title: Microbial succession under freeze–thaw events and its potential for hydrocarbon degradation in nutrient-amended Antarctic soil

authors: de Jesus, HE

subjects: Science & Technology

abstract: The polar regions have relatively low richness and diversity of plants and animals, and the basis of the entire ecological chain is supported by microbial diversity. In these regions, understanding the microbial response against environmental factors and anthropogenic disturbances is essential to understand patterns better, prevent isolated events, and apply biotechnology strategies. The Antarctic continent has been increasingly affected by anthropogenic contamination, and its constant temperature fluctuations limit the application of clean recovery strategies, such as bioremediation. We evaluated the bacterial response in oil-contaminated soil through a nutrient-amended microcosm experiment using two temperature regimes: (i) 4 C and (ii) a freeze–thaw cycle (FTC) alternating between −20 and 4 C. Bacterial taxa, such as Myxococcales, Chitinophagaceae, and Acidimicrobiales, were strongly related to the FTC. Rhodococcus was positively related to contaminated soils and further stimulated under FTC conditions. Additionally, the nutrient-amended treatment under the FTC regime enhanced bacterial groups with known biodegradation potential and was efficient in removing hydrocarbons of diesel oil. The experimental design, rates of bacterial succession, and level of hydrocarbon transformation can be considered as a baseline for further studies aimed at improving bioremediation strategies in environments affected by FTC regimes. ◦ ◦

text: Microbial succession under freeze–thaw events and its potential for hydrocarbon degradation in nutrient-amended Antarctic soil The polar regions have relatively low richness and diversity of plants and animals, and the basis of the entire ecological chain is supported by microbial diversity. In these regions, understanding the microbial response against environmental factors and anthropogenic disturbances is essential to understand patterns better, prevent isolated events, and apply biotechnology strategies. The Antarctic continent has been increasingly affected by anthropogenic contamination, and its constant temperature fluctuations limit the application of clean recovery strategies, such as bioremediation. We evaluated the bacterial response in oil-contaminated soil through a nutrient-amended microcosm experiment using two temperature regimes: (i) 4 C and (ii) a freeze–thaw cycle (FTC) alternating between −20 and 4 C. Bacterial taxa, such as Myxococcales, Chitinophagaceae, and Acidimicrobiales, were strongly related to the FTC. Rhodococcus was positively related to contaminated soils and further stimulated under FTC conditions. Additionally, the nutrient-amended treatment under the FTC regime enhanced bacterial groups with known biodegradation potential and was efficient in removing hydrocarbons of diesel oil. The experimental design, rates of bacterial succession, and level of hydrocarbon transformation can be considered as a baseline for further studies aimed at improving bioremediation strategies in environments affected by FTC regimes. ◦ ◦

year: 2021-03-01

title: Cultivating microbial dark matter - A combination of traditional and molecular techniques

authors: King, Charlotte

abstract: The services bacteria offer the world are immense and impact people's lives daily. They were first described by Antoni van Leeuwenhoek in 1676, but bacteria have been utilised in the medical, agricultural, and food and beverage industries even before they were formally identified. Microbes’ roles in the environment for cycling of nutrients, climate regulation and pollution are becoming more well understood, and they are also utilised to answer a wide variety of research questions in disparate fields. Traditional cultivation techniques were initially developed to study bacteria and understand their functions as isolates. Such cultivation techniques have become rarer as new molecular and bioinformatic tools have been developed. These cultivation- independent techniques have allowed the study of microbial life genetic data which led to the finding that 99% of all microbial species were yet to be cultured and studied in a laboratory. The applications of these unstudied organisms could be unprecedented, though their cultivation remains challenging due to the requirement of specific nutrients that are hard to identify, very slow growth rates, environmental conditions that are difficult to replicate in a laboratory setting, live in symbiosis with other organisms, or live in extreme climates. Therefore, much of the information may be overlooked and hidden in this microbial dark matter. Methods such as metagenomics, metagenome-assembled genomes (MAGs), and single amplified genomes (SAGs) have provided us with genomic data for previously uncharacterised microbes from diverse environments. However, such data is still limited. Omics tools can provide insights into what is living there and what it can potentially do. However, this data is regularly incomplete due to the processes involved in sequencing, and conclusions are often drawn from associations relating to cultured organisms. This can lead to potentially spurious conclusions as a gene may behave differently in different species, and the absence of genes may depend on how the data was analysed. Therefore, cultured isolates of the organism are vital. To combat this issue of an uncultivated majority, it is important to have a targeted approach to culture, what is wanted, not just what is easy.

