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Proton gradient occurs in the cytoplasmic membranes

A proton gradient occurs in the cytoplasmic membranes and membranous compartments e.g. mitochondria. A cytoplasmic membrane is selectively permeable to ions and organic molecules, controlling the movement of substances in and out of cells. The membrane surrounds the cytoplasmic matrix, splitting the inside of the cell i.e. organelles from the outer environment. The cytoplasmic membrane is a fluid mosaic model, which is made up of phospholipids and protein molecules, forming a phospholipid bilayer. Phospholipids are building blocks of cellular membranes; they are composed of a phosphate, glycerol, and fatty acids. The phosphate-glycerol segment of the molecule is water soluble (water-loving) and polar. The fatty acid fraction of the molecule is water insoluble (water-hating) and non-polar. As shown in figure 1 a phospholipid bilayer is made up of hydrophilic heads and hydrophobic tails with embedded proteins.

Figure 1: A phospholipid bilayer

The proteins in the membrane are involved in a variety of functions such as cell signalling, adhesion, transport, enzymatic activity, surface recognition.

The transport proteins are known as permeases, they assist substances to move across the membrane.

There are many transport mechanisms in bacteria which can uptake nutrients, eliminate waste/end products and remove toxic compounds.

Passive diffusion is the movement of small molecules and ions across a phospholipid bilayer membrane, from a region of high concentration to a region of low concentration. This type of diffusion does not require energy (ATP), it is the concentration gradient that drive the movement of molecules. Therefore, the size of the concentration gradient controls the rate of the reaction, so if more nutrients are taken in, the rate of the reaction will decrease, unless they are used immediately by the cell.

Facilitated diffusion is aided by a protein carrier; it facilitates the diffusion by moving a specific molecule down its concentration gradient, through the membrane into the cell. Depending on the concentration gradient, the diffusion can occur in both directions and this type of transport also does not require any energy input. Facilitated diffusion has a higher rate of diffusion across the cytoplasmic membrane than passive diffusion. Both passive and facilitated diffusion do not require a proton gradient in order for transport to take place.

Active transport is another mechanism, it involves the movement of molecules and ions across the membrane from a low concentration to a high concentration, transporting molecule against the concentration gradient. However, this process requires energy (ATP) and this is obtained from the proton motive force, hydrolysis of ATP. This process has the capacity to concentrate substances (can accumulate required substances such as, nutrients). At high solute, a carrier saturation effect takes place.

A proton motive force is the production of ATP from a proton gradient. In the electron transport chain there are three proton pumping sites where the protons (hydrogen ions) are pumped out to intermembrane space. An electrochemical gradient forms when there is a higher concentration of protons in the intermembrane than the inner membrane, so the protons then move back through the ATP synthase complex generating ATP. This helps to release the gradient. ATP synthesis is driven by the flow of protons, the more protons the more ATP is formed and less protons means less ATP is produced. The energy formed can be used for many cell activities such as active transport. There are certain compounds which can uncouple the tightly coupled ETC and ATP synthase. When these processes are uncoupled the ATP synthase stops but the ETC continues. When the ATP synthase is stopped this means no ATP can be made, so the energy is released as heat. This process (uncoupling) can be use for maintaining body temperatures during hibernation.

There are different types of proteins involved in the active transport system such as: the proteins of the ATP-binding cassette (ABC) transporters, the proteins occupied in group translocation, antiporters and symporters.

ATP-binding cassette (ABC) transporters (membrane spanning proteins), are protein which have a high affinity for substances found in the periplasm. The periplasm is positioned between the cell wall and the cytoplasmic membrane. So the periplasmic-binding protein binds to the substances to be transported across the membrane and carries them to the membrane-spanning protein/transporter. However, ATP energy is required for the transportation of substances across the membrane through the transport protein, so ATP binds to the ATP hydrolysing enzyme which breaks it down to ADP+Pi and energy. The energy produced facilitates the transportation. This method of transport is used by E. coli to transport sugars such as: maltose and galactose and amino acids like: glutamate and histidine.

Group translocation occurs in prokaryotes and is another type active transport. In the process of a molecule being transported across the membrane it chemically alters/modifies. After the molecule enters the cell, the cytoplasmic membrane becomes impermeable to that molecule and therefore, it remains within the cell. An example this transportation in bacteria is the phosphotransferase system.

Antiporters transport two substances simultaneously in opposite directions. Symporters also transport two substances simultaneously in the same direction. Both antiporters and symporters require energy; this is known as active transport. The energy can be obtained from ATP and/or the proton motive force.

Disruption of the proton gradient in a bacterial cell may lead to problems with solute transport such as active transport; this mechanism is important for accumulating nutrients against the concentration gradients. Without good functioning of the proton gradient; energy such as ATP would not be hydrolysed and without ATP many metabolic processes in the cell may come to a halt.

