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Secretome analysis of Rhodothermus marinus grown on different carbon sources

Malta Neri, Lara Chrystina LU (2023) KBTM01 20221
Biotechnology (M.Sc.Eng.)
Biotechnology (MSc)
Abstract
The project aimed to analyse by liquid chromatography coupled to high resolution mass spectrometry (LC-MS/HRMS) the extracellular proteins synthesized by Rhodothermus marinus strains DSM 4252T and DSM 16675 growing in medium containing different carbon sources. That is critical to find valuable enzymes for industrial uses in biomass conversion into value-added chemicals. Cells were grown in medium containing alginate, carboxymethyl cellulose, laminarin, xylan, and seaweed from two species: Laminaria digitata and Laminaria hyperborea. The growth on these different carbon sources needed to be followed to find the optimum time to take samples for LC-MS/HRMS analysis. As for start, envisioning sample preparation and optimization, xylan medium... (More)
The project aimed to analyse by liquid chromatography coupled to high resolution mass spectrometry (LC-MS/HRMS) the extracellular proteins synthesized by Rhodothermus marinus strains DSM 4252T and DSM 16675 growing in medium containing different carbon sources. That is critical to find valuable enzymes for industrial uses in biomass conversion into value-added chemicals. Cells were grown in medium containing alginate, carboxymethyl cellulose, laminarin, xylan, and seaweed from two species: Laminaria digitata and Laminaria hyperborea. The growth on these different carbon sources needed to be followed to find the optimum time to take samples for LC-MS/HRMS analysis. As for start, envisioning sample preparation and optimization, xylan medium was chosen as the first medium to be analysed as it yielded the highest cell density, in addition to exhibiting unique protein bands on an SDS- PAGE gel when compared to protein band profiles from control and other carbon source media. Protein precipitation by both TCA/acetone and acetone precipitation methods yielded protein pellets containing a high saccharide content that could interfere with the LC-MS/HRMS analysis and results. Hence, most of the project focused on obtaining protein pellets with lower saccharides content. Consequently, the xylan percentage was decreased from 1 to 0.2%, and the protein precipitation was performed with two volumes acetone per volume of sample. The LC-MS/HRMS is yet to be performed in later stages of the project, as well as transcriptomics and analysis of sugar usage. (Less)
Popular Abstract
In the past few years, awareness of environmental problems, waste management, and establishment of initiatives to mitigate clime issues have been widespread. The use of petroleum-based materials especially fuels responsible for the release of tons of greenhouse gases such as CO2 and the large output of bio-waste are of great concern. Therefore, efforts are being directed into converting complex biomass such as seaweed and bagasse into valuable products, an example would be the conversion of cellulose into glucose that can be fed to microorganisms to produce bioethanol. Enzymes, especially those that can withstand harsh conditions like high temperatures can be used as key tools for converting the biomass components into desirable products.
... (More)
In the past few years, awareness of environmental problems, waste management, and establishment of initiatives to mitigate clime issues have been widespread. The use of petroleum-based materials especially fuels responsible for the release of tons of greenhouse gases such as CO2 and the large output of bio-waste are of great concern. Therefore, efforts are being directed into converting complex biomass such as seaweed and bagasse into valuable products, an example would be the conversion of cellulose into glucose that can be fed to microorganisms to produce bioethanol. Enzymes, especially those that can withstand harsh conditions like high temperatures can be used as key tools for converting the biomass components into desirable products.

Hence, this work aimed to characterize and quantify the enzymes released by the thermophilic bacterium Rhodothermus marinus growing in medium supplied with different polysaccharides which are large chains of smaller sugars such as glucose. R. marinus needs to break those polysaccharides down releasing small sugars to be taken up by the cells for growth. As the bacterium grows at high temperatures, optimally at around 65 °C, it has been shown in studies that their enzymes can work at even higher temperatures of around 80 °C, which make them very robust and suitable for biomass conversion. Therefore, this project focuses on growing R. marinus in medium containing different polysaccharides such as xylan to analyse the extracellular proteins or enzymes released during late growth phase. (Less)
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author
Malta Neri, Lara Chrystina LU
supervisor
organization
course
KBTM01 20221
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Rhodothermus marinus, extracellular proteins, polysaccharides, xylan, xylanases, biotechnology
language
English
id
9124265
date added to LUP
2023-06-14 09:54:04
date last changed
2023-06-14 09:54:04
@misc{9124265,
  abstract     = {{The project aimed to analyse by liquid chromatography coupled to high resolution mass spectrometry (LC-MS/HRMS) the extracellular proteins synthesized by Rhodothermus marinus strains DSM 4252T and DSM 16675 growing in medium containing different carbon sources. That is critical to find valuable enzymes for industrial uses in biomass conversion into value-added chemicals. Cells were grown in medium containing alginate, carboxymethyl cellulose, laminarin, xylan, and seaweed from two species: Laminaria digitata and Laminaria hyperborea. The growth on these different carbon sources needed to be followed to find the optimum time to take samples for LC-MS/HRMS analysis. As for start, envisioning sample preparation and optimization, xylan medium was chosen as the first medium to be analysed as it yielded the highest cell density, in addition to exhibiting unique protein bands on an SDS- PAGE gel when compared to protein band profiles from control and other carbon source media. Protein precipitation by both TCA/acetone and acetone precipitation methods yielded protein pellets containing a high saccharide content that could interfere with the LC-MS/HRMS analysis and results. Hence, most of the project focused on obtaining protein pellets with lower saccharides content. Consequently, the xylan percentage was decreased from 1 to 0.2%, and the protein precipitation was performed with two volumes acetone per volume of sample. The LC-MS/HRMS is yet to be performed in later stages of the project, as well as transcriptomics and analysis of sugar usage.}},
  author       = {{Malta Neri, Lara Chrystina}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Secretome analysis of Rhodothermus marinus grown on different carbon sources}},
  year         = {{2023}},
}