This thesis aimed to provide an easy-to-follow, straightforward workflow to cultivate target environmental organisms using standard molecular tools and methods readily accessible in any standard microbiological laboratory. The idea is to isolate and culture what is specifically wanted or needed, not just what is easy. Specifically, it combined classical cultivation and molecular techniques to target a sulphur-reducing, obligate anaerobe from an environmental sample— namely a Desulfurella sp. from a geothermal pool in Rotorua, New Zealand.
In order to provide the desired targeted approach, specific primers needed to be designed and assessed to first find the right sampling site for the initial inoculum and second to ensure the target is not lost throughout the culturing process. I designed and evaluated primers for targeting specific taxa at different stringency levels. Initially, I tested published primers (EPS_F/EPS_R, Gittel et al., 2012) aimed at Epsilonproteobacteria, which were reclassified into the Campylobacterota phylum (including Desulfurella sp.). These primers did not target Desulfurella sp., so I modified them (EPS_FM/EPS_RL) to produce a longer amplicon for broader Campylobacterota detection. However, these also amplified non-target species (e.g., Desulfobacterota). Finally, I designed a third set of primers (DS_F/DS_R) specific to Desulfurella sp., confirmed by in silico testing. These primers allow assessment of Desulfurella sp. and broader Campylobacterota before sequencing.
The second experimental chapter focuses on enriching the target using the previously described primers, classical cultivation techniques, and molecular tools. Site selection was based on the 1000 Springs Project (Power et al., 2018). Culturing was performed originally with DSMZ Desulfurella medium and later modified, investigating pH (4 and 6) and temperature (50°C and 30°C) variations and adapting it for solid media by replacing elemental sulphur with sodium polysulfide. I used a vacuum food sealer to cultivate anaerobes, an accessible and portable alternative to expensive anaerobic chambers. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to assess axenicity before sending samples for 16S rRNA gene and whole genome sequencing. Desulfurella was detected in each culture using species-specific primers (DS_F/DS_R), enabling its phylogenetic placement. However, despite Desulfurella presence in the 16S rRNA gene amplicon sequencing, a metagenome-assembled genome (MAG) could not be produced. This thesis discusses lessons learned and suggests improvements for future studies.

text: Cultivating microbial dark matter - A combination of traditional and molecular techniques The services bacteria offer the world are immense and impact people's lives daily. They were first described by Antoni van Leeuwenhoek in 1676, but bacteria have been utilised in the medical, agricultural, and food and beverage industries even before they were formally identified. Microbes’ roles in the environment for cycling of nutrients, climate regulation and pollution are becoming more well understood, and they are also utilised to answer a wide variety of research questions in disparate fields. Traditional cultivation techniques were initially developed to study bacteria and understand their functions as isolates. Such cultivation techniques have become rarer as new molecular and bioinformatic tools have been developed. These cultivation- independent techniques have allowed the study of microbial life genetic data which led to the finding that 99% of all microbial species were yet to be cultured and studied in a laboratory. The applications of these unstudied organisms could be unprecedented, though their cultivation remains challenging due to the requirement of specific nutrients that are hard to identify, very slow growth rates, environmental conditions that are difficult to replicate in a laboratory setting, live in symbiosis with other organisms, or live in extreme climates. Therefore, much of the information may be overlooked and hidden in this microbial dark matter. Methods such as metagenomics, metagenome-assembled genomes (MAGs), and single amplified genomes (SAGs) have provided us with genomic data for previously uncharacterised microbes from diverse environments. However, such data is still limited. Omics tools can provide insights into what is living there and what it can potentially do. However, this data is regularly incomplete due to the processes involved in sequencing, and conclusions are often drawn from associations relating to cultured organisms. This can lead to potentially spurious conclusions as a gene may behave differently in different species, and the absence of genes may depend on how the data was analysed. Therefore, cultured isolates of the organism are vital. To combat this issue of an uncultivated majority, it is important to have a targeted approach to culture, what is wanted, not just what is easy.