Question 2a

Growth conditions and the history of the growth culture are important because they ensure a short lag-phase. For a short or no lag-phase, you would inoculate a sample from the exponentially growing culture to the same medium; nutrient broth and the same conditions of growth; incubation at 30oC. The bacterial culture would have an immediate exponential growth because there was no adaptations or regeneration of essential nutrients required. A lag phase will occur if the inoculum was taken from the stationary phase, old culture, and transferred to the same medium and growth conditions. So a lag phase occurs due to the depletion of essential components such as enzymes and intermediates in their metabolic pathways. Therefore, time is required for regeneration and adaptation for dormancy and protection.

Optimum growth rate occurs in the exponential phase. Environmental conditions such as temperature, medium and the organism’s genetic characteristics can influence the rate of growth. So to ensure an optimum growth rate the organism can be incubated at its optimum temperature, continuous supply of the essential nutrients and removal of any overflow this can be achieved by using a chemostat. A steady-state is maintained for the nutrient level and the cell number to remain constant. Therefore, an optimum growth rate and the population density are controlled.

Question 2b

When the cells are in their optimum state of growth they are in their exponential phase. They divide by binary fission at a maximum rate repeatedly and rapidly. It is the best studied phase because it is the most reproducible physiological state, consistent functioning of the bacteria. Growth can be related to any measurement of cell component. Only with exponential phase you can take any time point and associate it to any other point.

Question 2c

Cell mass, cell activity and cell number are the three different ways of measuring the growth of microorganisms. Another method of measuring the biomass can be the turbidity measurement. Turbidity is a method of assessing the number of bacteria in a sample, by the measurement of light scatter. A spectrophotometer or photometer can be used to measure unscattered light. The light passed through the suspension, can measure and quantitate the cell number indirectly. Increase in cell number can increase the absorbance and decrease the transmission hence, more light is scattered which means more turbid the suspension.

Turbidity measurement has many advantages such as: it is simple, fast to use, non-destructive and can also be reproducible; only after the calibration of a standard curve. The standard curve needs to be constructed in order to approximately calculate the cell mass. However, the sensitivity in this experiment is limited and also not suitable for bacteria that are clumped or aggregated together, this may lead to a low cell count.

Viable plate count only measures viable (living) cells and is very sensitive; these are the advantages of this experiment. There are two types of methods: spread plate method and a pour plate method. It is the transfer of diluted samples to plates for incubation. The difference between a spread and a pour plate is that colonies can form on the surface of the agar as well as within the agar, in the pour plate method. Disadvantages of this experiment can be the preparations of the dilutions, media and the time required for incubation before getting the results. Comparison between viable plate count and turbidity measurement is shown in table 1.

Table 1: Comparisons:

Viable plate count

Turbidity measurement

Direct measurement of microbial growth

Indirect measurement of microbial growth

Time required for preparation of the experiment

Time required for obtaining the results, preparation of a standard curve

Only measures viable cells

Measures both dead and viable cells

Sensitive

Limited sensitivity

Number of colonies obtained depends on the incubation conditions and time, the size of the inoculum

Turbidity depends also depends on the incubation conditions and time, the size of the inoculum

Question 3

Fermentation is the breakdown of carbon containing compounds, to yield energy in the absence of oxygen by substrate-level phosphorylation and also the production of natural products which are useful to humans for maintaining health.

There are different microorganisms which have different requirements of oxygen. Aerotolerant anaerobes are microorganisms which gain their energy only by fermentation; they cannot grow in the presence of oxygen. Obligate anaerobes are the same as aerotolerant anaerobes; the only difference is that they can grow in both fermentative and anaerobic conditions. Facultative anaerobes are the most common bacteria found. They can obtain their energy through both aerobic and anaerobic or fermentation respiration, if oxygen is present or absent it does not make a difference to them, they can still make products using an appropriate pathway.

Lactic acid and ethanol fermenters are the two main types of fermentation found in bacteria. Lactic acid fermentation is when pyruvate, the product from glycolysis, reduces (NADH to NAD) to form lactate as shown in figure 2.

In ethanol fermentation, decarboxylation of pyruvate takes place (CO2 is the by-product, given off) and reduction acetaldehyde by NADH takes place to form ethanol also shown in figure 2.

Figure 2: Simple equations of the two types of fermentation.

Homolactic fermenters produce only lactic acid whereas; hetrolactic fermenters produce lactic acid, ethanol and carbon dioxide.

In the absence of oxygen, fermentation allows the continuation of glycolysis, by regenerating NAD. However, this pathway only yields to 2 ATP whereas, if the full aerobic respiration took place (in the presence of oxygen), the process would had yielded 36 ATP. Some of the products produced in fermentation can be useful for industrial, environmental and medical uses.

In food industries, lactic acid fermentation is used to produce dairy products such as milk, yogurt, cheese can be made and rye bread. Example of lactate fermenters can include: Lactobacillus and Streptococcus.

In alcohol fermentation, the products can be used to produce wine and beer for industrial and commercial use. Ethanol produced by fermentation can be used as an alternative renewable fuel source for environmental uses.

Some medical uses of ethanol can include common antibacterial hand sanitizers (i.e. used in hospitals) and medical wipes. It can also be used to treat poisoning by toxic alcohols (e.g. methanol). Examples of alcohol fermenters can include: bacteria and yeast such as Saccharomyces cerevisiae.

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