During the last decades many studies have been undertaken to investigate life in extreme environments, leading to the discovery of novel organisms and novel habitats previously though to be unapproachable for life. Microbes are key players in a number of ecological processes such as mineral dissolution, soil genesis, plant growth promotion (PGP) and bioremediation of polluted sites and they are the main responsibles for element cycles both in conventional and extreme ecosystems. The biotechnological potential of extremophiles is well recognized, and the aim of this PhD project was to give further insight on the possible exploitation of the microbiome naturally adapted to cope with extreme values of one or more environmental parameters to develop sustainable strategies in agriculture and ecosystem management with a particular focus on arid and saline lands. Mineral-microbe interactions have been studied in detail, particularly regarding the importance of bioweathering bacteria in the ambit of soil fertility promotion in arid lands. Specific sites within the Midtre Lovénbreen glacier moraine (Svalbard, Norway), where pyritic rocks were present, hosted an active acidophilic iron-oxidizing bacterial community involved in the bioweathering of pyrite supplied by the rock disaggregation due to winter freezing. A decreased iron concentration and acidification were observed along the wheathered area departing form the pyrite-rich rock, where the oxidation of ferrous iron led to the accumulation of ferric oxy-hydroxides in the above soil. These ferric compounds were linked to the increase of soil physico-chemical properties that in turn determined a higher water holding capacity (WHC) and nutrient content in the surrounding vegetated area, densely colonized by mosses and small vascular plants. At the outer border of the vegetated area, the rest of the moraine hosted typical first colonizer bacteria, mainly belonging to the class Cyanobacteria, that are capable of nitrogen and carbon fixation. Thus, compared to the rest of the moraine, the enhancement of soil formation processes and plant colonization in the vegetated area was driven by the synergy between acidification and leaching activity of a chemolitotrophic community and the cyanobacteria-mediated primary productivity. A detailed description of the bacterial communities colonizing the weathered area, the vegetated area, and the barren moraine was obtained through the construction of 16S rRNA gene libraries. The statistical ∫-Libshuff analysis indicated these areas as three different ecological niches. The microbiome of the weathered area was dominated by few bacterial taxa due to the low pH value of the biological soil crust (BSC) whereas the vegetated area and the moraine displayed higher biodiversity. The most abundant phylogenetic groups in these BSCs were nevertheless different and in the case of the vegetated area they corresponded to those typical of mature and rhizospheric soils. The ability of microorganisms to interact with minerals is an essential factor that influence plant nutrition by providing nutrients, such as phosphorous, that are generally present in the soils as insoluble forms. The capability to solubilize poorly bioavailable nutrients is one of the PGP activities that have been investigated in the microbiome associated to different plant species living in arid hypersaline soils in Central and South Tunisia (Olea europea and Salicornia spp.) or acid soils located in a volcanic area in Mexico. A large collection of bacterial isolates has been constituted, identified and characterized for the in vitro PGP potential. Halophilic bacteria were isolated from the rhizosphere of Salicornia plants on oligotrophic media enriched with NaCl. The isolates obtained from 15% NaCl enriched media mainly belonged to the Halomonas genus, whereas the bacteria isolated at 10% NaCl showed a higher phylogenetic diversity at the genus level. Most of the bacteria comprised in the halophiles collection exhibited high resistance to drought, temperature and salt stresses. PGP activities were also widespread, especially the ability to produce indol-3-acetic acid (IAA), which promotes lateral roots developement. Furthermore, high percentage of the halophilic bacteria produced ammonium (94%) and were able to solubilize phosphate (64%) while the ability to produce protease, an activity involved in biocontrol processes, was less frequent. The comprehensive study realized on the culturable halophilic fraction of the rhizospheric bacteria associated to Salicornia spp. allowed the identification of 20 isolates as suitable candidate for developing a bacterial inoculum aimed to promote plant growth under saline stress. The diversity of the microbiome inhabiting different fractions of the Olea europea root system was investigated by applying a cultivation-independent method (Denaturing Gradient Gel Electrophoresis). The interior root tissues, the rhizosphere, the root surrounding soil and the bulk soil were colonized by a rich and diverse microbiome, shaped both by interaction with the plant and the environmental parameters of the collection