title: Sheet extrusion and plasticisation of Novatein thermoplastic protein

authors: Uitto, Jussi Matias

subjects: sheet extrusion

abstract: The purpose of this study was to develop a relationship between protein structure, rheological properties and plasticization of Novatein. Novatein is a biomass-based thermoplastic in which the main constituent is blood meal, a highly aggregated protein-rich biopolymer, which is a by-product from the meat industry. It can be processed with industrially scalable thermo-mechanical processing methods such as extrusion and injection molding. However, its properties makes more challenging processing methods such as sheet extrusion limited. This is considered a rheology problem and is related to the protein’s structural characteristics that can be modified by using polyol plasticizers such as ethylene glycol (EG), glycerol (GLY), propylene glycol (PG) and triethylene glycol (TEG).

Rheological characterisation revealed the apparent shear viscosity of highly plasticized Novatein and polypropylene (PP) to be very similar even though only PP can be sheet extruded. Novatein’s extensional viscosity was significantly higher and its entrance pressure accounted for up to 80% of the total pressure drop with no-polyol plasticized Novatein. Increasing polyol content and temperature were found to decrease the extensional viscosity but simultaneously increased the shear viscosity due to better flow development in the capillary. In other words, poor elongational properties of the no-polyol composition led to flow behavior closer to plug flow. Thus, with longer capillaries apparent shear viscosity of polyol plasticized samples (at the same water content) could become even higher but also raises the question whether fully developed flow is a desired property for processing Novatein. 

Synchrotron-based FT-IR measurements with support of XRD explained the difference in the rheological performance further. Blood meal’s protein structure was highly aggregated, consisting of up to 50% -sheets that do not melt into a fully amorphous state at suitable processing temperatures. The behavior of Novatein was considered to be closer to filled polymers, consisting of nano-crystallite aggregates, rather than a semi-crystalline polymer that has reached a molten state. This made sufficient plasticization of the amorphous fraction crucial for processability. 

Plasticization was classified into primary and secondary plasticization. In primary plasticization, the plasticizer interacts directly with the protein network by replacing the protein’s hydrogen bonding sites with water. In secondary plasticization, the polymer network becomes saturated, leading to phase separation and increases hygroscopicity. The effect is substrate and plasticizer dependant and secondary plasticization was dominant with Novatein due to its aggregated structure. The plasticizer content at which the equilibrated moisture content (EMC) became equal to that of no-plasticizer compositions was called the point of equivalence (POE). This is also the point at which primary plasticization turns into secondary plasticization. 

The POE is unique for all plasticizers and dependant on molecular characteristics such as size and theoretical hydrogen bonding sites. The EMC is a result of these changes (including secondary structure) and strongly correlates with mechanical properties and the brittle-to-ductile transformation. Water provided the ability to form ideally mixed phases, explaining the applicability of the free volume-based plasticizer theory. The Novatein network consisted of protein-rich, plasticizer-rich and an intermediate phase, and the fractional composition and the relative magnitude of each phase was determined by using the Couchman-Karasz model. The role of the intermediate phase was found to make the biggest difference in plasticizer performance and behaved in accordance to the observed secondary structure changes. 

Of the selected plasticizers, GLY clearly showed the highest tendency to phase separate followed by TEG, PG, and EG. Of the tested plasticizers the POE values varied between 20 pphBM to 29 pphBM in the order of GLY, TEG, PG, and EG. However, TEG had the lowest POE on a molar basis and nearly three times the molar amount of EG was required to reach the POE in comparison to TEG, whereas for GLY it was only 1.3 times. TEG’s plasticization is based on its ability to interact efficiently with the protein network and modify the secondary structure sufficiently. GLY, instead, showed a strong tendency to phase separate leading to the highest hydrogen bonding potential above the POE, and therefore also raised the EMC above TEG level. Despite the increase in ordered secondary structures, GLY led to the highest strain at break, which was attributed to phase separation. EG and PG as smaller sized molecules were able to diffuse into polymer network more efficiently.

