title: ¹⁵N stable isotope probing of pulp and paper wastewaters

authors: Addison, Sarah Louise

subjects: Stable isotope probing

abstract: Stable isotope probing (SIP) is an established technique that can be applied to identify the metabolically active micro-organisms within a microbial population. The SIP method utilises an isotopically-labelled substrate and PCR techniques to discern the members of a microbial community that incorporate the isotope into their DNA or RNA. The current literature gap around using ¹⁵N isotopes with RNA-SIP offers real potential and advantages for targeting and identifying active members from mixed communities involved in global biogeochemical nitrogen cycling.

This study specifically investigated whether nitrogen based compounds ncan be used as substrates in RNA-SIP methodologies and whether they can in turn be used to probe mixed community environments known to be actively fixing nitrogen. The nitrogen-limited systems targeted represented an ideal opportunity to assess the suitability of ¹⁵N-RNA-SIP approaches due to their known high nitrogen fixation rates. Identifying these nitrogen-fixing bacteria could provide a better representation analysis of the community, leading to an improved prediction on how to manage and optimise the treatment performance of target waste systems and to exploit the unique bioconversion properties of these types of organisms.

Initially, the project undertook methodological proof of concept by using a soluble nitrogen source, ¹⁵NH₄Cl, to label the RNA of Novosphingobium nitrogenifigens and a mixed microbial community. Successful separation of the ¹⁴N- (control) and ¹⁵N-RNA was achieved for both pure and mixed communities using isopycnic caesium trifluoroacetate (CsTFA) gradients in an ultracentrifuge. The usefulness of this technique to identify active diazotrophs in real environmental samples was tested using a nitrogen-fixing community from a pulp and paper wastewater treatment system. After growing the mixed culture with ¹⁵N₂ as the sole nitrogen source, the labelled RNA was extracted and fractionated using isopycnic centrifugation in CsTFA gradients.

The community composition of the active nitrogen-fixing community in the ¹⁵N₂ enriched fraction was analysed by establishing a 16S rRNA gene clone library containing over 200 members. These were analysed by comparison with published sequences and by phylogenetic analysis.

It was found that the more isotopic label substrate incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA. Novosphingobium nitrogenifigens gave an average BD shift of 0.03 + 0.004 g ml⁻¹ (95.0 atom % ¹⁵N) with ¹⁵NH₄Cl. For mixed communities the average BD shift was 0.02 + 0.004 g ml⁻¹ (80.0 atom % ¹⁵N) with 15NH4Cl and 0.013 + 0.002 g ml⁻¹ (32.6 atom % ¹⁵N) when using ¹⁵N₂. Clone library analysis of 16S rRNA genes present in the enriched ¹⁵N-RNA fraction of the mixed community was shown to consist of a diverse population of bacteria as indicated by a Shannon Weaver index value of >2.8. Three dominant genera (Aeromonas, Pseudomonas and Bacillus) were identified by comparison with published sequences and phylogenetic analysis. Many other groups not known as archetypal nitrogen-fixing bacteria were also identified, demonstrating that ¹⁵N₂-RNA-SIP provides a useful tool for the identification of important and previously unknown contributors to nitrogen fixation in a range of environments.

Overall, this project has established that nitrogen based RNA-SIP is a powerful tool that can be used successfully and reproducibly with both pure and complex mixed microbial communities to study active diazotrophs in environmental samples.

text: ¹⁵N stable isotope probing of pulp and paper wastewaters Stable isotope probing (SIP) is an established technique that can be applied to identify the metabolically active micro-organisms within a microbial population. The SIP method utilises an isotopically-labelled substrate and PCR techniques to discern the members of a microbial community that incorporate the isotope into their DNA or RNA. The current literature gap around using ¹⁵N isotopes with RNA-SIP offers real potential and advantages for targeting and identifying active members from mixed communities involved in global biogeochemical nitrogen cycling.

This study specifically investigated whether nitrogen based compounds ncan be used as substrates in RNA-SIP methodologies and whether they can in turn be used to probe mixed community environments known to be actively fixing nitrogen. The nitrogen-limited systems targeted represented an ideal opportunity to assess the suitability of ¹⁵N-RNA-SIP approaches due to their known high nitrogen fixation rates. Identifying these nitrogen-fixing bacteria could provide a better representation analysis of the community, leading to an improved prediction on how to manage and optimise the treatment performance of target waste systems and to exploit the unique bioconversion properties of these types of organisms.

Initially, the project undertook methodological proof of concept by using a soluble nitrogen source, ¹⁵NH₄Cl, to label the RNA of Novosphingobium nitrogenifigens and a mixed microbial community. Successful separation of the ¹⁴N- (control) and ¹⁵N-RNA was achieved for both pure and mixed communities using isopycnic caesium trifluoroacetate (CsTFA) gradients in an ultracentrifuge. The usefulness of this technique to identify active diazotrophs in real environmental samples was tested using a nitrogen-fixing community from a pulp and paper wastewater treatment system. After growing the mixed culture with ¹⁵N₂ as the sole nitrogen source, the labelled RNA was extracted and fractionated using isopycnic centrifugation in CsTFA gradients.

