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**Taken from my research thesis on ecological microbiology: Lotka Voltera Model Analysis**

The topic of developing a strategy for a rural village of about 500 people to create a community water supply that was safe to drink would be met without having to spend such a vast amount of capital in building large water treatment facilities that spend millions of dollars annually to treat water via UV light as well as chlorination. One logical way for this rural community to have a community water supply that is affordable for about 500 people is building a local reservoir system that will take advantage and collect rainwater, ground water.

To accommodate the community, plumbing systems will be built to every household and instead of the typical iron and metal-based plumbing systems, which are known to rust; the plumbing systems will be composed of a plastic inner layer allowing the collected water from the reservoir to pass tow every single house and thus minimize the amount of water’s interaction with water and any pathogens in the plumbing system. The outer layer of the plumbing system will be composed of platinum, which will provide structural support for the plumbing systems and due to its rust resistant capability, will be effective for the water. Now the plumbing systems will also have a local filter in each household that will prevent large bacteria and pathogen from entering. This is only the primary part of the water system, the second part is hiring workers to operate the local dam water reservoir and have the specialized workers that have a four water system treatment before being sent to the plumbing system to each household. The first phase is the boiling phase, which will boil water to up to 85 degrees and after the samples are boiled, sent to the second phase which will apply algaecides, which will kill and inhibit any gree or blue algae that were inside the water. Then the water samples will be sent to the third phase, which will utilize the use of neutralizing agents, In order to neutralize acids and basics we use either sodium hydroxide solution or calcium carbonate to increase pH levels and as a result neutralization causes a rise in temperature levels. The fourth system will be utilizing fluoride removal from the water samples and after all these systems are met, the water will be then sent to the plumbing system and will go through the ultrafilter that will be installed in each house to prevent the entrance of small bacteria and some large pores.

The use of this water treatment facility will be effective and provides clean drinking water for the people because the treatment plant itself will be small;  about 1/5th the size of a water treatment plant/dam in large cities. This way its cost effective for the small community. Additionally, to apply this setting, we would also be requesting a massive contribution by the state or federal government to run this experiment thereby having financial factors to pay and operate the system for the benefit of the local community.

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1.   Many bacterial communities in soil and aquatic systems include diverse bacteria that each utilize different assimilatory or dissimilatory pathways for nutrients such as sulfur or nitrogen. We are given that one bacterium is able to fix nitrogen, meaning that it is a nitrogen fixing bacteria. By this process, a nitrogen fixing bacteria like azotobacter, klebsiella, clostridium, and methanococcus will reduce atmospheric gaseous nitrogen to ammonia. In this process, the reduction of nitrogen to ammonia is catalyzed by a nitrogenase enzyme. The process of nitrogen fixation is quite exergonic for these bacterial organisms needs and requires a large ATP expenditure, at least 8 electrons and 16 ATP molecules, 4 ATPs per pair of electrons are necessary. It can be seen in the procedure of:
N2+ 8H+ + 16 ATP
                2NH3 +H2+16ADP +16Pi

 Although nitrogen gas is numerous in the atmosphere, there are very few organisms that can fix nitrogen and some organisms must incorporate either ammonia or nitrate. Ammonia nitrogen can be incorporated into organic material relatively easily and directly because of its abundance and one major route for ammonia incorporation often is the formation of glutamate from ?-ketoglutarate. Microorganisms will then utilize transaminase, which when worked with glutamate dehydrogenase, will incorporate ammonia into a variety of amino acids. Another route of ammonia incorporation that some bacteria use is the use of the enzymes glutamine synthetase and glutamate synthase. Ammonia is used to synthesize glutamine from glutamate, and transfers amide nitrogen back to the ? ketoglutarate to make more glutamate molecules. In this pathway, glutamate acts as an amino donor in transaminase reactions, ammonia may be used to synthesize all common amino acids when suitable transaminase enzymes are present. This particular route is common for organisms such as E. coli and B. magaterium. Some other organisms also utilize the pathway called assimilatory nitrate reduction, which reduces nitrate to ammonia before the nitrogen can be converted to an organic form. The process takes place in the cytoplasm in the bacteria and the process is seen as:
         NO3- + NADPH + H+ ? NO2- + NADP+ + H20
Nitrite is next reduced to ammonia with a series of two electron additions catalyzed by nitrite reductase and possibly other enzymes. The ammonia is then incorporated into amino acids. Although there is another procedure, which is anaerobic based, called the dissimilatory nitrate reduction pathway, which is also used by organisms such as Paracoccus and Pseudomonas aeruginosa. The pathway can be seen as:
            NO3- + 2e- + 2H ? NO2- + H20
 
2.   Oxygen can pose as a problem in nitrogen fixation because nitrogenase, which is responsible for catalyzing the reduction of nitrogen to ammonia, is quite sensitive to O2 and must be protected from O2 inactivation within the cell. Poisoned by oxygen because molecular O2 looks like molecular N2 to an enzyme’s point of view; the stronger electron acceptor will oxidize the metal cofactors in the nitrogenase enzyme. Bacteria do have a protection from oxygen and is a structure called the heterocyst. The three strategies that the nitrogen fixing bacteria utilize to prevent oxygen from entering the nitrogen-fixing machinery include the use of temporal, spatial and symbiotic strategies. The temporal strategy, which is used by Synechococcus, will photosynthesize by day and fix nitrogen by night. The Spatial strategy, which is used by anabaena, utilizes the heterocyst, which is a protection from oxygen. The symbiotic strategy, which is utilized by Rhizobium, depends on a second organism, in the case of Rhizobium, it will get a plant to supply energy and O2 free environment is observed (leghemoglobin).

