, automotive upholstery, food storage, and others [55, 56].

Many synthesis methods have been designed to create blends with metallic NPs and enable the combination and/or synergism of their catalytic, electronic, and optical qualities. Therefore, synthesis of supported gold and silver NPs has attracted lots of attention, in view of their remarkable properties, which depend on the NP size and the amount of each material [57]; they have been used in oxidation reactions, tumor cell targeting and detection, H2 O2 production and catalytic applications [58–60].

Several studies have been directed to design and understanding the composition and structure of new hybrid polymers. These hybrid materials are made of Au and Ag NPs supported on a polymer grill; the matrix prevents NPs aggregation, provides mechanical backing and keeps biocompatibility. In this area, CTS appears as a unique material with polycationic, chelating, and film forming properties. Additionally, through NPs incorporation its antibacterial effect increases and it can either stimulate or inhibit human cells activity [61].

conformed by fibrous proteins and proteoglycans, ranging in size from 50 to 500 nm [44]. In this regard, collagen is an important source of ECM, present in the majority of connective tissues, such as bone, skin, and tendons. It is confirmed by a three-dimensional protein network by nano-sized fibers, with high resistance and adherence [29, 45], and recently, many studies have

The challenge in the research of materials able to replace the ECM is the recreation of a functional nanostructured network which allows cellular growth and differentiation. In fact, there are a lot of techniques for this task but there is one in particular that has been used more frequently in the recent years by researchers because it actually generates a fibrous structure like the ECM [46]. Electro spinning technique can produce nano-sized fibers from different sources, such as polymers, biocompatible systems, sol-gel, and nanocomposite materials. This technique generates three-dimensional porous fibers with high electrostatic attraction, associated with their high surface area/aspect ratio [47]. In this regard, this technique works from a solution (polymer, nanocomposite, and others) passed through a syringe, ending from a Taylor cone to control the efflux. A voltage source creates a drop and is collected at different distances to create variable morphology fibers. The surface tension produced between the col-

Chitosan (CTS) has been defined as one of the most common biopolymers and chemically is a linear polymer derived from the deacetylated process of chitin, which is obtained from crustaceans [48]. The main characteristics of CTS are their biocompatibility and degradability [49], and can be easily processed in many different structures such as films, scaffolds, and fibers. CTS has been studied as antibacterial, biocompatible material, as a carrier for specific drug delivery and wound healing dressings [50, 51]. Some of its chemical properties are its solubility in organic acids [52] and low solubility in water. In order to improve the biological behavior of CTS material, different authors propose the addition of nano-sized structures to increase the physic-chemical and antibacterial properties, such as silver nanoparticles

NPs of noble metals are some of the most promising materials, owing to their high surface area and their facility of functionalization or coordination with organic molecules. For example, AuNPs are easily prepared in colloidal solutions. Novel research has been done exploring the potential use of AuNPs as phototherapeutic agents, in the detection and treatment of cancer, in gene therapy and in the transport and selective vectorization of drugs and macromolecules [17, 54]. Otherwise, the AgNPs are widely applied to produce artificial skin, sterilized materials, functional contraceptive devices, antibacterial surgical instruments, bone prostheses, bone coating, surface cleaners, antimicrobial paints, automotive upholstery, food storage,

Many synthesis methods have been designed to create blends with metallic NPs and enable the combination and/or synergism of their catalytic, electronic, and optical qualities. Therefore, synthesis of supported gold and silver NPs has attracted lots of attention, in view of their remarkable properties, which depend on the NP size and the amount of each material [57];

O2

produc-

they have been used in oxidation reactions, tumor cell targeting and detection, H2

been focused to mimic this behavior and replace it with functional materials.

lector and the needle is created by the electrostatic forces of the fibers [47].

(AgNPs) [53] and gold nanoparticles (AuNPs) [54].

and others [55, 56].

32 Tissue Regeneration

tion and catalytic applications [58–60].

The AgNPs synthesis allows the production of stable metal NPs. When these NPs are incorporated in CTS electrospun fibers, it is possible to obtain high antimicrobial nanomaterials [62]. This behavior is generated due to the polycationic characteristics of CTS matrix and their interaction with the embedded AgNPs, linked by electrostatic attractions [63]. It has been reported that amine/hydroxyl groups presented in CTS matrix can interact with metal ions, in order to form stable complexes, and it is possible to in situ synthesize metal NPs in CTS solution, with high morphology control [64]. Moreover, AuNPs are also used due to their excellent biocompatibility and especially because it was found that CTS-AuNPs nanocomposites enhance the proliferation of human fibroblasts. This significant enhancement of biocompatibility may be due to the altered surface morphology. The size of the nanometric surface domains could have an impact on cellular responses [65].

The mechanical properties of CTS (e.g. swelling), are not good enough for medical applications; to solve this it was inserted into the structure, a natural synthetic polymer CTS-based, grafted with glycidyl methacrylate (CTS-g-GMA) [66]. This arrangement of polymers provided a new material with better biomedical applications.

Ag and Au NPs show a collective oscillation of their electrons from the conduction band when they interact with a specific electromagnetic field; this property is called surface plasmon resonance (SPR). After all the evidence collected from the interaction between noble metals and natural polymers, the results are the evident success in the aggregation of AuNPs and AgNPs. This behavior was confirmed by UV-vis analyses, where the SPR bands were used to identify the metallic elements. As result, characteristic SPR for the AgNPs was located at 427 nm, while the SPR peak of the AuNPs was located around 530 nm [58], as shown in **Figure 1**.

Fibers obtained by electrospinning have been synthesized modifying the film method, the viscosity did not allow the correct stretched from the solutions, then it was necessary to add to the mixture polyethylene oxide and a surfactant to enhance the viscosity in order to obtain nanometric fibers of the polymer with NPs.

