title: Characterization of a novel aerobic cellulolytic thermophilic bacterium and its cellulases

authors: Huang, Xiao Ping

abstract: A taxonomic position was established for a thermophilic, cellulolytic bacterium strain COMP.A2 on the basis of both phenotypic and phylogenetic characteristics. The bacterium was a Gram-positive, aerobic endospore-forming rod. Growth was observed between 60 and 70°C, with an optimum at 65°C. The optimum pH for growth was between 6.5 and 7.5. A wide range of substrates including Avicel and Sigmacell 20 could be utilised as sole carbon and energy source at 70°C. Lactate and acetate were produced when grown on cellobiose or amorphous cellulose medium. The organism was very sensitive to antibiotics such as penicilin G, streptomycin, and neomycin. Growth was inhibited by low concentrations of agar or agarose. A preference for uptake of cellobiose over glucose as the substrate was observed in a mixture of glucose and cellobiose medium. The bacterium was unable to grow autotrophically using hydrogen and carbon dioxide. The lack of alicyclic fatty acid in the cell membrane and the neutral growth pH indicates that the strain is not a member of the genus Alicyclobacillus. The G+C content of the DNA was 62.4 mol%. The 16S rRNA sequences showed that strain COMP.A2 is phylogenetically distinct from other members of genus Bacillus and forms a cluster with the genus Alicylobacillus and two facultatively chemolithoautotrophilic hydrogen-oxidizing Bacillus species, B. tusciae and B. schlegelii. Strain COMP.A2 is tentatively proposed as a new species of a new genus, namely Caldibacillus cellulovorans gen. nov., sp. nov. on the basis of its physiological and phylogenetic characters.

Two CMCases (CMCase A and CMCase B) and one β-glucosidase have been purified from the culture filtrate of strain COMP.A2 by Phenyl-Sepharose chromatography, ion exchange chromatography and chromatofocusing. CMCase A and β-glucosidase were shown to be electrophoretically homogeneous on PAGE and SDS-PAGE. CMCase B was partially purified.

The molecular weight of CMCase A was determined to be 85.1 kDa by SDS-PAGE and 174 kDa by size-exclusion chromatography. The isoelectric point of CMCase A was 4.12. With an assay time of 10 min, CMCase A displayed highest activity at 80°C. An optimum pH for activity was in the range of pH 6.5 to 7.0. An active energy was 46.6 KJ mol⁻¹. The enzyme did not lose activity over one hour at 70°C and still possessed 83% of the original activity after three hours incubation at 70°C. The half-life at 80°C was 32 min and the thermostability was increased by the addition of bovine serum albumin. The enzyme activity was inhibited by heavy metal ions (Zn²⁺ and Hg²⁺) and the thiol-specific inhibitor p-chloromercuribenzenesulphonic acid (pCMPS). The CMCase A showed greatest hydrolytic activity on CMC and low activity on Avicel and xylan. CMCase A hydrolyses CMC or amorphous cellulose in an exo-model of action following initial and random attack. The Km value of the enzyme for CMC was 3.4 mg ml⁻¹. The enzyme cleaved cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. Cellotetraose was cleaved to cellobiose. Cellotriose and cellobiose were not degraded.

The β-glucosidase was largely cell-associated. The enzyme has a molecular weight of 52.5 kDa as determin by SDS-PAGE and an isoelectric point of 4.43. With an assay time of 15 min, β-glucosidase displayed highest activity at 75°C. The enzyme showed optimum activity in the range of pH 5.5 to 7.0. An active energy was 35.4 KJ mol⁻¹. The enzyme was inhibited by the thiol-specific inhibitor pCMPS and heavy metal ions (Cd²⁺, Hg²⁺, Ag⁺). The thermostability of β-glucosidase was increased by the addition of trehalose. The half-life of β-glucosidase was less than 5 minutes at 80°C and 2.5 hours at 70°C in comparison with 24 minutes at 80°C and 11.75 hours at 70°C in the presence of trehalose. The enzyme was competitive inhibited by gluconolactone. The β-glucosidase showed very broad substrate specificity and was active against sophorose, cellobiose and pNPG with Km values of 2.0 mM, 3.4 mM and 0.36mM, respectively.