site. Moreover, from these four fractions a bacterial collection was obtained for the screening of ecological and PGP features of culturable bacteria associated to olive tree growing under drought stress. In addition to the abiotic stress resistance, bacterial isolates displayed a variety of PGP activities, such as potential nitrogen fixation, siderophores and exopolysaccharides production and phosphate solubilization. Overall, the obtained dataset highlighted the possibility to use the investigated PGP bacteria associated to olive tree as biofertilizer for supporting olive growth under drought stress. An extremophile plant living at high T (42°C) and low pH (4.1) was collected at El Chichón volcanic system (Mexico). The PGP activities of a collection of rhizobacteria isolated from the plant were explored through in vitro tests. Several strains were able to affect phytohormones balance by the production of indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate (ACC)-deaminase, the latter being involved in the decrease of ethylene level in plants. Remarkable percentage of the isolated bacteria displayed also additional potential PGP activities based on weathering activity. Volcanic habitats can hence be estimated as source of extremophile rhizobacteria potentialy able to help pioneer plants to cope with the severe condition of acidic soils. Microbe-environment interactions have been investigated also in deep hypersaline anoxic basins (DHABs) located in the eastern Mediterranean Sea, model environments to look for bacterial phylotypes specifically adapted to high salinity conditions. Mediterranean DHABs are far below the photic zone and contain brines originated by the dissolution of Messinian evaporites. Compared to other DHABs, Urania has very high concentrations of methane (5.56 mM) and levels of sulfide (up to 16 mM) that make it one of the most sulfidic marine water bodies on Earth. The interface between seawater and the anoxic hypersaline brine of DHABs is an oxic-anoxic interface containing an halocline with layers from seawater to brine typical salinity and is a hot-spot of microbial activity. Methanogenesis activity was detected along the first of the two environmental chemoclines present in the Urania basin and 16S rRNA gene libraries indicated the Euryarchaeota group MSBL1 as the primary candidates for methane production. Cultivation-independent analyses proved that sulfur cycling is a major driver in shaping the microbial communities, though other chemolithoautotrophic processes like manganese oxidation and anaerobic ammonium oxidation (ANAMMOX) are involved. The occurrence of ANAMMOX reaction was verified in other DHABs, namely L’Atalante and Bannock. Labelled dinitrogen gas production in 15N activity test demonstrated that anammox bacteria were active in the chemoclines of both the basins. Fluorescence in situ hybridization and 16S rRNA gene libraries using anammox-specific PCR primers unveiled the presence of the known marine anammox genus ‘Scalindua’, together with putatively novel operational taxonomic units (OTUs) closely affiliated to sequences retrieved in other marine environments where anammox activity were detected. Real Time PCR assay allowed to quantify anammox-related 16S rRNA genes in Bannock basin, which were highly abundant in correspondence of the oxic-anoxic boundary in the salinity range comprised between 6.4 and 12.1%. Cluster analysis of 16S rRNA gene libraries showed that chemoclines of Bannock and L’Atalante basins, having diverse geochemical settings, selected for different anammox phylotypes and that a shift in anammox population could be observed at increasing salinity values. The detection of putative novel phylotypes specifically adapted to peculiar salinity levels represent a key step for designing ad hoc inocula to be used in the remediation of saline wastewaters originated by industrial and agricultural processes. Actually the known freshwater anammox populations could only adapt to salt concentrations up to 3% if salinity is slowly increased, thus the selection of naturally adapted anammox strains would be of primary importance to enhance the exploitation of this process during the removal of nitrogen compounds from wastewater. The occurrence of microbe-plant positive associations was proved in different stressed soils, and their exploitation is likely the most promising approach to avoid or reduce the use of chemical fertilizer and to boost plant growth and crop productivity whitout the use of genetically modified organisms (GMO) in respect of the biodiversity. Similarly, the discovery of novel anammox phylotypes in hypersaline ecosystems shed a new light on the utilization of this functional group of bacteria for the removal of nitrogen from saline wastewaters, a critical step of treatment processes due to the environmental impact of nitrogen compounds and the severe legislation on wastewater discharges.

MICROBIAL DIVERSITY IN EXTREME ENVIRONMENTS: A RESOURCE FOR A SUSTAINABLE AGRICULTURE MANAGEMENT / F. Mapelli ; tutor: S. Borin ; coordinatore: G. Zocchi. Universita' degli Studi di Milano, 2012 Feb 10. 24. ciclo, Anno Accademico 2011.

