title: Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

authors: Addison, Sarah Louise

subjects: bacteria

abstract: RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.

text: Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.

year: 2010-01

title: A Real (Wo)man's Beer: gendered spaces of beer drinking in New Zealand

authors: Hardy, Nicole Amy

subjects: beer

abstract: This thesis examines the ways in which rural, national, and urban spaces become gendered through the practices and representations of beer drinking in New Zealand. 

Critical social theory combined with feminist poststructuralist debates on identities provides the theoretical framework for this research. Two focus groups with Pākehā beer drinkers aged between 18 - 30 years old were conducted; one consisting of six males and the other consisting of six females. Critical textual analysis was also undertaken on five beer advertisements representing the most popular beer brands in New Zealand; Tui, Lion Red, Waikato and Speight's. 

Three points frame the analysis. First, I examine rural and national gendered identities associated with beer drinking. New Zealand's beer drinking cultures are constructed within rural discourses of masculinity. There is not a single masculinity present in New Zealand's beer drinking cultures, rather there are multiple and conflicting masculinities. I suggest that through the need to constantly perform their identity, men create a rural hegemonic masculinity that is both hard, yet vulnerable. I argue that the femininities constructed within these spaces are used to enhance and further enable the hard, yet vulnerable, rural masculinity.

Second, within urban spaces of beer drinking - such as the office, nightclub, clubrooms and home - homosexuals, metrosexuals and women are 'othered'. These identities are defined in relation to the hegemonic norm - 'Hard Man' masculinity - in negative ways. Furthermore, some women perform a hyper masculine identity in order to be included in these beer-drinking spaces.

Finally, I examine the ways in which hegemonic gendered identities in rural, national and urban spaces may be resisted and subverted. I use contradictions from my focus group participants to unsettle the 'Hard Man' masculinity of New Zealand's beer drinking cultures.

text: A Real (Wo)man's Beer: gendered spaces of beer drinking in New Zealand This thesis examines the ways in which rural, national, and urban spaces become gendered through the practices and representations of beer drinking in New Zealand. 

Critical social theory combined with feminist poststructuralist debates on identities provides the theoretical framework for this research. Two focus groups with Pākehā beer drinkers aged between 18 - 30 years old were conducted; one consisting of six males and the other consisting of six females. Critical textual analysis was also undertaken on five beer advertisements representing the most popular beer brands in New Zealand; Tui, Lion Red, Waikato and Speight's. 

Three points frame the analysis. First, I examine rural and national gendered identities associated with beer drinking. New Zealand's beer drinking cultures are constructed within rural discourses of masculinity. There is not a single masculinity present in New Zealand's beer drinking cultures, rather there are multiple and conflicting masculinities. I suggest that through the need to constantly perform their identity, men create a rural hegemonic masculinity that is both hard, yet vulnerable. I argue that the femininities constructed within these spaces are used to enhance and further enable the hard, yet vulnerable, rural masculinity.

Second, within urban spaces of beer drinking - such as the office, nightclub, clubrooms and home - homosexuals, metrosexuals and women are 'othered'. These identities are defined in relation to the hegemonic norm - 'Hard Man' masculinity - in negative ways. Furthermore, some women perform a hyper masculine identity in order to be included in these beer-drinking spaces.

Finally, I examine the ways in which hegemonic gendered identities in rural, national and urban spaces may be resisted and subverted. I use contradictions from my focus group participants to unsettle the 'Hard Man' masculinity of New Zealand's beer drinking cultures.

year: 2007

title: Lake Rotokakahi water quality modelling

authors: Jones, Hannah Frances Emily

abstract: Lake Rotokakahi is an iwi-owned lake, administered by the Lake Rotokakahi Board of Control. The lake has a surface area of 4.4 km2, a mean depth of 17.5 m, and a catchment of 19.7 km2 of which most is in exotic forest (46.5%), with some pasture (27.8%) and regenerating native forest and scrub (25.7%). Lake Rotokakahi has only one permanent surface inflow and it is assumed that the lake must be predominantly groundwater fed. The lake outflow, Te Wairoa Stream, flows into Lake Tarawera. Monitoring indicates that water quality has recently declined in the lake, with a shift from a mesotrophic state in the 1990's, to currently being classified as eutrophic. Increased algal biomass has reduced water clarity and in May 2011 the first ever recorded algae bloom of a toxic cyanobacterium species resulted in a fish kill in Te Wairoa Stream. With the majority of the catchment in either exotic or native forest, the cause for the decline in water quality is unclear. However there is some concern that forestry harvesting operations may lead to increased surface runoff of sediment and associated phosphorus in ephemeral streams that feed into the lake. The objective of this study was to setup a water quality model for Lake Rotokakahi, which in the future may provide a decisionsupport tool for lake managers.
The model used, DYRESM-CAEDYM, is a one-dimensional (1D) coupled hydrodynamic-water quality model that has been widely used in New Zealand and overseas. Methodology for deriving model forcing data (e.g. meteorological data, inflow volumes and nutrient loads), including the application of catchment and lake water balances, is described in this report. The catchment water balance indicates that groundwater and rainfall account for 37% and 42%, respectively, of the total inflow volume to the lake, with surface and ephemeral inflows contributing a smaller proportion (c. 20% combined). Furthermore, it appears that groundwater contributes a significant proportion (c. 60 - 70%) of the total nutrient loads, although surface and ephemeral inflows also contribute a significant proportion (34%) of the total phosphorus load. It should be noted that the catchment water balance is based on a very limited dataset, and therefore subject to significant uncertainty. 
We simulated the period July 2009 to June 2012 with DYRESM-CAEDYM. Model performance statistics indicated reasonable simulation of water quality for the calibration period, but did not satisfactorily capture the magnitude and dynamics of chlorophyll a and some nutrient species over an independent model validation period. There may be a number of reasons for the poor model performance over the validation period. For example, it may be that zooplankton and/or freshwater mussels (kakahi), which were not included in the model configuration, may exert significant grazing pressure on the phytoplankton populations at certain times. Furthermore, the significant uncertainty in the catchment water and nutrient loads most likely will have affected model performance. Nutrient concentrations are routinely monitored only in the surface inflow. There are no measurements of nutrient and sediment loads in ephemeral streams, and there is very limited data on groundwater nutrient concentrations.
In its current form, the DYRESM-CAEDYM model is not suitable for scenario testing of lake management options. It is recommended that effort is directed at quantification of ephemeral and groundwater inflows and their associated nutrient and (particularly for ephemeral inflows) sediment loads. This may be particularly important for ephemeral inflows located close to the lake that may be influenced by forestry harvesting operations. Recent increases in measured total phosphorus and phosphate concentrations in the surface inflow and in the lake, combined with the degradation of water quality that has already occurred over the last 20 years, provides further impetus for addressing this critical information gap. Were new data to  become available, opportunities exist for improving the performance of the current model and for developing a three-dimensional coupled hydrodynamic-water quality model (e.g. ELCOM-CAEDYM) with representation of kakahi and/or zooplankton, which may be able to resolve questions pertinent to the functioning of the lake, and to lake and catchment management.