These results are promising, the combined UV-vis spectra from the materials show SPR in 432 nm for AgNPs and 532 nm for AuNPs. Transmission electron microscopy (TEM) allows to observe fibers with several particles inserted in the surface, as presented in **Figure 2**. In this case, is not possible to determine if observed NPs are Ag or Au using only TEM imaging (elemental analyses show the presence of both elements), but the presence of them indicates that the NPs synthesis was successful and the electrospinning method is an option to perform materials with the characteristics to be used in biomedical applications.

The major contribution of this research is that normally both metals, Au and Ag, are reduced chemically by separated and joined after these chemical reductions, nevertheless both nanostructured materials shown above as, films and fibers, underwent the chemical reduction *in situ*.

**4. Clinical applications**

thermal burns.

spherical AgNPs around 7 nm, as presented in **Figure 3**.

**Figure 3.** AgNPs synthesized by *in situ* chemical reduction in CTS matrix.

The novel approach to use nanomaterials in regenerative medicine has established the design of functional tools to simulate, diagnose and stimulate cell growth of tissue or organs [16].

Trends in Tissue Regeneration: Bio-Nanomaterials http://dx.doi.org/10.5772/intechopen.75401 35

Burn wounds are a critical issue due to the widespread deaths due to the constant bacterial resistance to conventional antibiotics. In this regard, novel nanomaterials such as topic antimicrobial systems have been obtained to produce combined antibacterial/tissue regeneration responses in thermal burns. Luna-Hernández et al. [68] report the use of nanocomposites based on CTS/AgNPs synthesized by *in situ* chemical reduction method, obtaining embedded

In this research, controlled thermal burns produced in rats were treated with nanocomposites with different NPs concentration deposited at wound areas. These results showed the combined antibacterial responses to *S. aureus* and *P. aeruginosa*, depending on NPs concentration and the mesh formation of hydrated chitosan, which allowed bacterial penetration. As a result, significant tissue regeneration was shown in the thermal burns treated with CTS/AgNPs nanocomposites in comparison with untreated one, as presented in **Figure 4**. Also, histological assays showed important tissue regeneration responses in contact with nanocomposites, suggesting the myofibroblasts regeneration and accelerated healing processes compared to uncovered

Chemotherapy and radiation exert their effects by inhibiting tumor cell growth and by blocking tumor reformation. However, some cancer patients present tumor relapse due to cancer

**Figure 1.** UV-vis absorption spectra of silver nanoparticles (AgNPs), gold nanoparticles (AuNPs), and their corresponding nanomaterials formulated by AgNPs/CTS and AuNPs/CTS-GMA.

Is clear that there are more possibilities for NPs and natural polymers, here we have offered a slight landscape of that, additionally to Au and Ag different metals such as copper (Cu) also could be used in biomedical applications, but the noble metals are a field with an extensive list of contributions elsewhere [67].

**Figure 2.** Fibers of CTS with AuNPs and AgNP on the surface of the polymer.


*Article* **A Di**ff**erent Approach to Estimate Temperature-Dependent Thermal Properties of Metallic Materials**

#### **Luís Felipe dos Santos Carollo, Ana Lúcia Fernandes de Lima e Silva and Sandro Metrevelle Marcondes de Lima e Silva \***

Heat Transfer Laboratory—LabTC, Institute of Mechanical Engineering—IEM, Federal University of Itajubá—UNIFEI, Campus Prof. José Rodrigues Seabra, Av. BPS, 1303, Itajubá 37500-903, MG, Brazil **\*** Correspondence<EMAIL_ADDRESS>Tel.: +55-35-3629-1069

Received: 19 July 2019; Accepted: 10 August 2019; Published: 13 August 2019

**Abstract:** Thermal conductivity, λ, and volumetric heat capacity, ρ*cp*, variables that depend on temperature were simultaneously estimated in a diverse technique applied to AISI 1045 and AISI 304 samples. Two distinctive intensities of heat flux were imposed to provide a more accurate simultaneous estimation in the same experiment. A constant heat flux was imposed on the upper surface of the sample while the temperature was measured on the opposite insulated surface. The sensitivity coefficients were analyzed to provide the thermal property estimation. The Broydon-Fletcher-Goldfarb-Shanno (BFGS) optimization technique was applied to minimize an objective function. The squared difference objective function of the numerical and experimental temperatures was defined considering the error generated by the contact resistance. The temperature was numerically calculated by using the finite difference method. In addition, the reliability of the results was assured by an uncertainty analysis. Results showing a difference lower than 7% were obtained for λ and ρ*cp,* and the uncertainty values were above 5%.

**Keywords:** temperature-dependent thermal properties; simultaneous estimation; optimization; sensitivity coefficients; uncertainty analysis


The mean PL20 for all samples was even higher than for the 16S rDNA profiles at 92.2 ± 3.8. There were no statistically significant differences neither for the factor season nor the factor ozone (when analyzed for the summer sampling). The dissimilarity between ambient spring and summer samples (*t(*spring*/*summer*)* = 7*.*2 ± 1*.*6) did not differ significantly from the dissimilarity between summer and autumn (*t(*summer*/*autumn*)* = 7*.*0 ± 3*.*2). Additionally, dissimilarity between ambient and ozone treated summer samples was in the same range at *t(*ambient*/*ozone*)* = 7*.*5 ± 2*.*3 indicating no influence of any of the factors. These observations were verified with PerMANOVA and for all factors, season, and ozone, no statistically significant differences were observed.

