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: Evaluation of granular media to wastewater treatment; advanced primary treatment utilising modified pumice, titanomagnetite and ceramic nozzles.

authors: Blättler, Anna

abstract: The treatment of wastewater to higher standards is becoming increasingly important as legislation and public perception demand reduced impact of human activity on the environment. Filtration of primary treated effluent (PTE) and secondary treated effluent (STE) offers a possible sustainable technology for meeting or partially meeting these demands.

Granular media filtration, if used at all, is typically incorporated in wastewater treatment as a polishing step following secondary biological treatment and flocculation. The use of filtration following primary sedimentation has not been practised widely. The objective of this research was to investigate the feasibility of filtering PTE using New Zealand’s indigenous volcanic deposits as filter media in a recently developed dual media filtration system incorporating porous ceramic nozzles. The two media types studied included titanomagnetite (TM) and Silicon Sponge (SS). Titanomagnetite is an ironsand concentrate with a very fine grain size (0.07 to 0.18mm). Silicon Sponge is abraded river pumice and was available in two sizes: SS 14/24 (0.6 to 1 mm) and SS 7 /14 (0.4 to 2 mm). The surface of TM grains is smooth and that of SS is highly porous due to the vesiculated structure of pumice, while maintaining a low abrasive factor.

In order to test filtration behaviour of TM and SS media, bench-scale experiments, using glass columns were performed. A model effluent was prepared from reconstituted dairy farm manure. Suspended particle sizes in farm dairy effluent (FDE) were similar to those measured for PTE. Most of the bench trials used FDE. Filtration trends were easy monitored by turbidity reduction. About 50% of FDE turbidity was removed by 100 mm TM columns. TM performance was improved by the addition of TM fines but flows were reduced. Unmodified TM retained most particles larger than 5 µm. This would likely to remove protozoa such as giardia and cryptosporidium. The effect of pH on turbidity removal by TM beds was significant. Turbidity removal increased from 40 to 80% upon the lowering of the effluent pH from 6.5 to 3.5. Raising the pH above 6.5 had no effect. No pH-induced change in particle size distribution of the effluent was observed. A pH of 3.5 is below the isoelectric point of TM and it is likely that the enhanced turbidity removal was due to electrostatic effects.

In bench-scale filtration of PTE at pH 6.5, a 100 mm depth of TM removed 30% of the turbidity. Increasing the bed depth for TM did not lead to significant further improvement in filter performance. In the case of filtration by a fine SS (14/24), 25% turbidity removal was achieved using a 500 mm bed. Increasing the bed depth to 1800 mm increased turbidity removal to 50%. In glass column experiments, a 1000 mm deep bed of coarse SS (7 /14) removed 30% of the turbidity. An increase to 35% occurred when the bed depth, using this medium was increased to 1600 mm. The same media when used in larger pilot plant filters removed 50% of the turbidity. A particular feature of the TM medium was its ease of fluidisation at flowrates < 10 m/h. This characteristics make it a suitable medium for pulsed bed operation. A feature of the SS medium was its large hydraulic conductivity and high solids holding capacity.

On the basis of the results obtained from the bench trials, evaluation filter units incorporating the essential features of the porous ceramic dual media (PCDM) system were constructed. The evaluation systems were equipped with under drains that incorporated a variety of purpose designed titanomagnetite ceramic (TMC) nozzles held in place by a tube bolt that also delivered air and water during filtration and backwashing. Investigation of the flow characteristics of the TMC nozzles showed that the major flow-regulating factor in the nozzle system was the water orifice in the bolt stem. Resistance in up-flow was consistently larger than resistance in down-flow. This was attributed to differences in the hydrodynamics of flow in each direction. Re-designing the bolt could lead to a 30% reduced flow-restriction during backwashing.

