title: Characterisation of Hydrocarbon Degrading Antarctic Pseudomonas species

authors: Rhodes, Phillippa

subjects: Antarctica

abstract: Hydrocarbon contamination of Antarctica poses a threat to the unique and fragile ecosystems of this continent. Bioremediation, utilising indigenous hydrocarbon-degrading bacteria, has been proposed as an environmentally friendly method for clean up of contaminated terrestrial sites in Antarctica. Design and implementation of a successful bioremediation process requires detailed information of the degradative bacteria present at the contaminated site(s). This study focused on toluene and naphthalene-degrading Pseudomonas species isolated from JP8 jet-fuel contaminated sites at Scott Base, Antarctica.
Isolates were assessed for their ability to grow on toluene and meta-toluate or naphthalene as a sole carbon source on solid and in liquid media at 6°C and 15°C. All the toluene degrading isolates could grow on 5 mM meta-toluate in liquid and on solid media at 6°C and 15°C. Toluene degrading isolates, with the exception of 8/43 and 8/51, were able to grow on solid media supplemented with toluene vapour but none could grow on toluene in liquid media. Naphthalene degrading isolates could grow on solid media, with naphthalene vapour as a carbon source, at 15°C but at 6°C two of the six isolates, 8/47 and 7/38, could not grow on solid media supplemented with naphthalene vapour. None of the toluene and naphthalene degrading isolate could grow on carbon-free solid or liquid media without the addition of a growth substrate. Presumptive plasmids were isolated from the following toluene degrading isolates, 8/46, 7/163, 7/167, 7/22, and the following naphthalene degrading isolates, 7/156, 7/38, ant5, ant9. Two toluene degrading isolates, 7/167 and 8/46, were studied in more depth. Both isolates grew in liquid media, with meta-toluate supplied as a sole carbon source, over a range of temperatures, 6°C, 1 DOC, 15°C, 2DoC, and 25°C. The optimum growth temperature for isolate 7/167 is between 20°C and 25°C and the optimum growth temperature for isolate 8/46 is 15°C. Isolates were able to sustain growth on meta-toluate at 6°C, 15°C, and 25°C. Isolate 7/167 could not sustain growth on meta-toluate at 30°C. Isolate 8/46 could sustain growth on meta-toluate at 30°C but could not grow at 35°C. All of the Antarctic isolates are psychrotrophic.
Degradative plasmids were isolated from 7/167 and 8/46. Restriction enzyme digest fragment patterns of plasmid DNA generated with Xhol showed isolates 7/167 and 8/46 contain different sized plasmids, 61 kb and 95 kb, respectively. Regions of toluene catabolic genes were amplified by the Polymerase Chain Reaction. Sequence data of the PCR products produced using xylB primers revealed isolate 7/167 to be 100% homologous to the xylB gene region amplified from the archetypal TOL plasmid, pWWO, isolated from the Northern Hemisphere, and 8/46 to be 92% homologous.
Assays of four enzymes, involved in toluene degradation, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, from isolates 7/167 and 8/46 showed catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and -benzaldehyde dehydrogenase to be present and active. No activity was detected for catechol 1,2-dioxygenase, indicating the presence of a meta cleavage pathway of toluene degradation, typical of that found on TOL type plasmids, and not ortho cleavage of catechol typical of chromosomal gene expression.
Isolates 7/167 and 8/46 contain large plasmids, similar to Northern Hemisphere isolated TOL degradative plasmids, and cleave meta-toluate via the meta cleavage pathway. This degradative mechanism is analogous to the toluene degradative pathway encoded by the archetypal TOL plasmid, pWWO, in the mesophilic bacterium P. putida mt-2. These indigenous psychrotrophic toluene degrading Antarctic bacteria have the ability to degrade meta-toluate at temperatures typical of the Antarctic soil at Scott Base in the summer months. These toluene degrading Antarctic bacteria have the potential to be used to develop a bioremediation programme to successfully clean up JP8 jet fuel contaminated soil at Scott Base, Antarctica.