## *Diversity of Actinobacteria harboring the PKS II gene*

Although no clear response of the Actinobacteria harboring the PKS II gene in response to the factors season and ozone was visible, a clone library was constructed to do a phylogenetic identification of major groups of Actinobacteria harboring the PKS II gene. Out of the 54 selected clones 51 sequences had polyketide type II KS-domains as closest hits when submitted to a database search (blastx). The remaining three did not show any homology to known proteins. The nucleic acid codes of all obtained sequences were translated into protein sequences and aligned. Upon translation it was discovered that two of the sequences included internal stop codons. These sequences were considered to be pseudogenes and were therefore excluded from the phylogenetic analysis (accession numbers: EU138920 and EU138948). A maximum-likelihood tree was calculated for the remaining clones and 32 reference protein sequences (**Figure 4**). References were obtained from GenBank database and included sequences of known actinobacterial PKS type II KS-domains. Two outgroups were included, *fabB* (beta-ketoacyl-ACP synthase I) from *Escherichia coli* involved in fatty acid synthesis and a PKS type II from *Photorhabdus luminescens* TTO1 (γ-Proteobacteria). When looking at the phylogenetic tree eleven groups could be differentiated on the basis of 95% similarity of the protein sequences. Sequences obtained from the clone library were very diverse as seen by the wide distribution of the different groups throughout the tree. Eight of the eleven groups clustered alone or close to known antibiotics producing PKS type II KS-domains (groups 1–7 and 11), while three groups clustered in close vicinity to known spore pigment producing KS-domains (groups 8, 9 and 10).

**protein sequences (185 amino acid positions) from cultured polyketide producers with known PKS Type II sequences (NCBI database) and sequences from beech rhizosphere clone library (accession numbers indicated).** Tree topology was supported by

of the reference PKS systems are given in parentheses. Groups are assigned on the basis of 95% similarity of the aminoacid sequences. The scale bar indicates 10% dissimilarity in aminoacid sequences.

## **DISCUSSION**

## **STRUCTURAL DIVERSITY OF ACTINOBACTERIAL COMMUNITIES IN THE RHIZOSPHERE OF BEECH**

Using T-RFLP analysis of 16S rRNA gene fragments as a culture independent method to monitor changes in the actinobacterial beech rhizosphere community, the overall variability observed between different samples was very low. For all major peaks detected, the differences observed were merely on the level of peak intensities and no differences could be seen based on the presence or absence of these peaks. It can therefore be concluded that none of the applied factors (O3 and season) had the capability of qualitatively changing the actinobacterial community concerning its major components and that the general influence of the factors applied could be considered small. Nevertheless quantitative differences between samples for some T-RFs could be observed and clearly assigned to the influence of certain factors.

The clearest separation of samples was detected to be caused by seasonal shifts. Unique profiles were observed for spring samples and the major T-RF 102 bp responsible for this separation could be assigned to represent genera from the suborder Catenulisporinae based on comparisons with the clone library. This suborder contains mycelium-forming Actinobacteria, which are globally distributed and often isolated from acidic soils (Busti et al., 2006a,b; Tamura et al., 2008). Interestingly, no study documenting details of their ecology has been published so far and therefore the seasonal variability and dominance of this group observed in this study is a first indication of their active role for nutrient mobilization in forest soils. However, since the same peak was observed for two different genera (e.g., *Catenulispora* and *Actinospica* for T-RF 102 bp) the observed shifts cannot be assigned to a certain species. The phenomenon that several species or even genera are represented by the same peak in T-RFLP and other fingerprinting techniques has been reported in many studies (e.g., Smalla et al., 2007), thus interpretations of the results have to be done in general cautiously.

The drastic increase of T-RF 102 bp from spring to summer was statistically significant and similar for ozone treated and control plants. Seasonal shifts of microbial rhizosphere communities have been demonstrated in several studies (Smalla et al., 2001; Thirup et al., 2001) and in some cases these responses were associated specifically with Actinobacteria. Smalla et al. (2001) recorded a strong seasonal shift at the beginning of the vegetation period for rhizosphere communities of strawberries, oil seed rape and potato plants. Based on DGGE analysis of the rhizosphere communities they found indications that the abundances of bacterial high G + C populations were different during the developmental stages of all plants studied. Successional changes have also been described for plant associated Actinobacteria based on CFU counts and quantitative PCR methods (Thirup et al., 2001). The authors could show that at later time points in the season the abundance of Actinobacteria in the vicinity of barley roots increased significantly. They concluded that Actinobacteria are persistent during microbial succession beyond the early stages of root growth in annual plants due to their capability to penetrate and solubilize dead root litter (Thirup et al., 2001). Additionally, the active role of Actinobacteria in rhizospheres of diverse plants has been demonstrated in numerous studies (Billings and Ziegler, 2005). In the case of European beech the process of decomposition of old roots is likely to be more constant throughout the growing season compared to annual plants. The seasonal effect observed can thus not be assigned to increasing decomposition processes throughout the year. Additionally, the T-RF in question (T-RF 102 bp) peaked in summer and exhibited a slight decrease toward the end of the growing season. If this part of the bacterial population was mostly dependent on dead root litter as opposed to root exudates, it would rather be expected to peak in autumn, when the balance between new fine root production and older dead roots is largely in favor of the latter (Hertel and Leuschner, 2002).

A second statistically significant separation was observed in summer, where ozone samples showed unique profiles due to the relative reduction of one major T-RF (579 bp). Yet, the effect was very subtle and in this case transient, since autumn harvest of ambient and O3 treatments could not be differentiated any more. Clones from the library possibly representing this peak could not be identified by the RDP classifier. Therefore, the identity and ecology of this putative O3 responsive group would be of great interest and efforts are being made to isolate corresponding organisms from the studied soil. Only then, their functionality within the soil and possible implications on suppression of soil borne diseases can be investigated.

Responses of the microbial community to elevation of tropospheric ozone have been shown in other studies, but as observed in this study, the effects were relatively small in most cases. Dohrmann and Tebbe (2005) reported that from neither general Bacteria nor group specific SSCP profiles an ozone effect could be seen for the rhizosphere communities of different ozonesensitive and insensitive herbaceous plants. The only exception in this study was the ozone sensitive composite *Sonchus asper* L., where changes were observed exclusively for the Actinobacteria specific profiles under elevated O3. In other studies effects on the fungal rhizosphere community were shown. Chung et al. (2006) demonstrated that elevation of O3 significantly altered the fungal community composition in a free-air enrichment experiment under three deciduous tree species utilizing DGGE. They also observed an increase in fungal relative abundance by PLFA analysis. In contrast to these result, Phillips et al. (2002) observed a decrease in fungal PLFAs while the relative proportion of Gram-positive and Gram-negative indicator PLFAs were not affected.