text: Characterization of a novel aerobic cellulolytic thermophilic bacterium and its cellulases A taxonomic position was established for a thermophilic, cellulolytic bacterium strain COMP.A2 on the basis of both phenotypic and phylogenetic characteristics. The bacterium was a Gram-positive, aerobic endospore-forming rod. Growth was observed between 60 and 70°C, with an optimum at 65°C. The optimum pH for growth was between 6.5 and 7.5. A wide range of substrates including Avicel and Sigmacell 20 could be utilised as sole carbon and energy source at 70°C. Lactate and acetate were produced when grown on cellobiose or amorphous cellulose medium. The organism was very sensitive to antibiotics such as penicilin G, streptomycin, and neomycin. Growth was inhibited by low concentrations of agar or agarose. A preference for uptake of cellobiose over glucose as the substrate was observed in a mixture of glucose and cellobiose medium. The bacterium was unable to grow autotrophically using hydrogen and carbon dioxide. The lack of alicyclic fatty acid in the cell membrane and the neutral growth pH indicates that the strain is not a member of the genus Alicyclobacillus. The G+C content of the DNA was 62.4 mol%. The 16S rRNA sequences showed that strain COMP.A2 is phylogenetically distinct from other members of genus Bacillus and forms a cluster with the genus Alicylobacillus and two facultatively chemolithoautotrophilic hydrogen-oxidizing Bacillus species, B. tusciae and B. schlegelii. Strain COMP.A2 is tentatively proposed as a new species of a new genus, namely Caldibacillus cellulovorans gen. nov., sp. nov. on the basis of its physiological and phylogenetic characters.

Two CMCases (CMCase A and CMCase B) and one β-glucosidase have been purified from the culture filtrate of strain COMP.A2 by Phenyl-Sepharose chromatography, ion exchange chromatography and chromatofocusing. CMCase A and β-glucosidase were shown to be electrophoretically homogeneous on PAGE and SDS-PAGE. CMCase B was partially purified.

The molecular weight of CMCase A was determined to be 85.1 kDa by SDS-PAGE and 174 kDa by size-exclusion chromatography. The isoelectric point of CMCase A was 4.12. With an assay time of 10 min, CMCase A displayed highest activity at 80°C. An optimum pH for activity was in the range of pH 6.5 to 7.0. An active energy was 46.6 KJ mol⁻¹. The enzyme did not lose activity over one hour at 70°C and still possessed 83% of the original activity after three hours incubation at 70°C. The half-life at 80°C was 32 min and the thermostability was increased by the addition of bovine serum albumin. The enzyme activity was inhibited by heavy metal ions (Zn²⁺ and Hg²⁺) and the thiol-specific inhibitor p-chloromercuribenzenesulphonic acid (pCMPS). The CMCase A showed greatest hydrolytic activity on CMC and low activity on Avicel and xylan. CMCase A hydrolyses CMC or amorphous cellulose in an exo-model of action following initial and random attack. The Km value of the enzyme for CMC was 3.4 mg ml⁻¹. The enzyme cleaved cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. Cellotetraose was cleaved to cellobiose. Cellotriose and cellobiose were not degraded.

The β-glucosidase was largely cell-associated. The enzyme has a molecular weight of 52.5 kDa as determin by SDS-PAGE and an isoelectric point of 4.43. With an assay time of 15 min, β-glucosidase displayed highest activity at 75°C. The enzyme showed optimum activity in the range of pH 5.5 to 7.0. An active energy was 35.4 KJ mol⁻¹. The enzyme was inhibited by the thiol-specific inhibitor pCMPS and heavy metal ions (Cd²⁺, Hg²⁺, Ag⁺). The thermostability of β-glucosidase was increased by the addition of trehalose. The half-life of β-glucosidase was less than 5 minutes at 80°C and 2.5 hours at 70°C in comparison with 24 minutes at 80°C and 11.75 hours at 70°C in the presence of trehalose. The enzyme was competitive inhibited by gluconolactone. The β-glucosidase showed very broad substrate specificity and was active against sophorose, cellobiose and pNPG with Km values of 2.0 mM, 3.4 mM and 0.36mM, respectively.

year: 1999

title: Investigating probability concepts of secondary pre-service teachers in a game context

authors: Dayal, Hem Chand

abstract: There is a rich literature on students' and teachers' intuitions and misconceptions about probability. However, less attention has been paid to the development of pre-service teachers' probabilistic thinking in teacher education. Based on this, the second author developed a lesson sequence for teaching probability. In particular, it demonstrates how a game context can be used to explore the relationship between experimental and theoretical probabilities in a collaborative learning setting. The lesson sequence integrates concepts and processes related to probability and is grounded in socio-cultural theory. We trialed the sequence with secondary pre-service teachers. This paper focuses on their understanding of the probability concepts embedded in the sequence. Video and audio data indicates that while teachers used a range of strategies and data displays to explain the ideas integrated in the lessons, some reverted to equiprobability bias. The findings also reveal that pre-service teachers can modify their thinking when engaged in rich teaching and learning tasks.

