Aller au contenu Aller au menu Aller à la recherche

accès rapides, services personnalisés
Laboratoire d'Océanographie Microbienne
UMR 7621

Offres stages Master2 2019

Assessing the quality and molecular composition of dissolved organic matter produced by marine prokaryotes: Role of nutrient availability

Heterotrophic bacterioplankton play a key role in organic matter processing in all aquatic ecosystems, with consequences on the cycling of carbon and other elements. Heterotrophic bacteria and archaea reprocess roughly 50% of organic carbon that is fixed by phytoplankton, the main organic matter input in the ocean (Azam et al., 1983), which is either respired to CO2 or used to build new biomass. However, the role of bacterioplankton as sources of dissolved organic matter has been overlooked, but it has been demonstrated that bacterioplankton also release dissolved organic matter (DOM). This bacterial-derived DOM can be resistant to further remineralization and it is sequestered in the ocean’s interior through the so-called microbial carbon pump (Jiao et. al. 2010). However, there are still many open questions regarding the underlying mechanisms that lead to DOM production by bacterioplankton. Within the broad range of environmental parameters that could eventually affect the microbial carbon pump, nutrient stoichiometry would be a key factor affecting these mechanisms. DOM production by phytoplankton is highest and less bioavailable for bacteria when they are P-limited (Obernosterer & Herndl, 1995); although the importance of phosphorous limitation in DOM production by bacteria has not been yet tested. However, it was shown in laboratory incubations that refractory DOM (i.e. not bioavailable) was highly depleted in phosphorus vs carbon or nitrogen. Based on these previous results, we hypothesize that P would play a key role in BDOM generation and lability; being BDOM higher and more recalcitrant in phosphorus-limited ecosystems (e.g. the Mediterranean Sea) than in nitrogen-limited ones; affecting DOM cycling in these ecosystems.

The Master 2 student will help elucidate whether DOM production by heterotrophic bacterioplankton is affected by the initial nutrient (nitrogen vs. phosphorous) limitation. For this purpose, the student will work with model bacterial strains in culture. Different strains of contrasting taxonomy and physiology will be selected and grown using glucose as a single carbon source and under different nutrient-limiting conditions (nitrogen vs. phosphorus limitation). The growth of the bacterial cells will be followed by flow cytometry. Then, DOM produced in the cultures will be characterized by complementary methods: Fluorescent dissolved organic matter, amino acids, C:N:P ratios. An important part of the internship will be invested in characterizing DOM molecular diversity by Fourier Transmission-Ion cyclotron resonance Mass Spectrometry (FT-ICR-MS). This has revealed to be a powerful technique to analyze marine complex DOM samples, providing valuable information on elemental compositions on a molecular scale. Although FT-ICR-MS does not allow assigning molecular structures, molecular diversity and abundance patterns can be obtained giving information about its potential bioavailability. These analyses will be conducted at the national FT-ICR-MS facility located in the University of Rouen. The student will work to implement the protocol specifically for marine samples. Then, bacterial DOM fingerprints obtained from the cultures (12 samples) will be compared with those of natural samples (fresh vs aged seawater samples taken from an open sea observatory).

Responsable de l’encadrement : Eva ORTEGA-RETUERTA
Téléphone : 04.68.88.73.53
E-mail : ortegaretuerta @ obs-banyuls.fr

Texte complet de la proposition de stage

 

The use of genetic tools in order to understand how heterotrophic bacteria can cope under Fe-limitation.

Fe-limitation within the ocean

Iron (Fe) is an essential element for marine microbial growth but is present in trace amounts (<0.1 nM) in surface waters of the ocean. Little is known about how heterotrophic bacteria can cope under such low Fe-conditions which particularly impacts ATP production as Fe is an essential co-factor of enzymes involved in the electron-transport chain as well as the tricarboxylic acid (TCA) cycle (Tortell, 1996). Fe-limitation can therefore drastically reduce both bacterial growth and respiration, consequently affecting the efficiency of organic carbon remineralization. Heterotrophic bacteria, however, can possess various strategies to cope with Fe-limitation. The objective of this master thesis, is to use a combination of genetic tools (such as the use of knockout mutants and bioreporters) in order to gain further insight into the various strategies heterotrophic bacteria may use under Fe-limitation.

