Chemostat

One of the bioreactors used by the Microbial Physiology Group. In here, microbes grow on nitrous oxide (NO). (© Max Planck Institute for Marine Microbiology, Boran Kartal)
One of the bioreactors used by the Microbial Physiology Group. In here, microbes grow on nitrous oxide (NO). (© Max Planck Institute for Marine Microbiology, Boran Kartal)

What is continuous cultivation?

Most microorganisms live under substrate limitation, as the supply of their substrates depends on other organisms or physicochemical properties of their environment. In conventional microbiological approaches one or more substrate(s) are supplied to samples in excess, as a result the fastest growing species is brought to culture. This does not reflect what happens in the environment. In the Microbial Physiology group, we use continuous cultivation bioreactors, which closely mimic the environments that microorganisms live in – in the laboratory, under controlled conditions. That allows us to culture environmentally-relevant microorganisms to study their physiological and biochemical properties in molecular detail.

In our laboratory, we use chemostats and other types of continuous cultivation techniques. In a chemostat, fresh medium is continuously added to the culture vessel while a corresponding volume of culture liquid is removed at the same rate.

How does a chemostat work?

A chemostat usually consists of a closed (glass) vessel with a volume between 1 ml and a few liters. Fresh medium is supplied continuously with an influent pump. A second pump removes liquid from the vessel at the same rate. This way a constant volume is maintained. The microorganisms added to the vessel can only consume what is supplied by the influent pump. Their growth rate (per hour) is defined by the ratio of the influent rate (liter or milliliters per hour) and the vessel volume (L). This state can be maintained indefinitely. Among the substrates and growth factors added to the medium, one is the so-called controlling substrate, which limits growth. 

The others are present in surplus. The concentration of the controlling substrate determines the concentration of cells in the vessel and in the effluent. In most cases the energy or carbon source serves as the controlling substrate, but it can be adjusted depending on your research question. Furthermore, parameters such as pH, temperature and mixing can be easily and accurately controlled and manipulated.

Chemostats in action

The Microbial Physiology Group uses a variety of bioreactor setups to answer different research questions (left to right, up to down): Sand reactor, fed-batch reactor, annamox chemostat, series of chemostats. (© Max Planck Institute for Marine Microbiology, Boran Kartal)
The Microbial Physiology Group uses a variety of bioreactor setups to answer different research questions (left to right, up to down): Sand reactor, fed-batch reactor, annamox chemostat, series of chemostats. (© Max Planck Institute for Marine Microbiology, Boran Kartal)

A chemostat is an excellent tool to cultivate microorganisms, as the conditions are defined and constant compared to batch cultures (e.g., shake flasks). This makes experiments easier to reproduce. Furthermore, the set-up generates a continuous flow of microbial culture, much like your tap generates a continuous flow of clean water, that can be conveniently harvested for further experiments. The chemostat can be used for pure cultures to study the kinetics of microbial growth, as well as for more detailed -omics approaches. It can also be used for competition experiments. In such experiments two or three different microorganisms with comparable niches are released in the vessel under varying conditions, at high or low growth rates, at high or low oxygen concentrations, distinct pH or temperature values, with or without growth factors, etc.

There are different kinds of chemostat setups, allowing for different research questions to be posed and different organisms to be targeted. For example, some microorganisms might prefer to grow as biofilm or biofilm aggregates. They can be catered to e.g. by introducing more surface area to the bioreactor for microorganisms to attach or by adding a settler unit. Slow-growing microorganisms can be retained in chemostats using membrane filters that keep the microorganisms in the bioreactor, while letting spent medium through (so-called retentostats). In order to grow microorganisms directly from sandy sediments, we can use cylindrical cores equipped with precise pump units. Here, part of the control over the enrichment culture is lost as microniches can form throughout the (sandy) sediment core.