Biofilm growth on the TM and SS filtration media and the TMC used to manufacture the ceramic nozzles was studied by prolonged immersion in partially digested effluent. SS media showed a high degree of vesicular space and under aerobic conditions, affinity for loose attachment of organic material. TM showed most anaerobic film growth, accompanied by formation of a red iron oxide deposit. Significantly little biofilm growth on the ceramic material was observed. Such growth, if it occurred, could block the nozzles.
Pilot plant evaluation trials using PTE showed that for SS, filtration efficiency increased steeply with bed depth up to a bed depth of 500 mm, irrespective of the grade of SS used. At depths greater than 500 mm, filtration efficiency increased more slowly with depth. Run times were typically of 50 h duration by which time flow rates under a constant head of 2700 mm had decreased from 12 m/h to 1 m/h. Bed loadings were as high as 100 kg/m³ at the top of the bed with a mean of 22 kg/m³. In the case of TM, a minimum bed depth of 100 mm was required in order to accommodate the TMC tube nozzles. Deeper beds blocked after very short run times. Flowrates of 1 m/h or less and run times of 1 h or less were observed.

For both types of media, solids were easily dislodged during backwashing.

Pulsed backwashing greatly reduced backwash water requirements and in the case of SS beds yielded sludge with solids content suitable for anaerobic digestion. 

Flow-dynamics through PCDM filter nozzles showed increased resistance in upflow being caused by designs in the nozzle-bolt. Vena contracta effects provide an explanation. 

PCDM filtration using SS as the major medium offers a viable technology for the continuous removal of much of the TSS from PTE. A potential energy saving can be envisaged from the reduced aeration required for reduced BOD loadings going to secondary biological treatment. In addition, the concentrated backwash water from bed backwashing, can be transferred directly to anaerobic digesters. Reduced CO₂ generation and an increase in methane production would off-set installation and running costs of the filter in about 15 years.

text: Evaluation of granular media to wastewater treatment; advanced primary treatment utilising modified pumice, titanomagnetite and ceramic nozzles. The treatment of wastewater to higher standards is becoming increasingly important as legislation and public perception demand reduced impact of human activity on the environment. Filtration of primary treated effluent (PTE) and secondary treated effluent (STE) offers a possible sustainable technology for meeting or partially meeting these demands.

Granular media filtration, if used at all, is typically incorporated in wastewater treatment as a polishing step following secondary biological treatment and flocculation. The use of filtration following primary sedimentation has not been practised widely. The objective of this research was to investigate the feasibility of filtering PTE using New Zealand’s indigenous volcanic deposits as filter media in a recently developed dual media filtration system incorporating porous ceramic nozzles. The two media types studied included titanomagnetite (TM) and Silicon Sponge (SS). Titanomagnetite is an ironsand concentrate with a very fine grain size (0.07 to 0.18mm). Silicon Sponge is abraded river pumice and was available in two sizes: SS 14/24 (0.6 to 1 mm) and SS 7 /14 (0.4 to 2 mm). The surface of TM grains is smooth and that of SS is highly porous due to the vesiculated structure of pumice, while maintaining a low abrasive factor.

In order to test filtration behaviour of TM and SS media, bench-scale experiments, using glass columns were performed. A model effluent was prepared from reconstituted dairy farm manure. Suspended particle sizes in farm dairy effluent (FDE) were similar to those measured for PTE. Most of the bench trials used FDE. Filtration trends were easy monitored by turbidity reduction. About 50% of FDE turbidity was removed by 100 mm TM columns. TM performance was improved by the addition of TM fines but flows were reduced. Unmodified TM retained most particles larger than 5 µm. This would likely to remove protozoa such as giardia and cryptosporidium. The effect of pH on turbidity removal by TM beds was significant. Turbidity removal increased from 40 to 80% upon the lowering of the effluent pH from 6.5 to 3.5. Raising the pH above 6.5 had no effect. No pH-induced change in particle size distribution of the effluent was observed. A pH of 3.5 is below the isoelectric point of TM and it is likely that the enhanced turbidity removal was due to electrostatic effects.

In bench-scale filtration of PTE at pH 6.5, a 100 mm depth of TM removed 30% of the turbidity. Increasing the bed depth for TM did not lead to significant further improvement in filter performance. In the case of filtration by a fine SS (14/24), 25% turbidity removal was achieved using a 500 mm bed. Increasing the bed depth to 1800 mm increased turbidity removal to 50%. In glass column experiments, a 1000 mm deep bed of coarse SS (7 /14) removed 30% of the turbidity. An increase to 35% occurred when the bed depth, using this medium was increased to 1600 mm. The same media when used in larger pilot plant filters removed 50% of the turbidity. A particular feature of the TM medium was its ease of fluidisation at flowrates < 10 m/h. This characteristics make it a suitable medium for pulsed bed operation. A feature of the SS medium was its large hydraulic conductivity and high solids holding capacity.

