Environmental control of metabolism

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Growing microorganisms has been a fundamental element of much of experimental biology, as well as the underpinning of many modern molecular biology techniques. Perhaps we have a sample of earth that we want to analyse to find a new microorganism with antibacterial properties. Perhaps we are testing a patient sample for an infectious agent. Most likely, we are culturing a safe strain of E. coli that has been genetically modified to produce a protein of interest like human insulin that we can isolate from it and administer to patients.

This is how the metabolism of microorganisms can be used to obtain specific biological products. Culturing them is the go-to method because they are very adaptable, easy to use and have fast growth. Therefore, a variety of environmental factors can be fine-tuned in the lab to obtain specific outcomes or products from various types of microorganisms including archaea, bacteria and eukaryotes.

Secondary metabolism of microorganisms represents the non-basic pathways in metabolism that may not be entirely necessary for survival. Products of this include antibiotics produced by e.g. fungi against bacteria, and pigments.

Secondary metabolism can be advantageous as it doesn't focus on growth needs, and can instead provide better adaptations in the environment i.e. ecological advantage through better longer term outlook, reproduction and other metabolites non-essential to survival with an otherwise significant effect on the overall outcome of a species' ability to thrive in its environment. On the other hand, some secondary metabolites may not make a difference either way at a given time.


Aseptic technique

Aseptic means free of contamination. There are hundreds of fungal spores in the air we breathe at all times. There are bacteria and viruses everywhere. If we are to culture Escherichia coli (bacteria, prokaryote), archaea (such as species that can produce methane by metabolising CO2) or perhaps Pichia pastoris (yeast, eukaryote), we're going to be feeding them some nice nutrients, and chances are, loads of other microorganisms will jump at the opportunity to feast.

We don't want contamination, we just want our specific species that we are culturing and nothing else. The various techniques employed to this end have evolved through time and can even differ between labs and scientists:

A flame (Bunsen burner) can be used in the close vicinity of handling the target microorganism and related equipment and reagents, in order to make the surrounding air warm up and rise higher, carrying away any contaminants that might be present in the air close to our working space.

The equipment we use can be sterile as bought (e.g. plastic loops in sealed bags) or sterilised by passing it through the flame after dipping it in ethanol (e.g. reusable metal loop). Similarly, the lids and necks of bottles of liquids can be passed through the flame briefly upon opening and closing.

The working area can be cleaned with a 70% ethanol (now slightly changed and called IMS-industrial methylated spirit to make it unfit for human consumption) solution before and after the procedure is done, and to clean any other items as necessary, such as gloves, other items and surfaces, etc.




Sidenote: what even is that giant safety pin???

A step up from using a flame is using biological safety cabinets that provide a larger, fully controlled and enclosed working area, which filters the air mechanically to maximise safety and minimise contamination. This also needs to be maintained sterile with ethanol and other cleaning agents, and all samples and equipment kept inside must be separately sterilised with the ethanol solution as they are being used, taken in and out of the cabinet.


Basically, spray this stuff everywhere.




When finished with the samples and equipment, another round of safe disposal and sterilisation takes place, even if the microorganism you are dealing with is supposedly safe. You know, precautions and all that.


Yummy food

Ok, so we have our glorious sample or microorganism or whatever that we're about to grow. We grow it using special media, such as LB (lysogeny broth) for bacteria and YPD (yeast extract peptone dextrose) for yeast. These media contain basic nutrients like sugars and amino acids, and encourage microorganisms to thrive.

They can be formulated into liquid form (broth) and incubated in flasks, or into gel (agar) form and incubated in Petri dishes (a.k.a. plates). Selective media exist that specifically stimulate or inhibit a certain type or microorganism, making it easier to identify and isolate what we're growing.




Microorganisms need an energy source such as chemicals and light, as well as the raw materials to make biological products, They can be capable to constructing all the products needed e.g. carbohydrates and proteins (biosynthesis) from a simple pool of chemicals.

Some species may need additional compounds for their metabolic activity. These can be fatty acids or vitamins, and are added to their basic nutrient mix.

Depending on the experiment or application, extra compounds must be added to the growth medium. For example, metabolic precursors would be added to enable a specific reaction to take place under certain conditions. Say we are testing if a sugar-consuming species is able to evolve or activate a protein-metabolising pathways instead as a source of energy in extreme conditions. We might need to add specific proteins that we expect them to potentially break down.

Alternatively, we might be testing if a new enzyme they produce that we genetically engineered into them is capable of catalysing a reaction to produce a coloured chemical. The metabolic precursor here would be the substrate that precedes the coloured chemical.


Conditions

Temperature, oxygen provision and pH of the growth medium are two other key environmental factors determining microorganism metabolism.

Temperature is a master regulator of metabolism, and different species have different default temperatures. Many experiments are run at human metabolic conditions i.e. 37 degrees C because human proteins are being expressed, or the metabolic process investigated must work in human conditions if it is to be developed into a drug, etc. Microorganisms such as bacteria can also have the same optimal temperature as humans because they infect humans.