Even though the plasticization mechanism varied significantly, EMC was the dominant factor determining mechanical properties with a brittle-to-ductile transformation observed at 8%. However, it is important to understand that an EMC of 8% required different amounts of plasticizer for each polyol. In accordance to the constraint theory, the amount of theoretical hydrogen bonding sites of plasticizer was most appropriate to predict changes EMC. The role of water was significant in forming a ternary system that behaves in accordance to the free volume theory despite the protein’s heterogeneous structure. For dried samples, TEG and GLY formed clusters in the polymer network in the absence of water, whereas for hydrated samples the plasticizer was well distributed through the polymer network, albeit in micro-separated regions.

The concept of primary and secondary plasticization provided a better understanding of rheological behavior as well. Elongational flow was dominated by primary plasticization of the protein-rich and intermediate phases whereas secondary plasticization played a significant role in the reduction of the shear viscosity. Flow without secondary plasticization was characterised as a plug flow. PG showed the most efficient plasticization in both shear and elongational viscosity, which was attributed to the combination of its small molecular size and ability to enhance both primary and secondary plasticization. GLY acted mostly as a secondary plasticizer and had the higher elongational and the lowest shear viscosity. TEG as an efficient primary plasticizer with an ability to modify secondary structure performed exceptionally well in terms of extensional viscosity considering its high molecular weight. Higher shear viscosity levels were comparable to EG that was shown to diffuse very efficiently in the polymer network. 

With a fundamental understanding of plasticization and rheology, significant process improvements were made by increasing temperature and combining PG and TEG leading to efficient sheet extrusion.

text: Sheet extrusion and plasticisation of Novatein thermoplastic protein The purpose of this study was to develop a relationship between protein structure, rheological properties and plasticization of Novatein. Novatein is a biomass-based thermoplastic in which the main constituent is blood meal, a highly aggregated protein-rich biopolymer, which is a by-product from the meat industry. It can be processed with industrially scalable thermo-mechanical processing methods such as extrusion and injection molding. However, its properties makes more challenging processing methods such as sheet extrusion limited. This is considered a rheology problem and is related to the protein’s structural characteristics that can be modified by using polyol plasticizers such as ethylene glycol (EG), glycerol (GLY), propylene glycol (PG) and triethylene glycol (TEG).

Rheological characterisation revealed the apparent shear viscosity of highly plasticized Novatein and polypropylene (PP) to be very similar even though only PP can be sheet extruded. Novatein’s extensional viscosity was significantly higher and its entrance pressure accounted for up to 80% of the total pressure drop with no-polyol plasticized Novatein. Increasing polyol content and temperature were found to decrease the extensional viscosity but simultaneously increased the shear viscosity due to better flow development in the capillary. In other words, poor elongational properties of the no-polyol composition led to flow behavior closer to plug flow. Thus, with longer capillaries apparent shear viscosity of polyol plasticized samples (at the same water content) could become even higher but also raises the question whether fully developed flow is a desired property for processing Novatein. 

Synchrotron-based FT-IR measurements with support of XRD explained the difference in the rheological performance further. Blood meal’s protein structure was highly aggregated, consisting of up to 50% -sheets that do not melt into a fully amorphous state at suitable processing temperatures. The behavior of Novatein was considered to be closer to filled polymers, consisting of nano-crystallite aggregates, rather than a semi-crystalline polymer that has reached a molten state. This made sufficient plasticization of the amorphous fraction crucial for processability. 

Plasticization was classified into primary and secondary plasticization. In primary plasticization, the plasticizer interacts directly with the protein network by replacing the protein’s hydrogen bonding sites with water. In secondary plasticization, the polymer network becomes saturated, leading to phase separation and increases hygroscopicity. The effect is substrate and plasticizer dependant and secondary plasticization was dominant with Novatein due to its aggregated structure. The plasticizer content at which the equilibrated moisture content (EMC) became equal to that of no-plasticizer compositions was called the point of equivalence (POE). This is also the point at which primary plasticization turns into secondary plasticization. 

The POE is unique for all plasticizers and dependant on molecular characteristics such as size and theoretical hydrogen bonding sites. The EMC is a result of these changes (including secondary structure) and strongly correlates with mechanical properties and the brittle-to-ductile transformation. Water provided the ability to form ideally mixed phases, explaining the applicability of the free volume-based plasticizer theory. The Novatein network consisted of protein-rich, plasticizer-rich and an intermediate phase, and the fractional composition and the relative magnitude of each phase was determined by using the Couchman-Karasz model. The role of the intermediate phase was found to make the biggest difference in plasticizer performance and behaved in accordance to the observed secondary structure changes. 