text: Investigating probability concepts of secondary pre-service teachers in a game context There is a rich literature on students' and teachers' intuitions and misconceptions about probability. However, less attention has been paid to the development of pre-service teachers' probabilistic thinking in teacher education. Based on this, the second author developed a lesson sequence for teaching probability. In particular, it demonstrates how a game context can be used to explore the relationship between experimental and theoretical probabilities in a collaborative learning setting. The lesson sequence integrates concepts and processes related to probability and is grounded in socio-cultural theory. We trialed the sequence with secondary pre-service teachers. This paper focuses on their understanding of the probability concepts embedded in the sequence. Video and audio data indicates that while teachers used a range of strategies and data displays to explain the ideas integrated in the lessons, some reverted to equiprobability bias. The findings also reveal that pre-service teachers can modify their thinking when engaged in rich teaching and learning tasks.

year: 2020

title: Monitoring te mana o te wai: Intergrating an indigenous cultural health index with conventional biomonitoring tools for improved freshwater management

authors: Edgecombe, Shana Jade Te Iaa Tarere

subjects: Freshwater

abstract: Globally, there are universal efforts to quantify and address human impacts on ecosystems. In particular, changes in land use and river regulation have led to drastic declines in stream and river ecosystem health. These human impacts are driving the global freshwater biodiversity crisis and degrade ecosystem services provided by aquatic habitats. In Aotearoa New Zealand there is widespread concern about the state of our Nation’s freshwater ecosystems. Coupled with a renaissance in Māori culture, there has been a drive to develop indicators that help articulate cultural values, assess ecosystem health from a cultural perspective, and provide greater agency for Māori in environmental monitoring and management. In my thesis, I sought to integrate an established cultural monitoring framework with conventional biomonitoring tools for improved freshwater management. I hypothesized that using a suite of monitoring approaches would help detect the impacts of human land use on stream ecosystem health, and that cultural indicators would be consistent with conventional measures in diagnosing land use impacts. I also hypothesized that there could be discrepancies between cultural monitoring and conventional approaches, as each represent different perspectives and ways of knowing streams and rivers. 
To test my hypotheses, I selected ten stream sites across the catchment of the Kuratau River, a major tributary of Lake Taupō. Based on River Environment Classification land cover types, I used a balanced study design with five sites draining indigenous forest, and five sites draining pastoral land. I measured key stream physicochemical properties and collected water samples for determination of nutrient concentration and faecal coliform counts. I also characterised instream and riparian habitat. I collected benthic macroinvertebrate samples and sampled water for environmental DNA (eDNA) to assess different facets of biodiversity. I measured decomposition using the Cotton Strip Assay and quantified periphyton biomass using a portable fluorometer. In combination with these approaches, I also used a Cultural Health Index, an established cultural monitoring framework that assesses three components: customary significance, taonga species/mahinga kai (food gathering), and the Cultural Stream Health Measure (CSHM) to assess identified attributes for stream health.
I found that pastoral land uses lead to a decline in stream ecosystem health when compared to sites draining indigenous forest. Pastoral sites had higher specific conductivity and concentrations of total nitrogen and nitrate. Habitat variables changed, with increased sedimentation and more degraded riparian zones in pastoral streams. Decomposition rates as measured by the cotton strip assay increased in the pasture sites, which was likely a response to increased nutrient availability. There were shifts in macroinvertebrate composition, with key indicator taxa for Indigenous forest sites including pollution-sensitive mayflies and stoneflies. Total taxa, EPT (Ephemeroptera, Plecoptera, Trichoptera), and aquatic insect richness all significantly declined in pastoral sites. Increases in primary production were less pronounced, but did increase alongside abundances of grazing caddisflies and snails in the sites draining pastoral catchments. 
The Cultural Health Index indicated that sites in the Kuratau River catchment had customary and mahinga kai values. CSHM responded negatively to pastoral land uses, and the effect size it described was congruent with other responses. The CSHM was strongly correlated with an indicator of riparian habitat condition (the Riparian Condition Index) and had weak negative correlations with sedimentation and stream temperatures. The CSHM was not correlated with macroinvertebrate indices, which may have been because of the relatively few sites sampled, the narrow impact gradient in the Kuratau catchment, and the lack of temporal replication. However, cultural monitoring approaches should be seen to complement conventional environmental monitoring methods for assessing freshwater and not be expected to duplicate them. The strength of the Cultural Health Index approach comes from establishing Māori agency to assess the environment through a Te Ao Māori lens, and to support and foster inclusion for tangata whenua epistemologies in environmental monitoring and resource management. Future research should consider greater hapū involvement and further explore the mechanisms driving changes in stream health and biodiversity.