text: Characterisation of Hydrocarbon Degrading Antarctic Pseudomonas species Hydrocarbon contamination of Antarctica poses a threat to the unique and fragile ecosystems of this continent. Bioremediation, utilising indigenous hydrocarbon-degrading bacteria, has been proposed as an environmentally friendly method for clean up of contaminated terrestrial sites in Antarctica. Design and implementation of a successful bioremediation process requires detailed information of the degradative bacteria present at the contaminated site(s). This study focused on toluene and naphthalene-degrading Pseudomonas species isolated from JP8 jet-fuel contaminated sites at Scott Base, Antarctica.
Isolates were assessed for their ability to grow on toluene and meta-toluate or naphthalene as a sole carbon source on solid and in liquid media at 6°C and 15°C. All the toluene degrading isolates could grow on 5 mM meta-toluate in liquid and on solid media at 6°C and 15°C. Toluene degrading isolates, with the exception of 8/43 and 8/51, were able to grow on solid media supplemented with toluene vapour but none could grow on toluene in liquid media. Naphthalene degrading isolates could grow on solid media, with naphthalene vapour as a carbon source, at 15°C but at 6°C two of the six isolates, 8/47 and 7/38, could not grow on solid media supplemented with naphthalene vapour. None of the toluene and naphthalene degrading isolate could grow on carbon-free solid or liquid media without the addition of a growth substrate. Presumptive plasmids were isolated from the following toluene degrading isolates, 8/46, 7/163, 7/167, 7/22, and the following naphthalene degrading isolates, 7/156, 7/38, ant5, ant9. Two toluene degrading isolates, 7/167 and 8/46, were studied in more depth. Both isolates grew in liquid media, with meta-toluate supplied as a sole carbon source, over a range of temperatures, 6°C, 1 DOC, 15°C, 2DoC, and 25°C. The optimum growth temperature for isolate 7/167 is between 20°C and 25°C and the optimum growth temperature for isolate 8/46 is 15°C. Isolates were able to sustain growth on meta-toluate at 6°C, 15°C, and 25°C. Isolate 7/167 could not sustain growth on meta-toluate at 30°C. Isolate 8/46 could sustain growth on meta-toluate at 30°C but could not grow at 35°C. All of the Antarctic isolates are psychrotrophic.
Degradative plasmids were isolated from 7/167 and 8/46. Restriction enzyme digest fragment patterns of plasmid DNA generated with Xhol showed isolates 7/167 and 8/46 contain different sized plasmids, 61 kb and 95 kb, respectively. Regions of toluene catabolic genes were amplified by the Polymerase Chain Reaction. Sequence data of the PCR products produced using xylB primers revealed isolate 7/167 to be 100% homologous to the xylB gene region amplified from the archetypal TOL plasmid, pWWO, isolated from the Northern Hemisphere, and 8/46 to be 92% homologous.
Assays of four enzymes, involved in toluene degradation, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, from isolates 7/167 and 8/46 showed catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and -benzaldehyde dehydrogenase to be present and active. No activity was detected for catechol 1,2-dioxygenase, indicating the presence of a meta cleavage pathway of toluene degradation, typical of that found on TOL type plasmids, and not ortho cleavage of catechol typical of chromosomal gene expression.
Isolates 7/167 and 8/46 contain large plasmids, similar to Northern Hemisphere isolated TOL degradative plasmids, and cleave meta-toluate via the meta cleavage pathway. This degradative mechanism is analogous to the toluene degradative pathway encoded by the archetypal TOL plasmid, pWWO, in the mesophilic bacterium P. putida mt-2. These indigenous psychrotrophic toluene degrading Antarctic bacteria have the ability to degrade meta-toluate at temperatures typical of the Antarctic soil at Scott Base in the summer months. These toluene degrading Antarctic bacteria have the potential to be used to develop a bioremediation programme to successfully clean up JP8 jet fuel contaminated soil at Scott Base, Antarctica.