Of the selected plasticizers, GLY clearly showed the highest tendency to phase separate followed by TEG, PG, and EG. Of the tested plasticizers the POE values varied between 20 pphBM to 29 pphBM in the order of GLY, TEG, PG, and EG. However, TEG had the lowest POE on a molar basis and nearly three times the molar amount of EG was required to reach the POE in comparison to TEG, whereas for GLY it was only 1.3 times. TEG’s plasticization is based on its ability to interact efficiently with the protein network and modify the secondary structure sufficiently. GLY, instead, showed a strong tendency to phase separate leading to the highest hydrogen bonding potential above the POE, and therefore also raised the EMC above TEG level. Despite the increase in ordered secondary structures, GLY led to the highest strain at break, which was attributed to phase separation. EG and PG as smaller sized molecules were able to diffuse into polymer network more efficiently.

Even though the plasticization mechanism varied significantly, EMC was the dominant factor determining mechanical properties with a brittle-to-ductile transformation observed at 8%. However, it is important to understand that an EMC of 8% required different amounts of plasticizer for each polyol. In accordance to the constraint theory, the amount of theoretical hydrogen bonding sites of plasticizer was most appropriate to predict changes EMC. The role of water was significant in forming a ternary system that behaves in accordance to the free volume theory despite the protein’s heterogeneous structure. For dried samples, TEG and GLY formed clusters in the polymer network in the absence of water, whereas for hydrated samples the plasticizer was well distributed through the polymer network, albeit in micro-separated regions.

The concept of primary and secondary plasticization provided a better understanding of rheological behavior as well. Elongational flow was dominated by primary plasticization of the protein-rich and intermediate phases whereas secondary plasticization played a significant role in the reduction of the shear viscosity. Flow without secondary plasticization was characterised as a plug flow. PG showed the most efficient plasticization in both shear and elongational viscosity, which was attributed to the combination of its small molecular size and ability to enhance both primary and secondary plasticization. GLY acted mostly as a secondary plasticizer and had the higher elongational and the lowest shear viscosity. TEG as an efficient primary plasticizer with an ability to modify secondary structure performed exceptionally well in terms of extensional viscosity considering its high molecular weight. Higher shear viscosity levels were comparable to EG that was shown to diffuse very efficiently in the polymer network. 

With a fundamental understanding of plasticization and rheology, significant process improvements were made by increasing temperature and combining PG and TEG leading to efficient sheet extrusion.

year: 2018

title: Improved determination of nitrate concentrations and flow rates in freshwater using ‘Diffusive Gradients in Thin-films’

authors: Corbett, Thomas D. W.

