CHAPTER 5 DISCUSSION

Microorganisms associated with the rhizoplane of rock-dwelling plants have been reported for their rock-weathering potential (Leyval and Bethelin 1991; Puente et al. 2009). Knowledge on rock-weathering bacteria will help in understanding their microbial diversity, functional potential and mechanisms that mediate bioweathering of rocks and genesis of soil. In the present investigation, rhizoplane bacteria isolated from three different plant species such as Barleria acuminata Nees., Ficus nervosa Heyne ex Roth. and F. mollis Vahl. growing within the rocks lacking visible soil were studied for their functional potential. These rhizoplane bacteria that exhibited rock-weathering potential belonged to various genera such as Enterobacter, Burkholderia, Pseudomonas, Cronobacter and Klebsiella. All the bacteria reported in this study exhibited rockweathering potential. This result was in correlation with the reports of Puente et al. (2009) and Uroz et al. (2011) who documented the ability of bacteria such as Klebsiella sp., Acinetobacter sp., Pseudomonas sp., Bacillus sp., Burkholderia and Staphylococcus sp. to weather an array of rocks and minerals such as granite, basalt, perlite and biotite. However, in this study for the first time, we identified the strains of Enterobacter had the potential to weather different types of rocks such as igneous (granite), sedimentary (limestone) and metamorphic rocks (marble).

Present investigation on rock weathering bacteria has 2 important implications such as pedogenesis and CO2 sequestration. Soil production from rock weathering is a vital process that supports life forms on the earth. Weathering in nature occurs by two processes namely physical and chemical weathering. These two processes go hand in hand complementing 98

each other resulting in soil formation and maintenance of soil productivity. Also, soil is removed by various agents of erosion. In nature a dynamic balance exits between soil formation by weathering and its removal by erosion to maintain a layer of regolith (Dixon and von Blanckenburg 2012), which in turn is essential to support life forms on earth. In natural environments, weathering is accelerated by microbes because they inhabit a wide range of surface and subsurface environments and influence various mineral transformation reactions which need to be elucidated further.

The inorganic carbon stored in soils is known to be in the range of 780 and 940 Gt (Dart et al. 2007) and thus constitutes to around one third of the total terrestrial carbon pool (Aure´lie Violette et al. 2010). The weathering of silicate rocks has an impact on global environmental changes. During silicate weathering reactions, CO2 is consumed leading to the sequestration of atmospheric CO2 on a long term (Cockell et al. 2011). Thus, weathering of silicate minerals, acts as an irreversible CO2 sink, a process which is accelerated naturally by microbes. Microbe induced effects on bioremediation of polluted sites, ore recovery from mines are well characterized.

Many studies have documented microbial weathering and their diversity in temperate and frigid zones (Uroz et al. 2011; Cockell et al. 2009; Frey et al. 2010). However, it should be noted that tropical regions are the places where soil production and removal are considered to occur at a faster pace. The type and nature of vegetation and microbes inhabiting rock environments may vary and needs to be addressed. In the present study, the role of microbes on different rock types such as felsic granulite, gabbro and biotite granite has been reported. Techniques such as XRD, particle size analyzer, SEM-EDAX and ICP-MS have been successfully employed to study weathering of rocks by bacteria.

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Further, in the present investigation, under in vitro conditions all the chosen bacteria grew readily in the presence of rock powders as a trace nutrient source. XRD analysis revealed a decrease in the concentration of microcline, albite and removal of biotite from biotite granite and removal of almandine garnet and reduction in albite and orthoclase from felsic granulite, while a change in crystal lattice of different minerals is observed in the case of gabbro, due to bacterial action. The SEM-EDAX image analysis also confirms the above observation. As felsic granulite and biotite granite contains more quartz compared to gabbro, the reduction in grain size is more pronounced in gabbro than that of biotite granite and felsic granulite. This may be explained by the inert nature of quartz to bacterial action and since it does not contain any essential elements required for bacterial and plant growth. The elemental mobility is significant in the case of biotite granite and felsic granulite compared to gabbro analyzed in this study which is in accordance with the earlier reports for granite and found to depend on the mineral composition and abundance (Frey et al. 2010; Welch and Mc phail 2003; Song et al. 2010). In the case of gabbro, the percentage of major elements is relatively high and hence, relative percentage reduction due to bacterial action is not visible. Also, the abundance of nutrients would have probably resulted in secondary precipitations of the released elements as evidenced by the acicular and feathery structures observed in SEM-EDAX.

Results achieved in the present investigation revealed a new insight to the importance of microbes in soil production and elemental mobility suggesting that microbes have a direct effect on rock weathering leading to mineral transformations. In nutrient poor environment, microbes with rock weathering ability play a dominant role in plant nutrition.

Rhizoplane bacteria reported in this study exhibited an array of plant growth-promoting traits. A total of 21 bacteria were found to be phosphate solubilizers. Phosphate

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solubilization is an important trait of bacteria inhabiting rocks as this helps them to solubilize phosphate to meet out their nutrient requirement as well as that of the associated plants (Kim et al. 1997). Moreover, inorganic phosphate solubilization has been reported to be associated with the uptake of metals (Jiang et al. 2008).