MICROBIAL DIVERSITY IN EXTREME ENVIRONMENTS: A RESOURCE FOR A SUSTAINABLE AGRICULTURE MANAGEMENT

F. Mapelli
2012

Abstract

During the last decades many studies have been undertaken to investigate life in extreme environments, leading to the discovery of novel organisms and novel habitats previously though to be unapproachable for life. Microbes are key players in a number of ecological processes such as mineral dissolution, soil genesis, plant growth promotion (PGP) and bioremediation of polluted sites and they are the main responsibles for element cycles both in conventional and extreme ecosystems. The biotechnological potential of extremophiles is well recognized, and the aim of this PhD project was to give further insight on the possible exploitation of the microbiome naturally adapted to cope with extreme values of one or more environmental parameters to develop sustainable strategies in agriculture and ecosystem management with a particular focus on arid and saline lands. Mineral-microbe interactions have been studied in detail, particularly regarding the importance of bioweathering bacteria in the ambit of soil fertility promotion in arid lands. Specific sites within the Midtre Lovénbreen glacier moraine (Svalbard, Norway), where pyritic rocks were present, hosted an active acidophilic iron-oxidizing bacterial community involved in the bioweathering of pyrite supplied by the rock disaggregation due to winter freezing. A decreased iron concentration and acidification were observed along the wheathered area departing form the pyrite-rich rock, where the oxidation of ferrous iron led to the accumulation of ferric oxy-hydroxides in the above soil. These ferric compounds were linked to the increase of soil physico-chemical properties that in turn determined a higher water holding capacity (WHC) and nutrient content in the surrounding vegetated area, densely colonized by mosses and small vascular plants. At the outer border of the vegetated area, the rest of the moraine hosted typical first colonizer bacteria, mainly belonging to the class Cyanobacteria, that are capable of nitrogen and carbon fixation. Thus, compared to the rest of the moraine, the enhancement of soil formation processes and plant colonization in the vegetated area was driven by the synergy between acidification and leaching activity of a chemolitotrophic community and the cyanobacteria-mediated primary productivity. A detailed description of the bacterial communities colonizing the weathered area, the vegetated area, and the barren moraine was obtained through the construction of 16S rRNA gene libraries. The statistical ∫-Libshuff analysis indicated these areas as three different ecological niches. The microbiome of the weathered area was dominated by few bacterial taxa due to the low pH value of the biological soil crust (BSC) whereas the vegetated area and the moraine displayed higher biodiversity. The most abundant phylogenetic groups in these BSCs were nevertheless different and in the case of the vegetated area they corresponded to those typical of mature and rhizospheric soils. The ability of microorganisms to interact with minerals is an essential factor that influence plant nutrition by providing nutrients, such as phosphorous, that are generally present in the soils as insoluble forms. The capability to solubilize poorly bioavailable nutrients is one of the PGP activities that have been investigated in the microbiome associated to different plant species living in arid hypersaline soils in Central and South Tunisia (Olea europea and Salicornia spp.) or acid soils located in a volcanic area in Mexico. A large collection of bacterial isolates has been constituted, identified and characterized for the in vitro PGP potential. Halophilic bacteria were isolated from the rhizosphere of Salicornia plants on oligotrophic media enriched with NaCl. The isolates obtained from 15% NaCl enriched media mainly belonged to the Halomonas genus, whereas the bacteria isolated at 10% NaCl showed a higher phylogenetic diversity at the genus level. Most of the bacteria comprised in the halophiles collection exhibited high resistance to drought, temperature and salt stresses. PGP activities were also widespread, especially the ability to produce indol-3-acetic acid (IAA), which promotes lateral roots developement. Furthermore, high percentage of the halophilic bacteria produced ammonium (94%) and were able to solubilize phosphate (64%) while the ability to produce protease, an activity involved in biocontrol processes, was less frequent. The comprehensive study realized on the culturable halophilic fraction of the rhizospheric bacteria associated to Salicornia spp. allowed the identification of 20 isolates as suitable candidate for developing a bacterial inoculum aimed to promote plant growth under saline stress. The diversity of the microbiome inhabiting different fractions of the Olea europea root system was investigated by applying a cultivation-independent method (Denaturing Gradient Gel Electrophoresis). The interior root tissues, the rhizosphere, the root surrounding soil and the bulk soil were colonized by a rich and diverse microbiome, shaped both by interaction with the plant and the environmental parameters of the collection site. Moreover, from