On the basis of the results obtained from the bench trials, evaluation filter units incorporating the essential features of the porous ceramic dual media (PCDM) system were constructed. The evaluation systems were equipped with under drains that incorporated a variety of purpose designed titanomagnetite ceramic (TMC) nozzles held in place by a tube bolt that also delivered air and water during filtration and backwashing. Investigation of the flow characteristics of the TMC nozzles showed that the major flow-regulating factor in the nozzle system was the water orifice in the bolt stem. Resistance in up-flow was consistently larger than resistance in down-flow. This was attributed to differences in the hydrodynamics of flow in each direction. Re-designing the bolt could lead to a 30% reduced flow-restriction during backwashing.

Biofilm growth on the TM and SS filtration media and the TMC used to manufacture the ceramic nozzles was studied by prolonged immersion in partially digested effluent. SS media showed a high degree of vesicular space and under aerobic conditions, affinity for loose attachment of organic material. TM showed most anaerobic film growth, accompanied by formation of a red iron oxide deposit. Significantly little biofilm growth on the ceramic material was observed. Such growth, if it occurred, could block the nozzles.
Pilot plant evaluation trials using PTE showed that for SS, filtration efficiency increased steeply with bed depth up to a bed depth of 500 mm, irrespective of the grade of SS used. At depths greater than 500 mm, filtration efficiency increased more slowly with depth. Run times were typically of 50 h duration by which time flow rates under a constant head of 2700 mm had decreased from 12 m/h to 1 m/h. Bed loadings were as high as 100 kg/m³ at the top of the bed with a mean of 22 kg/m³. In the case of TM, a minimum bed depth of 100 mm was required in order to accommodate the TMC tube nozzles. Deeper beds blocked after very short run times. Flowrates of 1 m/h or less and run times of 1 h or less were observed.

For both types of media, solids were easily dislodged during backwashing.

Pulsed backwashing greatly reduced backwash water requirements and in the case of SS beds yielded sludge with solids content suitable for anaerobic digestion. 

Flow-dynamics through PCDM filter nozzles showed increased resistance in upflow being caused by designs in the nozzle-bolt. Vena contracta effects provide an explanation. 

PCDM filtration using SS as the major medium offers a viable technology for the continuous removal of much of the TSS from PTE. A potential energy saving can be envisaged from the reduced aeration required for reduced BOD loadings going to secondary biological treatment. In addition, the concentrated backwash water from bed backwashing, can be transferred directly to anaerobic digesters. Reduced CO₂ generation and an increase in methane production would off-set installation and running costs of the filter in about 15 years.

year: 2002

title: A literary and linguistic critique of a Māori lullaby

authors: August, Tipene

subjects: waiata Māori

abstract: Māori song poems surviving from pre-colonial times can provide a valuable source of information about the artistic and cultural heritage of Māoridom. However, they present a number of challenges for the contemporary analyst. On the basis of a stylistic analysis of a Māori lullaby, a number of hypotheses about Māori song poems in general, and the lullaby in particular, are put forward and it is suggested that these hypotheses could be tested in relation to a corpus of song poems. It is hoped that the approach to stylistic analysis adopted here will be of use to scholars of Māori song poems and, perhaps, also to others who are interested in indigenous art forms more generally.

text: A literary and linguistic critique of a Māori lullaby Māori song poems surviving from pre-colonial times can provide a valuable source of information about the artistic and cultural heritage of Māoridom. However, they present a number of challenges for the contemporary analyst. On the basis of a stylistic analysis of a Māori lullaby, a number of hypotheses about Māori song poems in general, and the lullaby in particular, are put forward and it is suggested that these hypotheses could be tested in relation to a corpus of song poems. It is hoped that the approach to stylistic analysis adopted here will be of use to scholars of Māori song poems and, perhaps, also to others who are interested in indigenous art forms more generally.

year: 2001-02