The community composition of the active nitrogen-fixing community in the ¹⁵N₂ enriched fraction was analysed by establishing a 16S rRNA gene clone library containing over 200 members. These were analysed by comparison with published sequences and by phylogenetic analysis.

It was found that the more isotopic label substrate incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA. Novosphingobium nitrogenifigens gave an average BD shift of 0.03 + 0.004 g ml⁻¹ (95.0 atom % ¹⁵N) with ¹⁵NH₄Cl. For mixed communities the average BD shift was 0.02 + 0.004 g ml⁻¹ (80.0 atom % ¹⁵N) with 15NH4Cl and 0.013 + 0.002 g ml⁻¹ (32.6 atom % ¹⁵N) when using ¹⁵N₂. Clone library analysis of 16S rRNA genes present in the enriched ¹⁵N-RNA fraction of the mixed community was shown to consist of a diverse population of bacteria as indicated by a Shannon Weaver index value of >2.8. Three dominant genera (Aeromonas, Pseudomonas and Bacillus) were identified by comparison with published sequences and phylogenetic analysis. Many other groups not known as archetypal nitrogen-fixing bacteria were also identified, demonstrating that ¹⁵N₂-RNA-SIP provides a useful tool for the identification of important and previously unknown contributors to nitrogen fixation in a range of environments.

Overall, this project has established that nitrogen based RNA-SIP is a powerful tool that can be used successfully and reproducibly with both pure and complex mixed microbial communities to study active diazotrophs in environmental samples.

year: 2008

title: Learning as spirituality and nurture - Pacific indigenous peoples' perspectives of lifelong learning

authors: Vaioleti, Timote Masima

subjects: Pacific

abstract: This paper discusses characteristics of an adult education practice for peoples in the Pacific. There is no one Pacific way as the Pacific population is diverse consisting of many cultures, languages, social structures and differing colonial experiences. For many Pacific peoples learning is holistic, is driven by cultural motivation, which is often for the benefit of their extended families(the collective)rather than personal gains or self-actualisation. Learning is constantly intervened by spiritual matters, the same being true for most aspects of Pacific peoples’ day-to-day lives. Therefore spirituality is integral to learning at all levels formal and informal.

text: Learning as spirituality and nurture - Pacific indigenous peoples' perspectives of lifelong learning This paper discusses characteristics of an adult education practice for peoples in the Pacific. There is no one Pacific way as the Pacific population is diverse consisting of many cultures, languages, social structures and differing colonial experiences. For many Pacific peoples learning is holistic, is driven by cultural motivation, which is often for the benefit of their extended families(the collective)rather than personal gains or self-actualisation. Learning is constantly intervened by spiritual matters, the same being true for most aspects of Pacific peoples’ day-to-day lives. Therefore spirituality is integral to learning at all levels formal and informal.

year: 2012

title: Māori leaders wellbeing: A self-determination perspective

authors: Roche, Maree A.

subjects: Positive psychology

abstract: This research draws on interviews with 18 Māori leaders from various leadership positions within business, community, political and marae organisations, to garner an understanding of how their leadership roles interact with their own well-being. Analysis of interviews revealed that crosscultural developments in self-determination theory could be gained by incorporating Māori tikanga and values into a model of well-being for Māori leaders. Largely, the principles of tino rangatiratanga (autonomy and self-determination), mana (respect and influence), whānau (extended family), whakapapa (shared history) and whanaungatanga (kin relations, consultation and engagement), were united into a model of leader well-being. This ensured that mātauranga Māori (Māori knowledge) informed our model of Māori leader well-being, while also drawing on the burgeoning Western research in the area of well-being, specifically self-determination theory. Overall, we find that similarities exist with self-determination theory and Māori tikanga and values. However, in contrast to self-determination theory, autonomy and competence are developed within relationships, which means that ‘others’ underpin Māori leaders’ well-being. From this perspective, we present a view of the psychological and well-being resources that Māori leaders draw on to guide them through complex times.

text: Māori leaders wellbeing: A self-determination perspective This research draws on interviews with 18 Māori leaders from various leadership positions within business, community, political and marae organisations, to garner an understanding of how their leadership roles interact with their own well-being. Analysis of interviews revealed that crosscultural developments in self-determination theory could be gained by incorporating Māori tikanga and values into a model of well-being for Māori leaders. Largely, the principles of tino rangatiratanga (autonomy and self-determination), mana (respect and influence), whānau (extended family), whakapapa (shared history) and whanaungatanga (kin relations, consultation and engagement), were united into a model of leader well-being. This ensured that mātauranga Māori (Māori knowledge) informed our model of Māori leader well-being, while also drawing on the burgeoning Western research in the area of well-being, specifically self-determination theory. Overall, we find that similarities exist with self-determination theory and Māori tikanga and values. However, in contrast to self-determination theory, autonomy and competence are developed within relationships, which means that ‘others’ underpin Māori leaders’ well-being. From this perspective, we present a view of the psychological and well-being resources that Māori leaders draw on to guide them through complex times.

year: 2015