Genetic Tools

The use of genetic tools is key in elucidating such strategies. The construction of a bioreporter can allow us to track and measure gene expression in a non-invasive way over time while gene knockouts further identify the phenotype of the cell and whether the presence or absence of a gene can indeed provide a bacterium with an advantage under Fe-limiting conditions.

The Glyoxylate Shunt

Recently, these genetic approaches were used to elucidate one such strategy, namely the upregulation of the glyoxylate shunt under Fe-limitation (Koedooder et al., 2018 under review). Bacteria can redirect their metabolism away from the classic TCA cycle and towards the glyoxylate shunt which bypasses certain steps such as the production of carbon dioxide (CO2) and the formation of NADH, thereby reducing the use and dependency of an otherwise inefficient electron transport chain (Kornberg, 1966; Kornberg and Krebs, 1957).

The addition of light: proteorhodopsin

Interestingly, the glyoxylate shunt has also been shown to be induced in the addition of light due to the presence of proteorhodopsin (Palovaara et al., 2014). Proteorhodopsin is a light induced hydrogen pump which may be used to produce ATP or aid in the uptake of Fe within the cell. Therefore, the inclusion of light is crucial towards further understanding the ability of heterotrophic bacteria to remodel their metabolism under Fe-limitation.

Internship work

In this master thesis, the student will use several genetic tools (gene knockouts and bioreporters) to test whether the presence of proteorhodopsin and light can impact the glyoxylate shunt under Fe-limitation. The genetic tools needed in order to conduct the work have already been constructed. The student will learn how to conduct bacterial culture work under trace metal clean conditions and learn general skills required for the growth and maintenance of these cultures. Preliminary experiments will be constructed in order to verify the induction of the glyoxylate shunt under Fe-limitation. Students will obtain several different data including bacterial growth curves, derive bacterial growth and respiration rates from O2 measurements, measure gene expression over time, and ATP measurements. The student is expected to continue these experiments in order to include the role of light (and the use of proteorhodopsin). Data analysis will include the use of basic R-skills in order to present coherent graphs and their statistical analysis.

Responsable de l’encadrement : Stéphane BLAIN / Coco KOEDOODER
Téléphone : 04.68.88.73.44
E-mail : blain@obs-banyuls.fr (blain @ obs-banyuls.fr)/ koedooder @ obs-banyuls.fr

Texte complet de la proposition de stage

 

Biogeochemical conditions that control the formation and evolution of abnormal organic matter aggregations in areas sensitive to climate change

Climate change and anthropogenic pressure are increasingly affecting the trophic status of coastal areas and therefore their capacity for carbon (C) sequestration. It is known that  increasing sea surface temperatures and stratification as well as anomalies in nutrient proportions (Bronk, 2007) can lead to abnormal aggregations of marine snow, already seen in  some Patagonian fjords.

However, their effects on ecosystem function and health are still uncertain (Misic et al 2011) as organic matter aggregations can act as vehicles of carbon and nutrients, microbial diversity (Del Negro et al 2005) and pollutants.

The student will work on the frame of a Fondecyt grant focusing on studying the biogeochemical conditions that control the formation and evolution of abnormal organic matter aggregations in areas sensitive to climate change.

The specific objectives will be to estimate the variability of nitrogen and phosphorous (as  organic and inorganic dissolved nutrients) and to determine the occurrence (in frequency and  magnitude) of P deficiency in two sensitive coastal areas: the Puyuhuapi fjord and off the  coastal upwelling area of central Chile. We will tackle this objective by analyzing historical data and generating the first coupled inorganic and organic N and P database in southern Chile.

Another specific aim will be to explore the microbial diversity associated to organic matter formation caused by abnormal nutrient conditions and identify dominant groups (including fungi, cyanobacteria, archaea ad bacteria). We will generate sequence libraries of active phytoplankton, bacterioplankton and marine fungi and we will compare them with the surrounding environment.

Research will be funded by FONDECYT project 1150891.

Responsable de l’encadrement : Camila FERNANDEZ
Email: fernandez @ obs-banyuls.fr

Texte complet de la proposition de stage

01/10/18

Traductions :