## **PKS TYPE II**

This is the first study performed to investigate the effect of elevated ozone on genes potentially responsible for antibiotics production in the rhizosphere. PKS type II systems are of great interest when investigating the antibiotics production potential of actinobacterial soil populations (Wawrik et al., 2007). This especially holds true since a large portion of the actinobacterial community in the studied soil belongs to the newly described suborder Catenulisporinae. For this new lineage of Actinobacteria, Busti et al. (2006a,b) described a high potential to produce secondary metabolites with a polyketide scaffold. This is a feature they share with members of Streptomyces and related genera. All strains analyzed by Busti et al. (2006a,b) belonging to this group yielded distinct bands when checked with specific primers for PKS type I and II. For one of the strains, the production of a bioactive molecule similar to the well-studied antibiotic actinorhodin was demonstrated. This antibiotic is synthesized by a PKS type II system (Hopwood, 1997). However, when analyzing the diversity of PKS type II genes, an effect was discovered for neither season, nor an influence of the ozone treatment.

Since PKS type II genes do not follow the trend observed for 16S rRNA genes, it can be concluded that phylotypes responsible for the observed changes (e.g., T-RF 102 bp) do either not contain similar PKS genes or they do not possess PKS type II genes at all, which is very unlikely for members of the Catenulisporinae but cannot be excluded. Anyhow it can be stated that in this case no correlation could be seen between phylogenetic trends and the genotypical trait PKS type II. This is in line with the findings of Metsä-Ketelä et al. (2002) who observed that the phylogenies of 16S rRNA genes and PKS genes in Actinobacteria soil isolates were not congruent. They concluded therefore that the phylogenetic grouping of Actinobacteria is an inadequate predictor for the type of secondary metabolites they produce.

However, another explanation for a lack of a significant separation of PKS type II genes might be the high background noise of these fingerprints.. Almost 10% of all clones in the PKS type II clone library have to be considered "junk" DNA (e.g., pseudogenes and sequences with no homology). Their distribution is likely to be random since no selective pressure takes effect on them. Previous studies using this primer pair used presence-absence data rather than relative peak intensities (Wawrik et al., 2005). Yet, it is very likely that this will only increase the influence of minor peaks, since they would have the same impact on multivariate statistical analysis as major, well-reproducible peaks. Furthermore, an additional nine sequences (16.7%) were closely related to spore pigment producing genes. The necessity to differentiate between antibiotics and spore pigments producing PKS systems has been stressed by Metsä-Ketelä et al. (1999) due to different ecological functions of the resulting molecules. While the property of a molecule to act as a pigment does not yield any information about its chemical properties concerning bioactivity, a major difference is that spore pigments are presumably covalently attached to macromolecular components of spores (Lee et al., 2005). Furthermore precursors of pigments e.g., monomers could well act as antibiotics or have other functions, while their massive production leads to incorporation in pigment macromolecules (Kämpfer, 2006). Therefore, they might act as protective against grazing by microfauna of the soil, but are not very likely to be involved in antibiosis against competing microorganisms.

## **CONCLUSION**

On the basis of the performed analysis, the diversity of Actinobacteria possessing type II PKS genes was only slightly affected by seasonal changes. The applied ozone treatment did not have any effect on the distribution of these genes in the rhizosphere of beeches, although the ecophysiology of the plant was changed in response to the increased ozone levels in the atmosphere. Also the dynamics of the total phylum of Actinobacteria which were monitored in the presence study based on 16S rRNA gene fingerprints, was lower toward changes in the rhizosphere compared to other phylogenetic groups of bacteria, which showed significant changes in response to changes in the plant performance (e.g., bacteria of the genus Pseudomonas). This the large flexibility of Actinobacteria might be related their relatively large genome size and the possibility of this group of bacteria maybe to adapt faster and better to changing environmental conditions. Vice versa for the plant this indicates a huge stability of one important functional group of bacteria at the plant soil interface even if plant performance is changed. However, it has to be kept in mind, that changes were analyzed exclusively on DNA and not RNA levels. A transcriptomic approach might therefore yield further insights into the active part of the actinobacterial population. Furthermore the observed response pattern of Actinobacteria might change if beech trees of different age classes are studied or other stressors are investigated like increased drought periods or lack of nutrients.

## **AUTHOR CONTRIBUTION**

Felix Haesler, Alexandra Hagn, Marion Engel, and Michael Schloter were involved in the development of this study, in the planning of the experimental setup, data analysis and manuscript writing. Felix Haesler performed all experiments described in this study.

## **ACKNOWLEDGMENTS**

This study has been supported by the Deutsche Forschungsgemeinschaft (SFB 607 – Growth and Parasite Defense)

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**Conflict of Interest Statement:** The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

*Received: 08 November 2013; accepted: 20 January 2014; published online: 11 February 2014.*

*Citation: Haesler F, Hagn A, Engel M and Schloter M (2014) Impact of elevated atmospheric O*3 *on the actinobacterial community structure and function in the rhizosphere of European beech (Fagus sylvatica L.). Front. Microbiol. 5:36. doi: 10.3389/fmicb. 2014.00036*

*This article was submitted to Plant-Microbe Interaction, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Haesler, Hagn, Engel and Schloter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

**PERSPECTIVE ARTICLE** published: 03 December 2013 doi: 10.3389/fmicb.2013.00355

## Bespoke microbiome therapy to manage plant diseases

## *Murali Gopal\*, Alka Gupta and George V. Thomas*

*Microbiology Section, Central Plantation Crops Research Institute, Kudlu, Kasaragod, India*

## *Edited by:*

*Gabriele Berg, Graz University of Technology, Austria*

#### *Reviewed by:*

*Gabriele Berg, Graz University of Technology, Austria Henry Mueller, Graz University of Technology, Austria*

