Mutagenesis of Halanaerobium saccharolyticum subsp. saccharolyticum aiming for antibiotic sensitivity and improved hydrogen and 1,3- propanediol tolerant strains.
Sudhakaran, Santhosh (2015)
Sudhakaran, Santhosh
2015
Master's Degree Programme in Science and Bioengineering
Luonnontieteiden tiedekunta - Faculty of Natural Sciences
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
Hyväksymispäivämäärä
2015-09-09
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201508281555
https://urn.fi/URN:NBN:fi:tty-201508281555
Tiivistelmä
Keywords: Halanerobium sacchrolyticum subsp. sacchrolyticum, Multi dug ef-flux pump, novobiocin, UV mutation, hydroxylamine Hydrogen, 1,3 –propanediol.
Halanerobium sacchrolyticum subsp. sacchrolyticum is a halophilic anaerobic fer-mentative bacteria, grows in high salt condition. Based on the salt concentration halo-philes are classified into moderate halophiles and extreme halophiles. Hydrogen and 1, 3-propanediol was the essential product produced by this bacterium. Survivals of the H. sacchrolyticum on high salt concentrations were by salt in and salt out strategy, which maintains the osmotic balance.
Different antibiotics were tested for resistivity of H. sacchrolyticum; ampicillin, kana-mycin and chloramphenicol were resistance to these bacteria. Whereas novobiocins with > 1 µg/ml were sensitive, thus inhibited the growth of bacteria, novobiocin concen-tration of < 1 µg/ml was resistant. Transfomation efficiency of Plasmid pMDS133 which contains NovR gene were analysed by natural transformation method. H. sac-chrolyticum with < 1 µg/ml of novobiocin was subjected to two different types of muta-tion, UV and hydroxylamine mutagenesis. The multidrug efflux pump plays important role in excreting the toxic substances, since H. sacchrolyticum is a gram negative bacte-ria, the multi dug efflux pump is complex.
Mutation in H. sacchrolyticum increases the yield of hydrogen, were the optimal yield was obtained 2.4 mol/mol (glucose) and with initial 33 % of hydrogen tolerance 1.5 mol/mol (glucose) was obtained. 1, 3-propanediol tolerance was tested after mutagene-sis, which inhibits the growth and affects in production of end metabolites. Screening of mutants was the most challenging part of the study; replica plating technique was used in selection of mutants, initially experimented with E. coli KRX strain and later with mutated H. sacchrolyticum.
Halanerobium sacchrolyticum subsp. sacchrolyticum is a halophilic anaerobic fer-mentative bacteria, grows in high salt condition. Based on the salt concentration halo-philes are classified into moderate halophiles and extreme halophiles. Hydrogen and 1, 3-propanediol was the essential product produced by this bacterium. Survivals of the H. sacchrolyticum on high salt concentrations were by salt in and salt out strategy, which maintains the osmotic balance.
Different antibiotics were tested for resistivity of H. sacchrolyticum; ampicillin, kana-mycin and chloramphenicol were resistance to these bacteria. Whereas novobiocins with > 1 µg/ml were sensitive, thus inhibited the growth of bacteria, novobiocin concen-tration of < 1 µg/ml was resistant. Transfomation efficiency of Plasmid pMDS133 which contains NovR gene were analysed by natural transformation method. H. sac-chrolyticum with < 1 µg/ml of novobiocin was subjected to two different types of muta-tion, UV and hydroxylamine mutagenesis. The multidrug efflux pump plays important role in excreting the toxic substances, since H. sacchrolyticum is a gram negative bacte-ria, the multi dug efflux pump is complex.
Mutation in H. sacchrolyticum increases the yield of hydrogen, were the optimal yield was obtained 2.4 mol/mol (glucose) and with initial 33 % of hydrogen tolerance 1.5 mol/mol (glucose) was obtained. 1, 3-propanediol tolerance was tested after mutagene-sis, which inhibits the growth and affects in production of end metabolites. Screening of mutants was the most challenging part of the study; replica plating technique was used in selection of mutants, initially experimented with E. coli KRX strain and later with mutated H. sacchrolyticum.