text: Monitoring te mana o te wai: Intergrating an indigenous cultural health index with conventional biomonitoring tools for improved freshwater management Globally, there are universal efforts to quantify and address human impacts on ecosystems. In particular, changes in land use and river regulation have led to drastic declines in stream and river ecosystem health. These human impacts are driving the global freshwater biodiversity crisis and degrade ecosystem services provided by aquatic habitats. In Aotearoa New Zealand there is widespread concern about the state of our Nation’s freshwater ecosystems. Coupled with a renaissance in Māori culture, there has been a drive to develop indicators that help articulate cultural values, assess ecosystem health from a cultural perspective, and provide greater agency for Māori in environmental monitoring and management. In my thesis, I sought to integrate an established cultural monitoring framework with conventional biomonitoring tools for improved freshwater management. I hypothesized that using a suite of monitoring approaches would help detect the impacts of human land use on stream ecosystem health, and that cultural indicators would be consistent with conventional measures in diagnosing land use impacts. I also hypothesized that there could be discrepancies between cultural monitoring and conventional approaches, as each represent different perspectives and ways of knowing streams and rivers. 
To test my hypotheses, I selected ten stream sites across the catchment of the Kuratau River, a major tributary of Lake Taupō. Based on River Environment Classification land cover types, I used a balanced study design with five sites draining indigenous forest, and five sites draining pastoral land. I measured key stream physicochemical properties and collected water samples for determination of nutrient concentration and faecal coliform counts. I also characterised instream and riparian habitat. I collected benthic macroinvertebrate samples and sampled water for environmental DNA (eDNA) to assess different facets of biodiversity. I measured decomposition using the Cotton Strip Assay and quantified periphyton biomass using a portable fluorometer. In combination with these approaches, I also used a Cultural Health Index, an established cultural monitoring framework that assesses three components: customary significance, taonga species/mahinga kai (food gathering), and the Cultural Stream Health Measure (CSHM) to assess identified attributes for stream health.
I found that pastoral land uses lead to a decline in stream ecosystem health when compared to sites draining indigenous forest. Pastoral sites had higher specific conductivity and concentrations of total nitrogen and nitrate. Habitat variables changed, with increased sedimentation and more degraded riparian zones in pastoral streams. Decomposition rates as measured by the cotton strip assay increased in the pasture sites, which was likely a response to increased nutrient availability. There were shifts in macroinvertebrate composition, with key indicator taxa for Indigenous forest sites including pollution-sensitive mayflies and stoneflies. Total taxa, EPT (Ephemeroptera, Plecoptera, Trichoptera), and aquatic insect richness all significantly declined in pastoral sites. Increases in primary production were less pronounced, but did increase alongside abundances of grazing caddisflies and snails in the sites draining pastoral catchments. 
The Cultural Health Index indicated that sites in the Kuratau River catchment had customary and mahinga kai values. CSHM responded negatively to pastoral land uses, and the effect size it described was congruent with other responses. The CSHM was strongly correlated with an indicator of riparian habitat condition (the Riparian Condition Index) and had weak negative correlations with sedimentation and stream temperatures. The CSHM was not correlated with macroinvertebrate indices, which may have been because of the relatively few sites sampled, the narrow impact gradient in the Kuratau catchment, and the lack of temporal replication. However, cultural monitoring approaches should be seen to complement conventional environmental monitoring methods for assessing freshwater and not be expected to duplicate them. The strength of the Cultural Health Index approach comes from establishing Māori agency to assess the environment through a Te Ao Māori lens, and to support and foster inclusion for tangata whenua epistemologies in environmental monitoring and resource management. Future research should consider greater hapū involvement and further explore the mechanisms driving changes in stream health and biodiversity.

year: 2024