Temperature can be set using a water bath and thermometer, an automatic incubating "fridge", heat-controlled plates, shaker-incubators and others.



Oxygen is already present in the air which can be enhanced by shaking liquid flasks to introduce bubbles. In the case of archaea, however, the species cultured for their special methane-producing capability will not metabolise CO2 into methane (CH4) in the presence of oxygen.

This makes their culture trickier because it must be done in the absence of oxygen altogether. Reducing agents are used to ensure no oxygen is present, and special precautions must be taken to handle them without accidentally letting atmospheric air in which is already 21% oxygen.

pH affects enzyme activity and chemicals involved in metabolism, so maintaining optimal pH is key to enabling the right outcomes from microorganism culture. Growth media can be pH adjusted e.g. to pH 7.4 by using a pH meter, acid and alkali solutions. Commonly, sodium hydroxide (NaOH) is used as the alkali (providing the OH- groups), and hydrochloric acid (HCl) is used as the acid (providing the H+ ions).



If the measured solution is too low (on the pH scale of 1 -14; most acidic - most alkaline), pH can be increased by adding NaOH to make it more alkaline. If it's too high, HCl can be added to make it more acidic.


Measuring growth

Once the right environment is set up (often this means 37 deg. Celsius, shaking the flasks to introduce oxygen bubbles into the solution and optimise growth, leaving plates incubating overnight, etc.), growth can finally be monitored. There are many ways of doing this, such as cell countviable cell countmass and optical methods that detect turbidity.

Total cell counts involve pipetting a small volume from a liquid culture under a slide with a grid and looking at it under a microscope. The smaller sections of the grid can be used to count the number of cells, then multiply it by the number of sections and by the factor corresponding to the volume taken from the main solution, to obtain the total number of cells present.

For example, if we count 15 cells in one of 25 grid sections from a 10 microlitre sample of a total volume of 10 mililitres (there are 10,000 microlitres in 10 mililitres, so our sample is 10,000 / 10 = 1,000 times less than the total volume), we would compute:

15 cells x 25 sections x 1,000 = 375,000 cells in our total volume



Another method of measuring your cultured microorganism is viable cell count through dilution plating. This is done when the initial amount is unknown, as well as to find out various things about the organism. Sequential agar plates of increasingly diluted microorganism samples are set up, and the emerging colonies are counted (usually grown overnight). Colonies form from just a single or few cells, so are good for tracking growth and estimating numbers. On some plates nothing might grow, while others might have far too many colonies to easily count.





The mass of course reflects the grow of microorganisms too, and can be measured from liquid cultures usually after centrifugation to separate the cells from the liquid, disposing off of the liquid an then weighing the solid mass of cells that have grown.

Because this method enables the detection of those cells in culture that are alive (as opposed to total cell count), is is termed viable cell count.

Finally, and the most used measurement technique, is measuring the absorbance or optical density (OD) of a liquid sample. As cells grow, the solution becomes increasingly turbid (opaque, cloudy) so how much light can pass through is a measure of how many cells have grown. For example, 1 ml samples from large flasks (of around 1 or 2 litres) of E. coli culture are taken every half an hour. They are pipetted into special clear cuvettes that are placed into a spectrophotometer. This passes a beam of light through the sample and detects the light passing through on the other side at a wavelength of 600 nm, specific to E. coli.


A value of 0.1 shows the beginning of bacterial growth, while by 0.8 they are growing exponentially. The trajectory of their growth curve is highly reproducible, and indicates specific growth stages in a culture.




The lag phase represents the beginning of their growth where enzyme induction starts taking place. Once they get adjusted to their new environment and start thriving, they are ready to divide. This takes place actively during the log phase when their growth is exponential (because 2 cells become 4, and 4 become 8, and 8 become 16). Once their expansion into the media has reached its maximum potential, and they begin to run out of space and nutrients, they reach the stationary phase where division halts.

If the medium is left the same, with excretory products ever increasing and nutrients running out, they begin to die. This is the death phase.

For the purposes of high metabolic activity and production of desired protein e.g. enzyme, the log (exponential) phase is of key interest. This is where bacteria divide rapidly and along with that, produce whatever the desired chemical might be. When the product is genetically controlled to be activated and start production upon the presence of a specific chemical e.g. a lactose analogue inducer, production can be initiated externally by adding the chemical at a specific time.

Alternatively, an inhibitor might be added to halt a certain reaction or protein synthesis activity. This might be done to check the amount produced in a given time period, or prevent toxicity arising to the microorganism making too much of a certain chemical.

This time is during log phase. A microorganism culture is often monitored closely to see when growth reaches this stage.




Growth from zero to exponential looks as expected on the linear scale, with slow start where the lag phase is, followed by the rapid growth into the log phase. The same growth data can be represented onto a semi-log scale that uses one of the axes (the population axis i.e. y axis) as a log scale while the x axis remains the same, linear. This graph shows growth as a linearly increasing variable over time.

Essentially, the exponential growth seen on the linear graph becomes a linear growth on the semi-log graph. Both represent the rapid bacterial growth over time into the log phase. The doubling time here is 20 minutes.

Ok byeeeeeeee

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