#### *\*Correspondence:*

*Murali Gopal, Microbiology Section, Central Plantation Crops Research Institute, Kudlu, Kasaragod 671 124, India e-mail<EMAIL_ADDRESS>
Information gathered with advanced nucleotide sequencing technologies, small molecule detection systems and computational biology is revealing that a community of microbes and their genes, now termed "the microbiome," located in gut and rhizosphere, is responsible for maintaining the health of human beings and plants, respectively. Within the complete microbiome a "core-microbiome" exists that plays the pivotal role in well being of humans and plants. Recent studies in medicine have shown that an artificial mixture of bacteria representing the core gut microbiome of healthy person when transferred into gut of diseased person results in re-establishment of normal microflora in the latter leading to alleviation from diseased condition. In agriculture, though not exactly in similar manner as in medicine, success in plant disease management has been achieved through transfer of microbiome by mixing disease suppressive soils with disease conducive soils. A study more similar to artificial gut microbiome transfer in medical field has been recently reported in agriculture, in which transfer of microbiome via soil solutions (filtered and unfiltered) has shown ability to alleviate drought stress in *Arabidopsis thaliana.* However, the exact practice of transferring artificially cultivated core-microbiome as in medicine has not thus far been attempted in plant disease management. Nonetheless, as the gut and rhizosphere microbiome are known to share many common traits, there exists a good scope for accomplishing similar studies in agriculture. Based upon the information drawn from all recent works in microbiome studies of gut and rhizosphere, we propose that tailor-made core-microbiome transfer therapy can be a success in agriculture too and it could become a viable strategy for management of plant diseases in future.

**Keywords: microbiome, core-microbiome, plant, disease management, soil, rhizosphere, root, gut**

## **MICROBIOME IN RELATION TO HUMAN AND PLANT HEALTH**

The power of next generation sequencing technology is transforming today's biology (Mardis, 2008; Schuster, 2008). Combined with bioinformatics (Lee et al., 2012), it is prising open the microbial "dark matter" and revealing the diversity and functions of microbiome at resolutions unknown hitherto (Forde and O'Toole, 2013; Rinke et al., 2013). It is shedding new light on the role played by the gut microbiome in governing the human health (Turnbaugh et al., 2007; Kinross et al., 2011; Cho and Blaser, 2012; Ottman et al., 2012; Norris et al., 2013), reviving the Metchnikoffian paradigm: colonizing the gut with beneficial microflora could lengthen the human life. The gut microbiota is not only limiting its influence on the human health by its functions in the intestine, it also is impacting the human brain and behavior (Heitz et al., 2011; Cryan and Dinan, 2012; Mulle et al., 2013) as well as social development evidenced by studies in mice (Desbonnet et al., 2013). Remarkably, similar train of evidences is being uncovered in plant world; root microbiome is observed to be tightly linked with the health of the plants (Friesen et al., 2011; Chaparro et al., 2012; Bulgarelli et al., 2013; Gaiero et al., 2013; Mendes et al., 2013). In insects, too, the same story is unfolding (Engel and Moran, 2013). The microbial diversity associated within these ecosystems is being referred to as the "second genome" that is easily 10 times more in scale than the host genome (Grice and Segre, 2012; Turner et al., 2013) and its impact on regulating human and plant health is becoming more apparent.

## **CORE MICROBIOME**

From among the multitude microbial communities inhabiting the gut and root, there appears to be a clutch of them which constitute the core microbiome (Tschöp et al., 2009). Core microbome contains organisms common across the microbiome hypothesized to play a key role in ecosystem function within a habitat (Lederberg and McCray, 2001). Core microbiome of human gut (Turnbaugh and Gordon, 2009; Turnbaugh et al., 2009; Huse et al., 2012; Petrof et al., 2013a,b) and plant (Bulgarelli et al., 2012; Lundberg et al., 2012; Peiffer and Ley, 2013) have been determined at Operational Taxonomic Unit (OTU) levels with small subunit ribosomal RNA genes or random sequencing of all genes. Any changes in the core-microbiome composition or function leads to debilitative or destructive diseases in humans as well as plants (Kinross et al., 2011).

## **DISEASE SUPPRESSIVE SOILS AND THEIR MICROBIOME**

It is well known that farmers moved soil from one field to another to take advantage of its disease suppression abilities endowed by the soil microbial populations harbored in it (Weller et al., 2002). Soil microbial studies mainly based on cultivation dependent methods lead to the finding of several bacteria termed plant growth promoting rhizobacteria (PGPR) (Kloepper and Schroth, 1978), particularly the genus *Pseudomonas* spp., in imparting the disease suppressive ability to such soils (Schroth and Hancock, 1982; Haas and Defago, 2005; Mendes et al., 2013). Today, with advanced technologies, studies are generating evidence that it is not an individual or couple of microbes, rather it is "microbiome" (Forde and O'Toole, 2013; Rinke et al., 2013), the complete assemblage of microbial communities of a habitat and their functions, in rhizosphere that is determining plant health (Berendsen et al., 2012; Mendes et al., 2013; Rout and Southworth, 2013). In insects too, the same phenomenon is being observed (Hussa and Goodrich-Blair, 2013).

## **ROOT MICROBIOME TRANSFER TO MANAGE PLANT DISEASE**

In plant disease management, a simple method of transferring complete microbiome by mixing disease suppressive soils with disease conducive one is practiced. Mendes and colleagues (2011) showed that when soils suppressive to *Rhizoctonia solani*, an important fungal pathogen, is mixed with disease conducive soils at 1:9 ratio (w/w), it successfully suppressed the infection in sugar beet. Metagenomic analysis of the soils using PhyloChip revealed consistent involvement of 17 bacterial communities belonging to Proteobacteria, Firmicutes, and Actinobacteria, considered as core-microbiome, in disease suppression. Other works too, similarly point to the involvement of core-microbiome in soils suppressive to potato common scab (Rosenzweig et al., 2012) and tobacco black root rot (Kyselkova et al., 2009). In all the above mentioned works, Pseudomonadaceae group of bacteria has been suggested as a key player in disease suppressiveness within the core microbiome.

