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: Volunteering: A community response to the Rena oil spill in New Zealand

authors: Sargisson, Rebecca J.

subjects: Rena

abstract: We explore the experiences of people who volunteer to help remediate the effects of non-natural environmental disasters. Following the grounding of the Rena, volunteers were engaged to clean up the resulting oil spill on the beaches of Tauranga, New Zealand. Volunteers were later invited to respond to an online questionnaire about their experiences. More women than men responded, and respondents tended to be older, and engaged in the paid workforce or retired. Greater membership in community organizations was associated with a greater participation in clean-up events. Respondents were positive about the experience, and were more positive when they had actually participated in a clean-up event, with positivity remaining high even after multiple volunteer occasions. Recommendations are made for increasing volunteer participation.

text: Volunteering: A community response to the Rena oil spill in New Zealand We explore the experiences of people who volunteer to help remediate the effects of non-natural environmental disasters. Following the grounding of the Rena, volunteers were engaged to clean up the resulting oil spill on the beaches of Tauranga, New Zealand. Volunteers were later invited to respond to an online questionnaire about their experiences. More women than men responded, and respondents tended to be older, and engaged in the paid workforce or retired. Greater membership in community organizations was associated with a greater participation in clean-up events. Respondents were positive about the experience, and were more positive when they had actually participated in a clean-up event, with positivity remaining high even after multiple volunteer occasions. Recommendations are made for increasing volunteer participation.

year: 2012

title: Experiencing cultural tourism: Visitors at the Maori Arts and Crafts Institute, New Zealand

authors: Ryan, Chris

subjects: indigenous tourism

abstract: This study reports results derived from semistructured interviews with 40 visitors to the Maori Arts and Crafts Institute in Rotorua, New Zealand. The article discusses briefly the mode of data capture used and adopts an approach of identifying classifications of a primarily functional nature to list reasons why people visit such a site and what it is they seek. In general, visitors reported high levels of satisfaction, yet the cultural component of their experience was primarily reported in terms of the visit being informative and entertaining. Additionally, whereas modest levels of interest in things Maori seemed to motivate visits to other Maori sites, the actual list of other locations being nominated was few in number. The impression results that visits are made to Maori sites because this is a "done thing" when visiting New Zealand, but overall, the level of interest shown by most visitors in indigenous culture is modest.

text: Experiencing cultural tourism: Visitors at the Maori Arts and Crafts Institute, New Zealand This study reports results derived from semistructured interviews with 40 visitors to the Maori Arts and Crafts Institute in Rotorua, New Zealand. The article discusses briefly the mode of data capture used and adopts an approach of identifying classifications of a primarily functional nature to list reasons why people visit such a site and what it is they seek. In general, visitors reported high levels of satisfaction, yet the cultural component of their experience was primarily reported in terms of the visit being informative and entertaining. Additionally, whereas modest levels of interest in things Maori seemed to motivate visits to other Maori sites, the actual list of other locations being nominated was few in number. The impression results that visits are made to Maori sites because this is a "done thing" when visiting New Zealand, but overall, the level of interest shown by most visitors in indigenous culture is modest.

year: 2006