subjects: DGT

abstract: Nutrient pollution, particularly by nitrate, is a persistent issue in waterways. Numerous mitigation strategies have been developed to address this pollution, such as denitrifying bioreactors. The monitoring, however, of nutrient concentrations and mitigation strategy performance has relied on infrequent grab sampling, or expensive on-site equipment. There are numerous challenges with these techniques, such as sample preservation and transport, operational expense, and the ability to account for temporal changes in concentration. Diffusive Gradients in Thin Films (DGT) are capable of overcoming many of the challenges, however, to date their use has mainly been research based, and they are not widely used by monitoring authorities or communities. Diffusive Gradients in Thin Films are passive accumulative chemical monitoring devices, designed to be analyte specific. This thesis sought, firstly, to establish the efficacy of a modified DGT for the monitoring of nitrate, following deployment in chemically and physically complex denitrifying bioreactors. Secondly, the extension of the DGT methodology through (A) incorporation of colour reagents into the binding layer for rapid in-field analysis of nitrate concentrations, (B) developing a DGT method for the determination of flow rates, and (C) understanding the potential biases inherent in DGT methodology for determining concentrations and loads.
The first study focused on the utility of DGT for the determination of denitrifying bioreactor performance – via deployment in two denitrifying bioreactors. DGT overcame many of the challenges of grab sampling, such as more easily accounting for temporal nitrate concentration variation, and reduced analytical requirements. Diffusive Gradients in Thin Films determined nitrate concentrations and removal rates were in strong agreement with high frequency grab sampling, and data collection via DGT was considerably easier than high frequency grab sampling. The DGT, however, still required in-lab analysis for nitrate.
Development of colourimetric binding layers, as a hydrogel or liquid binding layer, might allow infield determination of analyte concentrations easily and accurately. Here, a chitosan-stabilised AuNP suspension, as a liquid binding layer was developed and tested for the colourimetric determination of nitrite concentrations (0 to 1000 mg L⁻¹) and masses (145 μg) in lab based colour development studies. Nitrate reduction to nitrite was achieved through the development of an Fe(0) impregnated poly-2-acrylamido-2-methyl-1-propanesulfonic acid/acrylamide copolymer hydrogel. The developments lay the foundations for further development of the colourimetric-DGT concept.
A novel bromide selective DGT (Br--DGT) was developed, using the Purolite Bromide Plus anion exchange resin, which provided stream flow rates when combined with the constant rate tracer-injection method. The Br--DGT flow method was tested infield at the Mangaharakeke Stream. Flow rates determined by DGT were between -14.7 and 6.5 % of the flow independently monitored weir flow rate. In comparison, grab sample flow rates diverged by 5.5 to 58.9 % from the weir flow rate.
Dynamic and coordinated changes in temperature, flow, and concentration, as potential sources of bias in concentration and load calculations using DGT and grab sampling were modelled. Large, dynamic, and highly correlated to concentration, temperature changes have minimal effect on the calculated DGT concentration. In contrast, as the correlation between concentration and flow increases the bias in calculated DGT loads becomes significant. This means that in systems where there is a high correlation of concentration and flow DGT may not be appropriate for determining loadings.
This thesis had important implications for the use of DGT for determining nutrient concentrations and loads, and stream flow rates – and the monitoring of nutrient pollution. Firstly, it demonstrated that DGT were a useful tool for the determination of nitrate removal rates in the chemically and physically challenging denitrifying bioreactor systems. Secondly, it established the foundation for the in-field determination of nitrate concentrations. Lastly, it extended the DGT methodology for the determination of bromide concentrations and stream flow rates, and provided a greater understanding of the issues when using DGT to determine nitrate loads. This research opened the possibility of DGT for large-scale nutrient monitoring and determination of nutrient mitigation strategy performance, given the quality of DGT data and ease-of-use compared to grab sampling.

text: Improved determination of nitrate concentrations and flow rates in freshwater using ‘Diffusive Gradients in Thin-films’ Nutrient pollution, particularly by nitrate, is a persistent issue in waterways. Numerous mitigation strategies have been developed to address this pollution, such as denitrifying bioreactors. The monitoring, however, of nutrient concentrations and mitigation strategy performance has relied on infrequent grab sampling, or expensive on-site equipment. There are numerous challenges with these techniques, such as sample preservation and transport, operational expense, and the ability to account for temporal changes in concentration. Diffusive Gradients in Thin Films (DGT) are capable of overcoming many of the challenges, however, to date their use has mainly been research based, and they are not widely used by monitoring authorities or communities. Diffusive Gradients in Thin Films are passive accumulative chemical monitoring devices, designed to be analyte specific. This thesis sought, firstly, to establish the efficacy of a modified DGT for the monitoring of nitrate, following deployment in chemically and physically complex denitrifying bioreactors. Secondly, the extension of the DGT methodology through (A) incorporation of colour reagents into the binding layer for rapid in-field analysis of nitrate concentrations, (B) developing a DGT method for the determination of flow rates, and (C) understanding the potential biases inherent in DGT methodology for determining concentrations and loads.
The first study focused on the utility of DGT for the determination of denitrifying bioreactor performance – via deployment in two denitrifying bioreactors. DGT overcame many of the challenges of grab sampling, such as more easily accounting for temporal nitrate concentration variation, and reduced analytical requirements. Diffusive Gradients in Thin Films determined nitrate concentrations and removal rates were in strong agreement with high frequency grab sampling, and data collection via DGT was considerably easier than high frequency grab sampling. The DGT, however, still required in-lab analysis for nitrate.
Development of colourimetric binding layers, as a hydrogel or liquid binding layer, might allow infield determination of analyte concentrations easily and accurately. Here, a chitosan-stabilised AuNP suspension, as a liquid binding layer was developed and tested for the colourimetric determination of nitrite concentrations (0 to 1000 mg L⁻¹) and masses (145 μg) in lab based colour development studies. Nitrate reduction to nitrite was achieved through the development of an Fe(0) impregnated poly-2-acrylamido-2-methyl-1-propanesulfonic acid/acrylamide copolymer hydrogel. The developments lay the foundations for further development of the colourimetric-DGT concept.
A novel bromide selective DGT (Br--DGT) was developed, using the Purolite Bromide Plus anion exchange resin, which provided stream flow rates when combined with the constant rate tracer-injection method. The Br--DGT flow method was tested infield at the Mangaharakeke Stream. Flow rates determined by DGT were between -14.7 and 6.5 % of the flow independently monitored weir flow rate. In comparison, grab sample flow rates diverged by 5.5 to 58.9 % from the weir flow rate.
Dynamic and coordinated changes in temperature, flow, and concentration, as potential sources of bias in concentration and load calculations using DGT and grab sampling were modelled. Large, dynamic, and highly correlated to concentration, temperature changes have minimal effect on the calculated DGT concentration. In contrast, as the correlation between concentration and flow increases the bias in calculated DGT loads becomes significant. This means that in systems where there is a high correlation of concentration and flow DGT may not be appropriate for determining loadings.
This thesis had important implications for the use of DGT for determining nutrient concentrations and loads, and stream flow rates – and the monitoring of nutrient pollution. Firstly, it demonstrated that DGT were a useful tool for the determination of nitrate removal rates in the chemically and physically challenging denitrifying bioreactor systems. Secondly, it established the foundation for the in-field determination of nitrate concentrations. Lastly, it extended the DGT methodology for the determination of bromide concentrations and stream flow rates, and provided a greater understanding of the issues when using DGT to determine nitrate loads. This research opened the possibility of DGT for large-scale nutrient monitoring and determination of nutrient mitigation strategy performance, given the quality of DGT data and ease-of-use compared to grab sampling.