## **GUT MICROBIOME TRANSFER TO MANAGE HUMAN HEALTH**

As with rhizosphere microbiome of plants in agriculture, in medical studies too, the gut microbiome has been found to control the health of the human beings (Turnbaugh et al., 2007; Cho and Blaser, 2012) with a core mainly involved (Tschöp et al., 2009; Turnbaugh et al., 2009; Huse et al., 2012). "Stool transplant" therapy (de Vos, 2013) is one of the several medical practices that is adopted wherein stool taken from healthy person is transferred to diseased person resulting in suppression of many important gastro-intestinal diseases. The principle here is to re-establish normal gut bacteria in the gut of diseased person and bring about positive changes in their health. However, the "stool transplant" therapy is not widely followed since the method is not acceptable to many patients, besides the apprehension that it can transfer pathogenic microbes too. Two recent studies (Petrof et al., 2013a; Ridaura et al., 2013) have found a way to overcome "stool transplant therapy" by using "stool substitute" in which a culturable consortium representing core microbiome is transferred and found to transmit the phenotype expression aimed for. Petrof and colleagues' (2013a) work was first of its sort successfully demonstrating that patients suffering from *Clostridium difficile* infection, a debilitative disease of intestine, can be cured when administered with stool substitute mixture comprising a multi-species community of bacteria (RePOOpulate sample) of a healthy individual exhibiting resistance to the disease. Post-treatment metagenomic analysis of the cured patients revealed that the OTU reads from their guts were similar to that of the RePOOPulate sample until six months after its administration even though the microbiota profiles were different. This work was quickly followed by Ridaura et al. (2013) in which they transplanted intact uncultured or cultured human fecal microbiota from each member of a discordant twin pair (one lean and other obese) into separate groups of recipient germ-free mice and found that the obese twin's fecal microbiota significantly increased the body biomass and adiposity in the germ free mice. It will not, therefore be, contrary to consider that the "stool substitute" transfer consisting of the core-microbiome is an extension of the "stool therapy" and is able to reproduce the expected microbial ecology with desired results. Such successful scientific endeavors are spurring development of new disease management paradigm termed MET: Microbial Ecosystem Therapy (Petrof et al., 2013b).

## **ROOT CAN FOLLOW THE GUT**

In agriculture, a system of manipulating the root environment by artificially inoculating plant and soil beneficial microbes has been followed for long time for improving crop yields. The PGPRs and other plant beneficial microbes (nitrogen fixing and phosphate solubilizing bacteria, *Trichoderma* spp., arbuscular mycorrhizae fungi etc.) isolated from rhizospheres were mass multiplied and artificially inoculated, either singly or in combination of twos, for disease management in plants (Berg, 2009; Lugtenberg and Kamilova, 2009; Chaparro et al., 2012; Qiu et al., 2013). Though this approach has been widely adopted, its success in field conditions have been limited (Bakker et al., 2012). With unequivocal reports coming out indicating that it is not a single taxon, but a consortium of microorganism that is responsible for bringing about diseases suppression in plants (Mendes et al., 2011; Rosenzweig et al., 2012; Trivedi et al., 2012; Klein et al., 2013), the stage is now set for the root to follow gut by adopting the strategy of using "stool substitute" for disease management. Transferring disease suppressive soils has been the only alternate method for transferring the complete rhizosphere/core microbiome in plant protection strategy. Coming closer to "stool substitute" therapy, transfer of microbiome via soil solutions (filtered and unfiltered) has shown ability to alleviate drought stress in *Arabidopsis thaliana*. Pyrosequencing analysis of soils revealed a core microbiome (*Burkholderia, Phormidium, Bacillus, Aminobacter, Acidiphilum* among others) involved in alleviating the abiotic stress (Zolla et al., 2013). However, the exact transfer of artificially cultivated core-microbiome as performed by Petrof et al. (2013a) and Ridaura et al. (2013) with gut environment is yet to be attempted in root environment. The possibilities of achieving success is high as there exists a striking similarity between the gut and root microbiota (Berendsen et al., 2012; Ramírez-Puebla et al., 2013). Also, the fact that soil type plays significant role in the selection and election of microbiome of rhizosphere and root compartment (Berg and Smalla, 2009; Bulgarelli et al., 2012; Lundberg et al., 2012), chances of success for the "rhizosphere substitute" is significantly augmented.

## **ROOT MICROBIOME CULTURING**

In an important meeting convened on topic "culturing a plantmicrobiome community" in Rhodes, Greece in 2012, a long-term future research strategy became apparent in which it was suggested that after an initial culture-independent survey of the plant microbiota, the corresponding community members are isolated in collections of pure cultures (Lebeis et al., 2012). Today, by converging information deduced on microbial diversity and functions using next-generation sequencing technologies and multi-species transcriptome analysis (Schenk et al., 2012), molecules/volatile involved in plant-microbe interaction using mass-spectral investigations (Watrous et al., 2012; Badri et al., 2013) combined with power of bioinformatics (Lee et al., 2012), it has become very much possible to culture the appropriate coremicrobiome and apply it successfully (Ridaura et al., 2013). To assemble a robust core microbiome of an ecosystem not limited to OTU records alone, Shade and Handelsman (2012) and Lozupone and colleagues' (2012) suggested collecting the data on (i) OTU membership/α diversity, (ii) OTU composition/β diversity, (iii) OTU persistence across time and space and (iv) communication/metabolic networking among the OTUs. Determination of models, particularly of root environment, in which the plants favor the recruitment of antibiotic-producing (and -resistant) bacteria by stimulating interference competition through production of abundant resources, can help improve establishment of the artificially introduced microbiomes(Scheuring and Yu, 2013).

## **BESPOKE MICROBIOME THERAPY FOR PLANT DISEASE MANAGEMENT**

Artificial core-microbiome transfers can decrease the noise intrinsic to any complex communities and are step in right direction in disease management, both for plants and humans, built upon the principles of binary plant/human-microbe interaction in an ecological perspective.

