The True Superheroes: Microorganisms Survive it All!

Did you know that microorganisms can live far up in the clouds and deep in the Earth’s crust? These tiny organisms have adapted to all special habitats on Earth. To study special habitats, scientists can focus on the microorganisms and their biological reactions, or on the non-living components, like minerals and chemical reactions. However, it can be difficult to separate the two! Since little was known about the best way to remove microorganisms from environmental samples without changing the non-living properties of the sample, we decided to perform an experiment to find out. We exposed marine sediments to three different methods of sterilization, to see which method was best for eliminating microorganisms. We also studied whether the three methods affected the chemical properties of our samples. We found that it is challenging to create sterile conditions because some microorganisms can survive high pressure, high temperature, radiation, and toxic chemicals!


MICROORGANISMS CAN LIVE EVERYWHERE!
Microorganisms were the earliest life forms on our planet. They are also the smallest! The term "microorganisms" describes a diverse group of single-celled organisms including bacteria, archaea, some fungi, algae, protozoa, and viruses. These organisms can only be observed using microscopes because of their tiny size. Microorganisms live in our stomachs and on our skin, but they also live everywhere else-from the deepest parts of the Earth's crust to high up in the clouds. Some even live under the ice of the Antarctic, or in . billion-year-old volcanic rocks, or in hot springs ( Figure A). They can be found at extreme temperatures, both hot and cold, in the extreme salinity of the Dead Sea, in the extreme radiation of nuclear reactors, under the high pressures of the deep sea, in the complete dryness of desserts, and in acidic and basic lakes. Although many of these extreme conditions are toxic to most other life forms, bacteria have evolved ways to resist the harsh conditions and survive.

SURVIVAL SECRETS OF MICROORGANISMS LIVING UNDER EXTREME CONDITIONS
Microorganisms have evolved several fascinating strategies to help them survive in extreme habitats ( Figure B). Here, we explain a few of them.

Metabolic Flexibility
Many microbes have evolved the ability to change which foods they eat, depending on what is available. Some can also adapt their metabolism to changing conditions, like heat or cold [ ].

METABOLISM
All the biochemical processes that maintain the life of an organism. In simple words, metabolism is the sum of eating, drinking, digesting, and growing.

Dormancy
When conditions are unfavorable, some microorganisms can enter a state of dormancy, which resembles "sleep mode." They slow down DORMANCY When microorganisms shut down their metabolism, which means when they take a deep sleep and their growth, development and physical activity are temporarily stopped. their metabolism so that they eat and grow less until conditions become favorable again.

Spores
When conditions are unfavorable, some microorganisms can form a special blanket around themselves, made of extraordinary proteins. These protected forms are called spores. Spores can help a Heat Resistance Some microorganisms have special proteins and lipids that can protect them from very high temperatures. The Greek prefixes thermo-and pyro-tell us that these thermo-and pyro-microorganisms can live close to heat and fire.

Radio-Resistance
Gamma-rays are a type of radiation produced by the decay of radioactive material. Because gamma-rays can penetrate tissues and cells, they are very dangerous for most organisms. Some kids.frontiersin.org August | Volume | Article | microorganisms produce extraordinary pigments that serve as a protective shield against gamma-rays. These radiation-resistant microorganisms also contain multiple copies of their genetic information, which helps them to survive if one copy is damaged by radiation.

WHAT IS THE BEST WAY TO KILL MICROORGANISMS?
Microorganisms have a huge impact on our environment-they make life on Earth possible. Most microbial habitats have not been well-studied yet. For example, % of the ocean is still not well-characterized. This means that researchers who explore marine habitats still have a lot of work to do! When researchers study samples from the environment, they often want to know whether the processes happening in those samples are biological processes, meaning that

BIOLOGICAL PROCESSES
All processes that are performed by living organisms. they are driven by the biological activity of living microorganisms, or whether they are geochemical processes, meaning they are driven

GEOCHEMICAL PROCESSES
All processes that happen in the environment that are purely driven by chemical reactions. by abiotic, chemical reactions, without the activity of living organisms ( Figure A). One way to know which kinds of processes are occurring is to eliminate all microorganisms from the environmental samples so that the geochemical processes can be studied on their own. Eliminating all microorganisms from a sample is called sterilization STERILIZATION A process that kills, removes, or deactivates microorganisms.
To e ectively investigate geochemical processes, the sterilization methods used on the samples should not a ect the geochemical processes. Before our experiment, not much was known about whether sterilization methods change the geochemical properties of environmental samples. So, we decided to study the e ects of commonly used sterilization techniques on marine sediment. We soon found out that this question was not as straightforward as it seems!