Rhizoplane bacteria that produce siderophores included strains belonging to different genera such as Enterobacter, Burkholderia, Pseudomonas and Cronobacter. Earlier studies suggested that siderophores play a significant role in bioweathering of rocks by chelating various divalent and trivalent cations and are found to increase the solubilization of ironcontaining minerals such as olivine, goethite and glauconite (Page and Huyer 1984; Kalinowski et al. 2000; Liermann et al. 2000). Siderophores also play a key role in the regulation of auxin level in plants growing in metal contaminated sites. Siderophores complex with toxic metals, decreasing concentration of free metals, thereby attenuating metal inhibiton of auxin synthesis (Dimkpa et al. 2008). Production of siderophores is another important mechanism adapted by bacteria to suppress pathogenic microbes. The role of siderophore and HCN in microbial antagonism has been documented in earlier studies (Ajay Kumar et al. 2012; Costa and Loper 1994). In the present study, production of HCN by strains of Enterobacter and Burkholderia was identified. In addition to playing a role in microbial antagonism, microbial production of HCN was also reported to enhance the dissolution of various cations and anions released due to weathering of rocks.

The plant growth-promoting phytohormone, indole-3-acetic acid (IAA) plays an important role in xylem and root formation, cell division, enlargement and differentiation. As reported earlier, (Torres-rubio et al. 2000; Tsavkelova et al. 2007) strains of Enterobacter and Pseudomonas reported in this study also produced IAA. Enhanced development of root system due to production of IAA by the rhizoplane bacteria of plants growing in rocks

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can indirectly help in weathering of rocks by the developing root system. 1-Aminocyclopropane-1-carboxylate (ACC) deaminase is an enzyme which lowers the levels of ethylene thereby delays senescence and results in longer roots which inturn induces tolerance of plants to various biotic and abiotic stresses. Production of ACC deaminase was reported earlier (Shah et al. 1998; Ravindra Naik et al. 2008). Present study identified a total of 21 strains of Enterobacter and Pseudomonas that produced ACC deaminase. Production of ACC deaminase by rhizobacteria helps the plant to withstand environmental stress such as extreme temperature, salinity, drought and pathogenecity which are essential traits of bacteria residing in the roots of plant growing within rocks.

The rhizoplane bacteria reported in this study also produced important industrial enzymes such as cellulase (4 strains), DNase (7 strains), protease (1 strain) and xylanase (5 strains). Extracellular enzymes such as protease and cellulase are reported to be important fungal cell wall-degrading enzymes (Pathma and Sakthivel 2013). Thus, production of such extracellular enzymes by the rhizoplane bacteria would play a role in the exhibition of antagonism against the phytopathogenic fungi. Rhizoplane bacteria (20 strains) showed antifungal potential against one or more phytopathogenic fungus while a total of 23 strains, showed antibacterial activity against one or more human microbial pathogen.

Present study identified the production of organic acids by rhizoplane bacteria. Production of organic acids has been reported to impact the mineral stability of the rock by acidolysis and chelation of divalent cations such as Ca, Mg, Mn, Zn, Sr and Ni resulting in biological weathering (Berthelin et al. 1974; Palmer et al. 1991; Hirsch et al. 1995a; Kim et al. 2005; Uroz et al. 2009).

Metal bioreduction activity may be considered as an important mechanism adopted by microorganisms to survive in metal dominated environments such as rocks. Interestingly, 102

the rock-weathering rhizoplane bacteria also exhibited the ability to synthesize biogenic gold and silver metal nanoparticles. The ability to reduce the metals helps the rhizoplane bacteria to grow in nutrient deficient and metal dominated environment such as rocks. Interestingly, strains RB1, RB2 and RB3 had the prospective to bioreduce the gold chloride into gold nanoparticles and strains, RB6, RB7, RB9 and RB11 synthesized silver nanoparticles from silver nitrate. Characterization of synthesized nanoparticles by XRD, FT-IR, SEM-EDAX and TEM confirmed the presence of face centered cubic crystalline structure of the gold and silver nanoparticles, stabilized by enzymes and proteins secreted by rhizoplane bacteria. The gold nanoparticles were found to be spherical with a size of 65 nm and while the silver nanoparticles were anisotropic in shape with an average size of 15 nm. Specific characteristics such as the ability to grow at high temperature (45oC), high salt (3%) tolerance and metal bioreduction are considered as vital virtues to survive in harsh environments. In addition, production of IAA, ACC deaminase, siderophores, HCN and fungal cell wall-degrading enzymes such as protease and cellulase plays an important role in plant growth-promotion and disease control. To conclude, present study highlights the presence of various bacterial strains harboring efficient rock weathering and plant growthpromoting potential in the rhizoplane of plants which grow on rock lacking visible soil. These inherent abilities of rhizoplane bacteria of rock-dwelling plants can be exploited for plant growth-promotion in arid and semi-arid environments, commercial production of enzymes and nanoparticles for industrial and medical applications.

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