Example projects

Greenhouse Gas Mitigation through Advanced Nitrogen Removal Technology - GREENT

Hu­man activ­it­ies have severe im­pacts on the bio­lo­gical car­bon and ni­tro­gen cycles, the most im­port­ant con­sequences of these are global warm­ing and wa­ter pol­lu­tion. Wastewa­ter treat­ment tech­no­logy, in par­tic­u­lar ni­tro­gen re­moval sys­tems, im­proved con­sid­er­ably in the last dec­ade. The ap­plic­a­tion of an­aer­obic am­monium ox­id­iz­ing (anam­mox) bac­teria in oxy­gen-lim­ited gran­ules has the po­ten­tial to turn wastewa­ter treat­ment plants into en­ergy-ef­fi­cient sys­tems with min­imal green­house gas emis­sions. Re­cently, mi­croor­gan­isms that couple the an­aer­obic ox­id­a­tion of meth­ane to de­ni­tri­fic­a­tion were dis­covered. An in­nov­at­ive in­teg­ra­tion of these mi­croor­gan­isms into certain sys­tems for wastewa­ter treat­ment of­fers an el­eg­ant and ef­fi­cient solu­tion to com­bat green­house gas emis­sions from wastewa­ter treat­ment plants. The aim of the GREENT pro­ject is to de­term­ine ni­trous ox­ide emis­sions from par­tial ni­trit­a­tion-anam­mox biore­act­ors and the para­met­ers that gov­ern these emis­sions, and to in­vest­ig­ate the re­spons­ible path­ways in mo­lecu­lar de­tail. Fur­ther­more, it explores the feas­ib­il­ity of an in­nov­at­ive biore­actor, which will re­move am­monium and meth­ane sim­ul­tan­eously through anam­mox and an­aer­obic meth­ane-ox­id­iz­ing mi­croor­gan­isms.

More Information: https://www.mpi-bremen.de/ERC-Project-GREENT.html

Mi­crobe hunters dis­cover long-sought-after iron-munch­ing mi­crobe

A microbe that ‘eats’ both methane and iron: microbiologists have long suspected its existence, but were not able to find it. In 2016, researchers at Radboud University and the Max Planck Institute for Marine Microbiology in Bremen discovered a microorganism that couples the reduction of iron to methane oxidation, and could thus be relevant in controlling greenhouse gas emissions worldwide.

Here you can find the press release: https://www.mpi-bremen.de/en/Microbe-hunters-discover-long-sought-after-iron-munching-microbe.html

One of the bioreactors, in which Kartal and his colleagues found the rust-munching microbes. (©Max Planck Institute for Marine Microbiology, B. Kartal)
One of the bioreactors, in which Kartal and his colleagues found the rust-munching microbes. (©Max Planck Institute for Marine Microbiology, B. Kartal)

Mi­crobes can grow on nitric ox­ide (NO)

Nitric oxide (NO) is a central molecule of the global nitrogen cycle. A study by Boran Kartal and colleagues reveals that microorganisms can grow on NO at concentrations that would be lethal to all other lifeforms. Their results, which were published in Nature Com­mu­nic­a­tions, change our view of the earth’s nitrogen cycle and how microorganisms regulate the release of greenhouse gases from natural and man-made environments.

Here you can find the press release: https://www.mpi-bremen.de/en/Microbes-can-grow-on-nitric-oxide-NO.html

Kuenenia stuttgartiensis, here seen under a transmission electron microscope, is a model anammox microorganism, which grows as single cells. It is a freshwater species also found in wastewater treatment plants. (© Max Planck Institute for Marine Microbiology, Laura van Niftrik)
Kuenenia stuttgartiensis, here seen under a transmission electron microscope, is a model anammox microorganism, which grows as single cells. It is a freshwater species also found in wastewater treatment plants. (© Max Planck Institute for Marine Microbiology, Laura van Niftrik)

Dis­cov­ery of an un­usual pro­tein

Scientists from Bremen discover an unusual protein playing a significant role in the Earth’s nitrogen cycle. The novel heme-containing cytochrome is involved in the anammox process, which is responsible for producing half of the dinitrogen gas in the atmosphere and important in greenhouse gas regulation.

Here you can find the press release: https://www.mpi-bremen.de/en/Discovery-of-an-unusual-protein.html

One of the bioreactors that Kartal and his colleagues used to grow cells of K. stuttgartiensis in the lab. Anammox bacteria are packed with heme-containing proteins, including the enzymes that perform the key reactions of the anammox process, making the cells remarkably red. (© Max Planck Institut for Marine Mikrobiology, Boran Kartal)
One of the bioreactors that Kartal and his colleagues used to grow cells of K. stuttgartiensis in the lab. Anammox bacteria are packed with heme-containing proteins, including the enzymes that perform the key reactions of the anammox process, making the cells remarkably red. (© Max Planck Institut for Marine Mikrobiology, Boran Kartal)

Please direct your queries to

Group leader

Microbial Physiology Research Group

Dr. Boran Kartal

MPI for Marine Microbiology
Celsiusstr. 1
D-28359 Bremen
Germany

Room: 

3126

Phone: 

+49 421 2028-6450

Dr. Boran Kartal
 
 
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