Environmental microorganisms: microsystem approaches to separation and analysis
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Three technologies were tested in this PhD project, all of which have potential for use in micro systems for environmental analyses: The Trilobite® technology for particle separation and purification, the analysis technique nucleic acids sequence-based amplification (NASBA), and POCNAD, a lab-on-a-chip system for automatic sample analysis. The Trilobite® microfluidic chip for concentration and separation of particles in fluids was tested for its use in applications for biological particles. Three species of microalgae were used to investigate the chip’s potential as an alternative dewatering technique in microalgae harvesting. The Trilobite® chip was promising for concentration of rigid diatoms, and for separation of cells based on their sizes. However, the various properties of the particles, such as size, flexibility and production of extracellular polymers, have great impact on efficiency of the chip. The chip was also tested for its application in analysis of environmental samples for waterborne protozoan parasites. These experiments suggested that the Trilobite® chip might not currently be optimal for concentration of cysts and oocysts for water samples, where efficient particle recovery is crucial for detection. The numbers of these particles in water are generally low, and few cysts or oocyst may be enough to cause infection if they are ingested by humans or animals. NASBA assays were developed for sensitive detection of a consortium of oil-utilizing bacteria and also for protozoan parasites. Cryptosporidium parvum and Cryptosporidium hominis are the two Cryptosporidium species most often associated with human cryptosporidiosis. By developing a NASBA assay targeting the MIC1 transcript of C. parvum and C. hominis, oocysts of these two species were successfully detected down to 5 oocysts. The assay was also able to distinguish C. parvum oocysts from C. hominis. mRNA, which is the target molecule for NASBA, has been suggested to be a suitable marker for viability, as mRNA is degenerated quickly after cell death. However, the assay did not seem to be suitable for using as a viability assay, as transcripts from inactivated oocysts could also be detected. NASBA primer sets were designed for the binucleated flagellate Giardia duodenalis, Assemblages A, B, and E. Unfortunately, due to time constraints and the lack of Giardia cysts of the appropriate Assemblages, these primer sets were not fully tested. This remains a subject for further studies. NASBA primer sets were also developed for a consortium of four strains of oil-utilizing bacteria. Using these, all strains were detected by NASBA. However, not all strains could be distinguished from each other using the primer sets. An additional intention was to develop primer sets that could provide information on the metabolism of these strains, but this was not achieved. Even when there are very few transcripts available from a down-regulated gene, these may still be sufficient for the sensitive amplification NASBA provides. However, the primer could be used in the NASBA assay component of the POCNAD lab-on-a-chip system. The NASBA reaction was carried out in the POCNAD system. The NASBA reaction itself did not seem to have any difficulties in the microfluidic POCNAD chip, but factors regarding pumping of reagents in the chip, and real-time detection proved to be challenging in the system. All the required components of the system, and a control instrument that was not sufficiently developed, made this system very complicated. Complete hands-off automatic analysis could not be conducted in the POCNAD system as it stood. These experiments provided a clear demonstration of the difficulty in developing usable systems of this type.
Består avPaper 1: Hønsvall, B.K., Altin, D., Robertson, L.J. (2016). Continuous harvesting of microalgae by new microfluidic technology for particle separation. Bioresource Technology, 200, 360-365. doi: 10.1016/j.biortech.2015.10.046
Paper 2: Hønsvall, B.K., Robertson, L.J. 2017. Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts. Experimental Parasitology, 172, 61-67.
Paper 3: Hønsvall, B.K., Robertson, L.J. 2017. From research lab to standard environmental analysis tool: Will NASBA make the leap? Water Research, 109, 389-397.
Paper 4: Hønsvall, B.K., Robertson, L.J. (submitted). Washed away; minimising RNA losses during isolation. Submitted to Journal of Biomolecular Techniques
Paper 5: Hønsvall, B.K., Ezkerra, A., Gulliksen, A., Dong, T., Karlsen, F. (2013). Detection of oilutilizing microorganisms by nucleic acid sequence-based amplification in a total analysis lab-on-a-chip device. in: 17th international conference on miniaturized systems for chemistry and life sciences, 2013, Freiburg, Germany, 27-31 Oct, 2013, Chemical and Biological Microsystems Society, pp. 1767-1769.