year: 2021

title: Telomere length in early childhood is associated with sex and ethnicity

authors: Ly, Kien

subjects: Science & Technology

abstract: Telomeres are repetitive DNA sequences at the end of chromosomes that function to protect chromosomes from degradation. Throughout the life course, telomere length decreases with age and is influenced by environmental factors and health conditions. This study aimed to determine the relative telomere lengths in a diverse cohort of about 4000 four-year-old children in New Zealand. Linear regression was used to investigate the relationship between telomere length, child gender, ethnicity, paternal age and deprivation. We observed substantial variation in telomere length according to sex and self-identified ethnicity. Telomere length was longer in females compared to males (coefficient of 0.042, 95% confidence interval (CI) 0.024–0.060). European children had shorter telomere than both the indigenous Māori (coefficient of 0.03, CI 0.007–0.055) and Pacific children (coefficient of 0.15, CI 0.12–0.18). The data suggest that telomere lengths are highly variable and variability between individuals arise from early age, influenced partly by sex and ethnicity. Longer telomeres in indigenous Māori and Pacific children may reflect the heritability of telomere length in genetically less complex populations. This study increases our understanding of telomere dynamics in young children since the majority of telomere studies are conducted in adults.

text: Telomere length in early childhood is associated with sex and ethnicity Telomeres are repetitive DNA sequences at the end of chromosomes that function to protect chromosomes from degradation. Throughout the life course, telomere length decreases with age and is influenced by environmental factors and health conditions. This study aimed to determine the relative telomere lengths in a diverse cohort of about 4000 four-year-old children in New Zealand. Linear regression was used to investigate the relationship between telomere length, child gender, ethnicity, paternal age and deprivation. We observed substantial variation in telomere length according to sex and self-identified ethnicity. Telomere length was longer in females compared to males (coefficient of 0.042, 95% confidence interval (CI) 0.024–0.060). European children had shorter telomere than both the indigenous Māori (coefficient of 0.03, CI 0.007–0.055) and Pacific children (coefficient of 0.15, CI 0.12–0.18). The data suggest that telomere lengths are highly variable and variability between individuals arise from early age, influenced partly by sex and ethnicity. Longer telomeres in indigenous Māori and Pacific children may reflect the heritability of telomere length in genetically less complex populations. This study increases our understanding of telomere dynamics in young children since the majority of telomere studies are conducted in adults.

year: 2019