This thesis aimed to provide an easy-to-follow, straightforward workflow to cultivate target environmental organisms using standard molecular tools and methods readily accessible in any standard microbiological laboratory. The idea is to isolate and culture what is specifically wanted or needed, not just what is easy. Specifically, it combined classical cultivation and molecular techniques to target a sulphur-reducing, obligate anaerobe from an environmental sample— namely a Desulfurella sp. from a geothermal pool in Rotorua, New Zealand.
In order to provide the desired targeted approach, specific primers needed to be designed and assessed to first find the right sampling site for the initial inoculum and second to ensure the target is not lost throughout the culturing process. I designed and evaluated primers for targeting specific taxa at different stringency levels. Initially, I tested published primers (EPS_F/EPS_R, Gittel et al., 2012) aimed at Epsilonproteobacteria, which were reclassified into the Campylobacterota phylum (including Desulfurella sp.). These primers did not target Desulfurella sp., so I modified them (EPS_FM/EPS_RL) to produce a longer amplicon for broader Campylobacterota detection. However, these also amplified non-target species (e.g., Desulfobacterota). Finally, I designed a third set of primers (DS_F/DS_R) specific to Desulfurella sp., confirmed by in silico testing. These primers allow assessment of Desulfurella sp. and broader Campylobacterota before sequencing.
The second experimental chapter focuses on enriching the target using the previously described primers, classical cultivation techniques, and molecular tools. Site selection was based on the 1000 Springs Project (Power et al., 2018). Culturing was performed originally with DSMZ Desulfurella medium and later modified, investigating pH (4 and 6) and temperature (50°C and 30°C) variations and adapting it for solid media by replacing elemental sulphur with sodium polysulfide. I used a vacuum food sealer to cultivate anaerobes, an accessible and portable alternative to expensive anaerobic chambers. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to assess axenicity before sending samples for 16S rRNA gene and whole genome sequencing. Desulfurella was detected in each culture using species-specific primers (DS_F/DS_R), enabling its phylogenetic placement. However, despite Desulfurella presence in the 16S rRNA gene amplicon sequencing, a metagenome-assembled genome (MAG) could not be produced. This thesis discusses lessons learned and suggests improvements for future studies.

year: 2024

title: Holding a mirror to society? Sociodemographic diversity within clinical psychology training programmes across Aotearoa.

authors: Scarf, Damian

subjects: Education, Medical

abstract: It has recently been demonstrated by Crampton et al that there has been a marked increase in the sociodemographic diversity of the University of Otago’s health professional programmes between 2010 and 2016.1 This change is evidenced by the fact that a historic 76 health professional Māori graduates crossed the Dunedin Town Hall stage in December 2018. A key driver of this change was the University of Otago’s Division of Health Sciences Mirror on Society selection policy. Here, by documenting the sociodemographic diversity of an equally important professional programme, clinical psychology, we highlight the need for similar selection policies to be implemented Aotearoa/New Zealand wide.

text: Holding a mirror to society? Sociodemographic diversity within clinical psychology training programmes across Aotearoa. It has recently been demonstrated by Crampton et al that there has been a marked increase in the sociodemographic diversity of the University of Otago’s health professional programmes between 2010 and 2016.1 This change is evidenced by the fact that a historic 76 health professional Māori graduates crossed the Dunedin Town Hall stage in December 2018. A key driver of this change was the University of Otago’s Division of Health Sciences Mirror on Society selection policy. Here, by documenting the sociodemographic diversity of an equally important professional programme, clinical psychology, we highlight the need for similar selection policies to be implemented Aotearoa/New Zealand wide.

year: 2019