Physiology of Caulobacter crescentus grown on different carbon sources

Abstract

Freshwater, obligate aerobic, Gram-negative bacterium Caulobacter crescentus strain CB2 is able to metabolise various types of carbon sources. In this degree project, growth characterization of C.crescentus was made on different substrates including glucose, xylose, and mannose using shake-flask cultivations and bioreactor experiments. Special emphasis was put on the xylose degradation pathway, the Weimberg pathway in C. crescentus. This pathway has recently been of interest for introduction into other hosts.
In shake-flask cultivations of C. crescentus strain CB2, the highest specific growth rates were found in 10 g/L glucose (0.142 h-1) followed by 10 g/L xylose (0.109 h-1). The organism also grew successfully in 10 g/L mannose but with a lower specific growth rate (0.053 h-1). The main product in cultivations in xylose was α-ketoglutaric acid, the last metabolite in the Weimberg pathway. The strain CB2 showed incomplete substrate consumption whereas another strain tested, CB15, showed complete substrate consumption in shake-flask cultivations using 5 g/L glucose or xylose substrates.
Bioreactor experiments with Caulobacter crescentus strain CB2 indicated that the optimal pH for growth was 6.5. The incomplete substrate consumption suggested that growth was limited (or inhibited) by sensitivity to aeration, limiting nutrients (present in yeast extract), or sensivity to some products formed.
Enzyme assays were made for three of the enzymes in the Weimberg pathway (xylose dehydrogenase (xylB), xylonate dehydrogenase (xylD), and α-ketoglutaric semialdehyde dehydrogenase (xylA). Activities of these enzymes were all found, 38.2, 25.4, and 50.9 μmol/(min*mg) protein, respectively, in medium containing only xylose as the carbon source. No activities were found in medium containing glucose as the carbon source.

Popular Abstract

The world desperately needs a solution to an ever increasing environmental problems connected to oil-based industries. Today, unfortunately, almost every material we come in contact with was produced directly or indirectly using petrochemicals. Teams of scientists all over the world have worked with the idea to produce necessary chemicals from other more environmental-friendly sources such as plant biomass – more specifically, the part of the plant which is disposed of by many agricultural and industrial processes. Since this is waste, it would not compete with food production because competition with food would be the last thing the society needs right now.
In order to convert this waste part of the plants into something useful for us, microorganisms such as certain harmless bacteria and yeasts are used. There are millions to choose from but one of the most ideal ones for industrial use is the fungus Saccharomyces cerevisiae, also known as Baker’s yeast. Yes, the one you use for baking! The problem is, Baker’s yeast can only work with around 40% of the waste part of the plant, the part composed of sugar with 6 carbon atoms. So teams of specialized scientists have been working on changing the genetic make-up of the yeast so that it could convert almost 100% of the waste part of the plant.
They do this by taking some important genes from other microorganisms that do convert the remaining component of the waste part of plants, the part composed of sugars with 5 carbon atoms. In this case, an organism called Caulobacter crescentus which naturally degrades xylose, a sugar with 5 carbon atoms. This organism is normally found in freshwater and thrives on decaying plant material. The perfect target!
Caulobacter crescentus was the main focus of the degree project. Getting to know its growth behaviour in different environments was investigated with the aim of shedding light on how this organism can be used to improve Baker’s yeast. Caulobacter crescentus was grown in shake-flasks and bioreactors (see figures below). It was found that this organism grows the best in glucose (sugar with 6 carbon atoms) followed by xylose. Also, it was found that when xylose is the only food for Caulobacter crescentus, an acid called α-ketoglutaric acid is formed, which is not the case when glucose is the only food. This organism was also shown to be quite smart. In fact, it changes its mechanism of survival depending on which sugar to eat. Caulobacter crescentus uses different sets of tools, in this case, enzymes, as a response to which type of food is available. For instance, the enzymes used for xylose do not appear when only glucose is available. For an organism that is about 170 million times smaller than yourself, that is indeed very intelligent!
The degree project has opened many doors for further research. There were some discoveries, but more questions were left unanswered such as why Caulobacter crescentus died before finishing all its food. The logic of the question may not be applicable to human beings but for a bacteria grown in a bioreactor, or shake-flasks for that matter, it does.
All in all, it was an interesting project and hopefully to some degree this would contribute to developing a more sustainable solution to some of the major problems the world is facing today.

• author: Jónsdóttir Glaser, Sara Lind
• supervisor: Henrik Almqvist
• organization: Chemical Engineering (M.Sc.Eng.)
• alternative title: Physical characterisation of Caulobacter crescentus grown on different carbon sources
• course: KETM01 20152
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: aerobic cultivation, physical characterization, lignocellulosic biomass, xylose degradation, Weimberg, bioreactors, enzyme assays, chemical engineering, kemiteknik
• language: English
• id: 8881240
• date added to LUP: 2016-06-21 15:59:16
• date last changed: 2017-06-01 04:09:55