3.   Bacteria use three different pathways for the oxidation of glucose to pyruvate. These three pathways include the Glycoloytic pathway (Embden-Meyerhoff pathway), Pentose phosphate pathway, and the Entner-Doudoroff pathway. From running the Glycolytic pathway, glucose (C6) will lead to 2 glyceraldehyde 3-P (3P) and will then lead to 2 pyruvate (C3) and yields 2 ATP and 4 NADH per glucose. This is the most common for glucose breakdown and can go in the presence or absence of O2, and this is quite common in all major groups of microorganisms. This pathway is rather is the most energetically efficient. The second pathway, called the Pentose phosphate pathway starts off with (C6)?3 ribulose 5P (C5) ? 2 C5 ? C3 + C7 ?C6(fructose) + C4; C4 + C5 ? C3 (glyceraldehyde 3P) + C6 (fructose) and yields 2 NADPH per glucose. The fructose is then recycled. The cells that do this for bio synthetic purposes and not for energy, on the contrary, organisms that use Pentose phosphate pathway also use the Glycoloytic pathway for energy. The Entner-Doudoroff pathway takes glucose? KDGP (2-keot-3-deoxy-6-phosphogluconate) ? 2 pyruvate; yields 1 ATP, 1 NADH, 1 NADPH per glucose; is substituted for glycolysis in Pseudomonas, Rhizobium, Azotobacter, Agrobacterium etc. The 1NADH is used for catabolic reactions and the 1 NADPH is used for anabolic reactions such as photosynthesis and to build things. And the reason, as explained, for choosing to run each pathway even if its not the most energy-yielding pathway is because these pathways are essential for either biosynthetic purposes (building), anabolic reactions, catabolic reactions; there is a reason for running each pathway.

4.   The reason why most amino acid encoding operons repressible while most sugar-metabolizing operons are inducible because enzymes whose amount of reduced by the presence of an end product are repressible enzymes, which in this case are amino acids for the amino acid encoding operons. An amino acid present in the surroundings may inhibit (repress) the formation of the enzymes responsible for its biosynthesis. This is why most amino acid encoding operons are repressible. However, sugar metabolizing operons are inducible, that is, its level rises in the presence of a small molecule called an inducer. This means that sugar metabolizing operons can be increased in growth or number in the presence of an inducer; the regulater gene, snynthesizes an active repressor that binds to the operator, and blocks RNA polymerase binding to the promoter, unless the inducer inactivates it, of course. As a result, in the presence of the inducer, the repressor protein is inactive and transcription will occur. In this case, sugar will be processed.
Sporulation genes are controlled by sigma factors rather than by inducible or repressible operons because the sigma factors (there are for kinds, the sigma e, sigma f, sigma g, and sigma k) are proteins that binds to the RNA polymerase and change the promoter specificity. The reason for this is that although the RNA polymerase core enzyme can transcribe any gene to produce a messenger RNA copy, it needs the assistance of a sigma factor to bind the promoter and initiate transcription. This provides a means for regulating gene expression; if a complex process requires a radical change in transcription, or the synthesis of several gene products in a precisely timed sequence, it may be regulated by a series of sigma factors; each sigma factor (there are four kinds), enables the RNA polymerase core enzyme to recognize a specific set of promoters and transcribe only those genes.  And as a result cannot be controlled by inducible or repressible operons. It is also critical to understand that sigma e and sigma f are responsible for controlling expression in the mother cell, the sigma g controls the expression in spore and the sigma k controls expression of mother cell lysis genes.
The process of sporulation becomes irreversible at a certain stage and this stage is in stage 0, in which there is miroorganismic decision to sporulate, which means that the mother cell will eventually die. Microorganism is motivated by environmental extremities such as starvation etc, and thus causes a signaling cascade in which kinase phosphorylates Spo0F protein. And as a result, Spo0F transfers PO4 to Spo0B, which transfers it to Spo0A, which in turn causes expression of sigma factors for sporulation. Once this occurs, it is impossible to reverse because once this happens, the sigma factors will then bind to RNA polymerase and change the promoter specificity. As a result of this, one will become a ‘mother cell’ and the other will become a ‘spore’. The latter stages such as stage I leads to the condensation of DNA into a nucleoid, , stage II is when the cell membrane engulfs DNA and forms forespore septum and forespore is the cell that is developing into the spore. Stage III is when the membrane surrounds forespore again, stage IV is when the cortex layer is formed between the membranes, thus helps remove water form the inside of the cell and protects from heat and radiation as well. Stage V is when the protein coats form around the cortex, stage VI is when the spore matures and lastly stage VII is when the mother cell lyses and thus releases the spore. From understanding these stages and steps, it is evident that stage O prepares the steps for sporulation and thus it is the stage where sporulation is irreversible because once the sigma proteins binds to the RNA polymerase and changes the promoter specificity, the outcome is essentially ? sporulation.