HOW CAN WE PROVE THAT AN ENVIRONMENTAL SAMPLE IS STERILE?
What does "sterile" mean, exactly? By definition, it means that something is totally clean and free of living microorganisms. When we want to remove microorganisms from our experimental samples, we must think about how we will test for the presence of microorganisms. How do we know if the microorganisms are living? Well-known properties of living cells include an organized cell structure, an intact cell membrane, the ability to respond to stimuli like chemicals or light, and the ability to grow and reproduce. We can easily measure some of these properties in the lab ( Figure C). We can examine the reproduction of microorganisms by counting the number of living cells under a microscope or by growing them in dishes called agar plates, which contain nutrient-rich food. We can examine microbial productivity, which means following microbial activity in the laboratory, by quantifying how much the microorganisms are eating, digesting, and producing. For example, we can measure how many amino acids microorganisms eat while producing biomass to grow. There are also laboratory techniques to measure the amounts of nucleic acids (RNA and DNA) in a sample providing information about the present microbial community and which microorganisms are active.

OUR STUDY WITH MARINE SEDIMENT
To determine the most e ective method to sterilize marine sediment samples, we looked at the impacts of di erent sterilization methods on marine sediment. Equipped with rubber boots, shovels, gloves, and buckets, our research team collected marine sediments in Denmark, on the Baltic Sea coast. We sterilized these samples using three methods: autoclaving, in which samples were exposed to high heat AUTOCLAVE An instrument that applies heat and pressure and is used to kill microorganisms.
( • C) and high pressure; a high dose of gamma-radiation; or a high concentration of a toxic chemical called sodium azide ( Figure  B) [ ].
kids.frontiersin.org August | Volume | Article | After the sterilization treatments, we looked for the presence of microorganisms in the samples by directly counting them on agar plates, detecting how much they were growing, and measuring the amounts of nucleic acids present. We took these measurements right after sterilization and again weeks later. At the same time points, we assessed the geochemical properties of the samples, to see if substances were released from the samples or if changes to the minerals occurred due to the sterilization processes ( Figure C).

NUCLEIC ACIDS ARE STILL PRESENT IN STERILIZED SAMPLES!
Surprisingly, we still found nucleic acids immediately after all sterilization procedures ( Figure A) [ ]. So far, it is known that DNA from dead microorganisms can persist in ocean sediments for weeks to years [ ]. RNA is normally found primarily in living microbial communities, but there are hints that RNA also persists long after the death of the organisms, and that RNA might be more stable in ocean sediments than previously assumed [ ]. Nucleic acids can be broken down by special proteins called enzymes, which work like molecular scissors. After sterilization by autoclaving, we knew that DNA-cutting enzymes were still working because the amount of DNA was reduced after weeks. Chemicals like sodium azide do not a ect most nucleic acid-cutting enzymes, which is illustrated by the degradation of DNA and RNA at the week time point in those samples. In gamma-irradiated samples, however, low levels of RNA were still present after weeks. It is possible that the RNA-cutting enzymes were damaged by the radiation and no longer able to degrade the RNA. Does the presence of nucleic acids mean that the samples are sterile, or not?

MICROORGANISMS SURVIVED, MINERALS CHANGED, SUBSTANCES WERE RELEASED
Autoclaving is widely used for sterilization because it inactivates most fungi and protozoans. But a lot of bacteria and archaea survived autoclaving in our experiment ( Figure A) [ ]. In addition, autoclaving changed the minerals in our sediment samples and caused the release of di erent kinds of substances from the samples. Sterilization by gamma-radiation eliminated most microorganisms, but it also caused the release of substances from our sediment samples and changed minerals ( Figure A) [ ]. Therefore, autoclaving and radiation might not be the best sterilization methods to use for environmental samples, since they changed the sample's geochemical properties. Sodium azide sterilization killed only a minority of microorganisms but inhibited microbial growth by the two-week time point [ , ]. Sodium azide did not change the release of substances from our samples or the minerals in those samples.

TAKE-HOME-MESSAGE: MICROBES SURVIVE IT ALL!
To clearly distinguish biological processes from geochemical processes in environmental samples, an optimal sterilization method must be chosen. However, all methods seem to have disadvantages, so researchers need to compromise depending on the research questions to be answered. Autoclaving should be used for studies that focus on biological properties, while chemicals and gamma-radiation might be better to sterilize samples that will be analyzed for geochemical properties.
Our experiments make it clear that microorganisms are unseen heroes, much stronger than we think ( Figure B)  . Soil microorganisms can overcome respiration inhibition by coupling intra-and extracellular metabolism: C metabolic tracing reveals the mechanisms. ISME J. : -. doi: . /ismej. .