The similarities between the gut and rhizosphere microbiota is striking in many aspects which can encourage emulating experiments carried out in gut with root environment and vice-versa. Based on the increasingly available body of evidences discussed in this article, we propose the model of transfer of bespoke coremicrobiome, rather than individual species, as a viable strategy for management of plant diseases in future.

## **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 October 2013; accepted: 08 November 2013; published online: 03 December 2013.*

*Citation: Gopal M, Gupta A and Thomas GV (2013) Bespoke microbiome therapy to manage plant diseases. Front. Microbiol. 4:355. doi: 10.3389/fmicb.2013.00355*

*This article was submitted to Plant-Microbe Interaction, a section of the journal Frontiers in Microbiology.*

*Copyright © 2013 Gopal, Gupta and Thomas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Beneficial effects of plant-associated microbes on indoor microbiomes and human health?

#### *Gabriele Berg1 \*, Alexander Mahnert 1 and Christine Moissl-Eichinger 2*

*1 Institute of Environmental Biotechnology, Graz University of Technology, Graz, Austria*

*2 Institute for Microbiology and Archaea Center, University of Regensburg, Regensburg, Germany*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Martin Grube, Karl-Franzens-University Graz, Austria*

#### *Reviewed by:*

*Parag Vaishampayan, California Institute of Technology, USA*

**Keywords: interplay of plant -and indoor microbiomes, management of beneficial microbes, next-generation sequencing, houseplants, omicstechnologies**

## **PLANT MICROBIOMES—AN INTRODUCTION**

Just like humans, plants have recently been recognized as meta-organisms, possessing a distinct microbiome and revealing close symbiotic relationships with their associated microorganisms (Berg et al., 2013; Mendes et al., 2013). Each plant harbor specific species to a certain degree but also cosmopolitan and ubiquitous microbial strains; the majority of them fulfill important host as well as ecosystem functions (rev. in Berg and Smalla, 2009). In addition to the microbe-rich rhizosphere, which has been studied extensively, the phyllosphere is of special interest for the study of indoor microbiomes due to its large and exposed surface area and its remarkable microbial diversity (Lindow and Leveau, 2002; Lindow and Brandl, 2003; Redford et al., 2010; Meyer and Leveau, 2012; Vorholt, 2012; Rastogi et al., 2013). In addition to the majority of beneficial and neutral inhabitants, all plantassociated microbiomes contain plant as well as human pathogens (Berg et al., 2005; Mendes et al., 2013). A broad spectrum of plant pathogens is well-known from disease outbreaks. Human pathogens belong mainly to the so called opportunistic or facultative human pathogens such as *Burkholderia cepacia*, *Pseudomonas aeruginosa* or *Stenotrophomonas maltophilia*, which cause diseases only in patients with predisposition or in hospital (Berg et al., 2005; Ryan et al., 2009).

Microbiomes of humans and plants are currently intensively studied using the same methods and addressing similar scientific questions (Ramírez-Puebla et al., 2013). However, knowledge about the microbiomes' interaction, microbial dynamics and exchange in a certain biotope or even indoor environment is very much limited. Although the composition and function of plant microbiomes is well-studied, there is still little to no information regarding their overlap, interaction with -and impact on other microbiomes or the microbiomeharboring hosts. Information is available about the connection of soil and rhizosphere microbial diversity, which share a selective sub-set (Smalla et al., 2001). The root-soil interface is the selection site for plant-associated bacteria by root exudates, which acts as chemo-attractants as well as repellents to which bacteria respond (Badri and Vivanco, 2009). In addition, plant defense signaling play a role in this process (Doornbos et al., 2012). For the phyllosphere we know that there is only a part of residents, while a substantial part of bacteria is shared with the air microbiome (Lindow and Brandl, 2003). Based on these data, a strong interaction and exchange of rhizosphere and phyllosphere microbiomes with other microbiomes is obvious. However, this opinion paper focuses on the question, if there is also a connection from plant–to indoor microbiomes as well as an impact on human health.

## **INDOOR MICROBIOMES— IMPORTANCE AND ORIGIN**

Despite the fact that the majority of our lifetime is spent in indoor environments such as home, work place, or public buildings, our knowledge of microbial diversity inside buildings is limited. We are not alone in these indoor environments: they provide new habitats and residence to numerous microbial communities comprising possibly hundreds of individual bacterial, archaeal and fungal species including diverse viruses. Recent studies analyzed potentially pathogenic and allergenic indoor microorganisms with mainly cultivation-based methods (Täubel et al., 2009; Yamamoto et al., 2011). Since the fraction of cultivable microbes on one specific medium is extremely low, information about specifically-adapted microorganisms, or those with special needs, remains inaccessible by standard cultivation assays. Recently, however, the application of molecular methods, including next generation sequencing (NGS) techniques has provided new insights into indoor microbial communities, revealing a generally high prokaryotic diversity including diverse bacterial, archaeal and fungal phyla (Flores et al., 2011, 2013; Moissl-Eichinger, 2011; Hewitt et al., 2012, 2013; Kembel et al., 2012; Dunn et al., 2013; Kelley and Gilbert, 2013; Meadow et al., 2013).