5.   Bacteria ferment even though it produces so much less ATP than respiring because bacteria have no external electron acceptors, thus it has to use its own electron acceptor; no electron transport; no electron transport chain (no electron acceptor) but it is necessary because NADH must be recycled for glycolysis to continue and survive. Certain bacteria ferment such as homolactic fermentors, which reduce pyruvate to lactate; the heterolactic fermentors, which allow the variety of end products such as lactate, EtOH, CO2 and there are the formic acid fermentors, which produce format? H2O + CO2; present in Escherichia, Salmonella, Proteus to name a few. It is also critical to understand that when dealing with respiration, there is some kind of external electron acceptor; and an electron transport chain; however in dealing with fermentation, there is no external electron acceptor; hence one has to use own electron acceptor; no electron transport. Considering the importance of fermentation and the end result of NADH and its recycling for the process of glycolysis to continue, it is clearly evident why bacteria ferment.
Anaerobic respiration produce less ATP than aerobic respiration because due to the lack of oxygen, pyruvate is not metabolized by cellular respiration, but by fermentation and unlike in aerobic respiration, the pyruvate is not sent to the mitochondia, but kept in the cytoplasm of the cell where it eventually converted to waste products. One has also to keep in mind that during aerobic respiration, oxygen is present and as a result during the process 36 ATPs per glucose is produced whereas in anaerobic respiration only 2 ATPs per glucose are produced. Both anaerobic and aerobic respiration share similar initial pathway of glycolysis, but aerobic metabolism continues with the Krebs cycle and oxidative phosphorylation while anaerobic does not. Microorganisms that utilize anaerobic respiration include obligate aerobes such as C. tetani and C. perfringens. The pathway of anaerobic respiration is seen as: C6H12O6 ---> 2C3H6O3 + 2 ATP.

6.   The restriction endonuclease and the restriction methylase systems protect a bacterium from viral inspection in that these are both restriction enzymes in that they are naturally present in bacteria to keep out unwanted DNA (viruses in particular) and restricts the entry of DNA into the cell. These restriction enzymes recongizes a 6-8 bp sequence , usually palindromic; the restriction enzymes will cut somewhere in the sequence and may leave overhanging nucleotides, which are called ‘sticky ends’; may even cut strands evenly called ‘blunt ends’. The cell must have a way to protect its own DNA thus restriction methylase and restriction endonuclease are critical. These restriction enzymes tage the genomic and plasmid DNA as its own and the methyl group causes steric hindrance, thus restriction enzymes cant bind and cut. It is also important to know that all commerical restriction enzymes are cloned from different bacterial species in example: EcoRI- E. coli, strain R, first enzyme discovered.
           EcorV-E. coli, strain R, fifth enzyme discovered
           Hinfl-Haemophilus influenzae

7.   The PCR process was not practical before the discovery of extremely thermophilic archaeans in that PCR (polymerase chain reaction) process involves the use of in vitro of fast working polymerase and involves in the idea that thermophilic bacteria have Pol proteins that can survive and endure extreme high heat. Primers bind DNA at ~65? C and strands denature at ~95?C, while the Pol elongates at around 72-80?C. The PCR process tests with varying temperatures requires the comprehension of the concept that enzyme doesn’t denature after repeating temperature cycles. So, without the discovery of extremely thermophilic archaeans, heat loving bacteria, then PCR would have never been tested because PCR was developed after the discovery of thermophilic bacteria such as Thermus aquaticus and Pyrococcus furiosus, both being vent dwelling thermophiles. After their discovery, it was needed to understand their primer binding temperature range and their denaturation range as well as their pol elongation range. One can say that the discovery of thermophilic archaeans was the catalyst for the invention and eventual use of the PCR (polymerase chain reaction) process by Kary Mullis to synthesize the large quantities of a DNA fragment without cloning it.

8.   The trp operon is regulated by attenuation, rather than by being inducible or repressible. At any given time, a cell will be producing trp mRNA, whether it needs to make tryptophan or not. One also has to consider that the synthesis of a leader peptide bya ribosome while RNA polymerase is transcribing the leader region regulates transcription therefore the tryptophan operon is expressed only when there is insufficient tryptophan available.  The trp operon’s usage of attenuation means that mRNA encodes the leader peptide before the first actually trip synthesis protein. From this, the leader peptide mRNA has 4 hairpin-side-forming regions and at the first side are 2 trp codons. If the cell is low on trp, then the RNA pol stalls here while waiting for trp-Trp-tRNA. This prevents first pair of sides from forming hair pin so that 2&3  form loose hairpin and translation continues and as a result tryptophan is made. If there are high presence of trip, RNA pol breezes through and 1&2 will form hairpin, thus allowing 3&4 to form hairpin. The 3&4 hairpin is quite tight and causes termination of transcription, which is called rho-independent terminator. This is why this ‘wasteful’ strategy works.


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