these four fractions a bacterial collection was obtained for the screening of ecological and PGP features of culturable bacteria associated to olive tree growing under drought stress. In addition to the abiotic stress resistance, bacterial isolates displayed a variety of PGP activities, such as potential nitrogen fixation, siderophores and exopolysaccharides production and phosphate solubilization. Overall, the obtained dataset highlighted the possibility to use the investigated PGP bacteria associated to olive tree as biofertilizer for supporting olive growth under drought stress. An extremophile plant living at high T (42°C) and low pH (4.1) was collected at El Chichón volcanic system (Mexico). The PGP activities of a collection of rhizobacteria isolated from the plant were explored through in vitro tests. Several strains were able to affect phytohormones balance by the production of indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate (ACC)-deaminase, the latter being involved in the decrease of ethylene level in plants. Remarkable percentage of the isolated bacteria displayed also additional potential PGP activities based on weathering activity. Volcanic habitats can hence be estimated as source of extremophile rhizobacteria potentialy able to help pioneer plants to cope with the severe condition of acidic soils. Microbe-environment interactions have been investigated also in deep hypersaline anoxic basins (DHABs) located in the eastern Mediterranean Sea, model environments to look for bacterial phylotypes specifically adapted to high salinity conditions. Mediterranean DHABs are far below the photic zone and contain brines originated by the dissolution of Messinian evaporites. Compared to other DHABs, Urania has very high concentrations of methane (5.56 mM) and levels of sulfide (up to 16 mM) that make it one of the most sulfidic marine water bodies on Earth. The interface between seawater and the anoxic hypersaline brine of DHABs is an oxic-anoxic interface containing an halocline with layers from seawater to brine typical salinity and is a hot-spot of microbial activity. Methanogenesis activity was detected along the first of the two environmental chemoclines present in the Urania basin and 16S rRNA gene libraries indicated the Euryarchaeota group MSBL1 as the primary candidates for methane production. Cultivation-independent analyses proved that sulfur cycling is a major driver in shaping the microbial communities, though other chemolithoautotrophic processes like manganese oxidation and anaerobic ammonium oxidation (ANAMMOX) are involved. The occurrence of ANAMMOX reaction was verified in other DHABs, namely L’Atalante and Bannock. Labelled dinitrogen gas production in 15N activity test demonstrated that anammox bacteria were active in the chemoclines of both the basins. Fluorescence in situ hybridization and 16S rRNA gene libraries using anammox-specific PCR primers unveiled the presence of the known marine anammox genus ‘Scalindua’, together with putatively novel operational taxonomic units (OTUs) closely affiliated to sequences retrieved in other marine environments where anammox activity were detected. Real Time PCR assay allowed to quantify anammox-related 16S rRNA genes in Bannock basin, which were highly abundant in correspondence of the oxic-anoxic boundary in the salinity range comprised between 6.4 and 12.1%. Cluster analysis of 16S rRNA gene libraries showed that chemoclines of Bannock and L’Atalante basins, having diverse geochemical settings, selected for different anammox phylotypes and that a shift in anammox population could be observed at increasing salinity values. The detection of putative novel phylotypes specifically adapted to peculiar salinity levels represent a key step for designing ad hoc inocula to be used in the remediation of saline wastewaters originated by industrial and agricultural processes. Actually the known freshwater anammox populations could only adapt to salt concentrations up to 3% if salinity is slowly increased, thus the selection of naturally adapted anammox strains would be of primary importance to enhance the exploitation of this process during the removal of nitrogen compounds from wastewater. The occurrence of microbe-plant positive associations was proved in different stressed soils, and their exploitation is likely the most promising approach to avoid or reduce the use of chemical fertilizer and to boost plant growth and crop productivity whitout the use of genetically modified organisms (GMO) in respect of the biodiversity. Similarly, the discovery of novel anammox phylotypes in hypersaline ecosystems shed a new light on the utilization of this functional group of bacteria for the removal of nitrogen from saline wastewaters, a critical step of treatment processes due to the environmental impact of nitrogen compounds and the severe legislation on wastewater discharges.
10-feb-2012
Settore AGR/16 - Microbiologia Agraria
BORIN, SARA
ZOCCHI, GRAZIANO
Doctoral Thesis
MICROBIAL DIVERSITY IN EXTREME ENVIRONMENTS: A RESOURCE FOR A SUSTAINABLE AGRICULTURE MANAGEMENT / F. Mapelli ; tutor: S. Borin ; coordinatore: G. Zocchi. Universita' degli Studi di Milano, 2012 Feb 10. 24. ciclo, Anno Accademico 2011.
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