Indoor microbial communities are an important component of everyday human health (Arundel et al., 1986; Lee et al., 2007; Kembel et al., 2012). Due to human activity and high emission rate of up to 106 bacteria per person-hour as measured via 16S rRNA gene quantification from aerosols (Qian et al., 2012), indoor environments are strongly influenced by typically human-associated bacteria (Fierer et al., 2008). Hence, built environments like hospitals are more easily colonized to a large extent by patient-associated microbes (Oberauner et al., 2013). As a result, many patients in hospitals and especially in intensive care units (ICUs) develop hospitalacquired "nosocomial infections" that compound their underlying severe disease (Vincent et al., 1995; Plowman, 2000). Moreover, these nosocomial infections remain among the leading causes of death in developed country hospitals. The risk to get nosocomial infections for patients in European ICUs was reported as 45% (Plowman, 2000). Hospital surfaces are often overlooked reservoirs for these bacteria (Hota, 2004; Gastmeier et al., 2005; Kramer et al., 2006). Apart from hospitals, indoor microorganisms affect human health as allergenic agents as well (Hanski et al., 2012). Indoor microorganisms are also involved in the development of the Sick Building Syndrome (SBS), which causes symptoms such as sensory irritation of the eyes, nose, and throat, neurotoxic or general health problems, skin irritation, non-specific hypersensitivity reactions, and odor and taste sensations (Godish, 2001).

Indoor microbiomes originate primarily from human skin, pets, or the outside air (Flores et al., 2011; Kembel et al., 2012; Meadow et al., 2013). Plants as a source of indoor microbes are so far less considered. However, air-borne microbes as substantial part—bacteria, fungi or microscopic algae—are scattered and can travel long distances such as in the wind or in clouds before returning to ground-level (Hamilton and Lenton, 1998). They have received more attention because they can serve as nuclei for condensation and as such influence our world climate as rainmaking bacteria. Interestingly, cloud and hailstone studies indicated plant-surface bacteria as the dominant source of these rain-making microbes (Morris et al., 2008; Šantl-Temkiv et al., 2013). In addition, little is known about the impact of houseplants and its microbes, although older studies indicate indoor plants as important source (Burge et al., 1982).

Comparing indoor with plant microbiomes, it is our opinion that both outside and inside plants are of importance for our indoor microbiome. Plants provide beneficial bacteria for indoor rooms and therefore can positively influence human health. The following facts support our opinion about the importance of plants as source for a beneficial microbial biodiversity:

highlights the beneficial balance, which is necessary between microorganisms and hosts.

5. Interestingly, Thaumarchaeota, originally described to be associated with ammonia-oxidation in soil and the rhizosphere of plants, have been found on human skin (Probst et al., 2013). Currently it is unknown, whether the human skin archaea have positive or negative effect on human health and whether they have different genomic capabilities compared to their soilrelatives. However, it becomes clear, that closely related microorganisms can exist in different microbiomes, based on a dynamic exchange or distribution and subsequent development of adaptation strategies.

Based on these facts, we speculate the following:

Enclosed environments and their microbiomes—like private/public buildings, hospitals, and clean rooms, which are more or less separated from outside, are especially shaped by human influence and human associated microbes (Hospodsky et al., 2012; Dunn et al., 2013). Hence, microbial diversity is altered and partially reduced compared to the outdoor environment. A reduction in microbial diversity is well known to facilitate dominant proliferations of certain strains, which might bear the risk to have a negative effect toward our health. To increase microbial diversity in an indoor environment we could simply open our windows instead of using air-condition (Hanski et al., 2012; Kembel et al., 2012; Meadow et al., 2013). Alternatively, we could use potted houseplants in built environments as a source of microbial biodiversity and possibly beneficial microorganisms.

Microbes, which live in close vicinity to human beings, are adapted to us as symbionts, commensals, or pathogens, whereas these life-styles are changeable dependent on the host-microbe balance. Indoors we share these microbes, which might get deposited on various surfaces by one person and afterwards get collected by another. Human-associated microbes e.g., skin associated, are confronted with totally new biotic and abiotic factors in the built environment. Here they have to adapt to new surface materials, compete with

houseplants and animals from plant microbiomes, presents an overview on typical and dominant bacterial groups occurring in the built environment. Schematic chart represents occurrence of the bacterial inhabitants indoors.

environments (red squares). Taxa highlighted in green are typical phyla detectable in plant microbiomes. This image has no demand of being complete.

others for scarce nutrients and withstand stresses associated to cleaning reagents etc. However, in the case of houseplants we allow them to proliferate in a protected environment. Plant associated microbes stay on the leave or stem surface, where they have adapted to and are sheltered from cleaning procedures. Although these phyllosphere communities are confronted with an absence of direct sun light and rain as well as other changed meteorological parameters like air/dust turbulences, their rhizosphere and surrounding soil communities stay in their natural habitat. Hence, these well balanced plant communities, which we bring inside, have the potential to balance an indoor microbiome, by increasing its diversity and filter airborne microbes.

## **CONCLUSION**

Members of the plant microbiome are an important source for indoor microbiomes. Both, plants from inside and outside can contribute to the microflora. Plant-associated bacteria could act as counterparts against pathogens within the microbial ecosystems. They stabilize the ecosystem, enhance biodiversity and avoid outbreaks of pathogens. However, more research is necessary to understand the microbiology of indoor environments. Currently used cleaning and hygiene strategies in built environments especially in hospitals and ICUs often promote multi-resistant pathogens instead of supporting beneficials. In future, it is important to re-think our understanding of necessary sterility and our relationship to our surrounding microbiomes. This "paradigm shift in ecology" is not only required for plants, humans (Jones, 2013) but also for our environment. Fortunately, "omics"-technologies guided by next-generation sequencing and microscopic techniques allow us now a much better assessment of them. Moreover, we can develop management strategies for beneficial interactions.

## **REFERENCES**

their survival time in intensive care units. *J. Hosp. Infect.* 62, 181186. doi: 10.1016/j.jhin.2005.08.010 Godish, T. (2001). *Indoor Environmental Quality.* Boca

*Indoor Air* 21, 521–530. doi: 10.1111/j.1600- 0668.2011.00732.x

*Received: 27 September 2013; accepted: 10 January 2014; published online: 29 January 2014.*

*Citation: Berg G, Mahnert A and Moissl-Eichinger C (2014) Beneficial effects of plant-associated microbes on indoor microbiomes and human health? Front. Microbiol. 5:15. doi: 10.3389/fmicb.2014.00015*

*This article was submitted to Plant-Microbe Interaction, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Berg, Mahnert and Moissl-Eichinger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*