year: 1999

title: Photodegradation leads to increased carbon dioxide losses from terrestrial organic matter

authors: Rutledge, Susanna

subjects: abiotic decomposition

abstract: CO₂ production in terrestrial ecosystems is generally assumed to be solely biologically driven while the role of abiotic processes has been largely overlooked. In addition to microbial decomposition, photodegradation – the direct breakdown of organic matter (OM) by solar irradiance – has been found to contribute to litter mass loss in dry ecosystems. Previous small-scale studies have shown that litter degradation by irradiance is accompanied by emissions of CO₂. However, the contribution of photodegradation to total CO₂ losses at ecosystems scales is unknown. This study determined the proportion of the total CO₂ losses caused by photodegradation in two ecosystems: a bare peatland in New Zealand and a seasonally dry grassland in California. The direct effect of solar irradiance on CO₂  production was examined by comparing daytime CO₂  fluxes measured using eddy covariance (EC) systems with simultaneous measurements made using an opaque chamber and the soil CO₂ gradient technique, and with night-time EC measurements under the same soil temperature and moisture conditions. In addition, a transparent chamber was used to directly measure CO₂ fluxes from OM caused by solar irradiance. Photodegradation contributed 19% of the annual CO₂ flux from the peatland and almost 60% of the dry season CO₂ flux from the grassland, and up to 62% and 92% of the summer mid-day CO₂ fluxes, respectively. Our results suggest that photodegradation may be important in a wide range of ecosystems with exposed OM. Furthermore, the practice of partitioning daytime ecosystem CO₂ exchange into its gross components by assuming that total daytime CO₂ losses can be approximated using estimates of biological respiration alone may be in error. To obtain robust estimates of global ecosystem–atmosphere carbon transfers, the contribution of photodegradation to OM decomposition must be quantified for other ecosystems and the results incorporated into coupled carbon–climate models.

text: Photodegradation leads to increased carbon dioxide losses from terrestrial organic matter CO₂ production in terrestrial ecosystems is generally assumed to be solely biologically driven while the role of abiotic processes has been largely overlooked. In addition to microbial decomposition, photodegradation – the direct breakdown of organic matter (OM) by solar irradiance – has been found to contribute to litter mass loss in dry ecosystems. Previous small-scale studies have shown that litter degradation by irradiance is accompanied by emissions of CO₂. However, the contribution of photodegradation to total CO₂ losses at ecosystems scales is unknown. This study determined the proportion of the total CO₂ losses caused by photodegradation in two ecosystems: a bare peatland in New Zealand and a seasonally dry grassland in California. The direct effect of solar irradiance on CO₂  production was examined by comparing daytime CO₂  fluxes measured using eddy covariance (EC) systems with simultaneous measurements made using an opaque chamber and the soil CO₂ gradient technique, and with night-time EC measurements under the same soil temperature and moisture conditions. In addition, a transparent chamber was used to directly measure CO₂ fluxes from OM caused by solar irradiance. Photodegradation contributed 19% of the annual CO₂ flux from the peatland and almost 60% of the dry season CO₂ flux from the grassland, and up to 62% and 92% of the summer mid-day CO₂ fluxes, respectively. Our results suggest that photodegradation may be important in a wide range of ecosystems with exposed OM. Furthermore, the practice of partitioning daytime ecosystem CO₂ exchange into its gross components by assuming that total daytime CO₂ losses can be approximated using estimates of biological respiration alone may be in error. To obtain robust estimates of global ecosystem–atmosphere carbon transfers, the contribution of photodegradation to OM decomposition must be quantified for other ecosystems and the results incorporated into coupled carbon–climate models.

year: 2009

title: He pao

authors: Roa, Tom

subjects: Pao

abstract: Ko Te Whare Wānanga O Waikato" te ingoa o tētehi pao nāku i tito hei waiata mā Te Whare Wānanga O Waikato, i te tau 2004.

text: He pao Ko Te Whare Wānanga O Waikato" te ingoa o tētehi pao nāku i tito hei waiata mā Te Whare Wānanga O Waikato, i te tau 2004.

year: 2012-05