text: Lake Rotokakahi water quality modelling Lake Rotokakahi is an iwi-owned lake, administered by the Lake Rotokakahi Board of Control. The lake has a surface area of 4.4 km2, a mean depth of 17.5 m, and a catchment of 19.7 km2 of which most is in exotic forest (46.5%), with some pasture (27.8%) and regenerating native forest and scrub (25.7%). Lake Rotokakahi has only one permanent surface inflow and it is assumed that the lake must be predominantly groundwater fed. The lake outflow, Te Wairoa Stream, flows into Lake Tarawera. Monitoring indicates that water quality has recently declined in the lake, with a shift from a mesotrophic state in the 1990's, to currently being classified as eutrophic. Increased algal biomass has reduced water clarity and in May 2011 the first ever recorded algae bloom of a toxic cyanobacterium species resulted in a fish kill in Te Wairoa Stream. With the majority of the catchment in either exotic or native forest, the cause for the decline in water quality is unclear. However there is some concern that forestry harvesting operations may lead to increased surface runoff of sediment and associated phosphorus in ephemeral streams that feed into the lake. The objective of this study was to setup a water quality model for Lake Rotokakahi, which in the future may provide a decisionsupport tool for lake managers.
The model used, DYRESM-CAEDYM, is a one-dimensional (1D) coupled hydrodynamic-water quality model that has been widely used in New Zealand and overseas. Methodology for deriving model forcing data (e.g. meteorological data, inflow volumes and nutrient loads), including the application of catchment and lake water balances, is described in this report. The catchment water balance indicates that groundwater and rainfall account for 37% and 42%, respectively, of the total inflow volume to the lake, with surface and ephemeral inflows contributing a smaller proportion (c. 20% combined). Furthermore, it appears that groundwater contributes a significant proportion (c. 60 - 70%) of the total nutrient loads, although surface and ephemeral inflows also contribute a significant proportion (34%) of the total phosphorus load. It should be noted that the catchment water balance is based on a very limited dataset, and therefore subject to significant uncertainty. 
We simulated the period July 2009 to June 2012 with DYRESM-CAEDYM. Model performance statistics indicated reasonable simulation of water quality for the calibration period, but did not satisfactorily capture the magnitude and dynamics of chlorophyll a and some nutrient species over an independent model validation period. There may be a number of reasons for the poor model performance over the validation period. For example, it may be that zooplankton and/or freshwater mussels (kakahi), which were not included in the model configuration, may exert significant grazing pressure on the phytoplankton populations at certain times. Furthermore, the significant uncertainty in the catchment water and nutrient loads most likely will have affected model performance. Nutrient concentrations are routinely monitored only in the surface inflow. There are no measurements of nutrient and sediment loads in ephemeral streams, and there is very limited data on groundwater nutrient concentrations.
In its current form, the DYRESM-CAEDYM model is not suitable for scenario testing of lake management options. It is recommended that effort is directed at quantification of ephemeral and groundwater inflows and their associated nutrient and (particularly for ephemeral inflows) sediment loads. This may be particularly important for ephemeral inflows located close to the lake that may be influenced by forestry harvesting operations. Recent increases in measured total phosphorus and phosphate concentrations in the surface inflow and in the lake, combined with the degradation of water quality that has already occurred over the last 20 years, provides further impetus for addressing this critical information gap. Were new data to  become available, opportunities exist for improving the performance of the current model and for developing a three-dimensional coupled hydrodynamic-water quality model (e.g. ELCOM-CAEDYM) with representation of kakahi and/or zooplankton, which may be able to resolve questions pertinent to the functioning of the lake, and to lake and catchment management.

year: 2014