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University of Waterloo
BIOL 241
Barbara Butler

Topic 7: Drinking Water Treatment - Goals: remove pathogenic microorganisms; decrease turbidity; eliminate taste and odor and reduce nuisance chemicals. - Potable – safe for human consumption. - Most of the drinking water is the ground water. - The process: o Raw Water is dumped into a sedimentation basin where anionic polymers, alum and chlorine are added. o Sedimentation – allows the large particles to settle down. Water is then pumped into coagulation basin. o Coagulation – removes floc, containing insoluble material and microorganisms. After mixing the large particles interact and form large, aggregated masses. As they settle (by gravity), they trap and remove microorganisms, absorb organic matter and sediment. o Filtration – removes of all remaining particulates, organic and inorganic compounds. Filter typically consists of thick layers of sand, activated charcoal and ion exchangers.  Critical to remove disinfectant-resistant microbes like Giardia and Cryptosporidium  Slow sand filtration: Pile of sand in a large tank (4-6 feet in depth). Water runs in there and filters through the sand pile. At the surface of the sand you eventually get a build up of microbes that form a biofilm. These are significant in removal of impurities.  Rapid filtration: Uses a series - solid media/mixed media present, sand and ionic media present. Partially filtration and partially absorption o Chlorination – kills remaining microorganisms and prevents growth of new inocula. Add enough chlorine to satisfy the demand. Anything that is oxidizable will be oxidized via the chlorine. This activity will kill the microbes. It can help in terms of taste and odor. Add enough to satisfy but also add enough to have some left over – residual. This will let you know whether all the organisms have been killed or not.  Chloramination: Since the gas is extremely volatile and dissipates within hours so ammonia gas is added to it. It is there to ensure that the residual chlorine levels are maintained through the distribution system. HOCl + NH3  NH2Cl + H2O o Ozonization and UV radiation are other strategies that can be used to avoid production of disinfection byproducts. Ex. Halomethanes. Can also kill oocytes and cysts. Physical process that introduces no chemicals into the water. The equipment can be used in existing flow systems. No disinfection by-products are formed with UV disinfection. o Distribution systems - Monitoring Water Quality: - Water courses – sources of raw water for drinking. This is crucial to monitor the presence of potential disease-causing agents. - A lot of pathogens cause GI Tract Infections like Gastric, Diarrhea and Colitis. Some are transmitted by the fecal-oral route (cycle), other’s are acquired by physical contact or ingestion of water contaminated by the disease-causing agent. - Potential Pathogens: o Bacteria, Viruses: hepatitis viruses, rotaviruses, noroviruses and Protozoa: Giardia, Cryptosporidum, Entamoeba, Balantidium, Naegleria - Indicator Organisms: o There are so many pathogens; their numbers are often low in test waters; technical issues. o Use them for monitoring purposes. The type of indicator one uses depends on purpose/goal. o It is easy to detect and deal with and it behaves more like the actual pathogen. You want more indicators – as a safety factor. o Ideally the indicator should be resistant to the environment insults, disinfection in water treatments, easily detectable via inexpensive methods, non pathogenic, can multiply in environments and is a member of intestinal microbiota of warm blooded animals. - Coliform group: facultatively aerobic, gram negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 h at 36 degrees Celsius. - Total Coliforms: subgroup of thermotolerant bacteria Enterobacter, E. coli, Klebsiella preumoniae and others. Less common pathogenic intestinal inhabitant. - Fecal Coliforms: all coliforms that can ferment lactose at 44.5 degree Celsius. Typically don’t grow under environmental conditions some will grow not all - Testing for coliforms: - MPN Procedure – Total coliform test – employs liquid culture medium in test tubes to which samples of drinking water is added. Growth indicates contamination. - MF Procedure – fecal coliform test – at least 100 mL of water is passed through a sterile membrane filter, trapping any bacteria on the filter surface. Filter placed on a plate of eosin-methylene blue (EMB) – culture selective for gram-negative, lactose-fermenting microorganisms. - Defined Substrate Technology: based on the ability of coliforms and E. coli to metabolize certain substrate. Based on expression of certain genes. - Coliforms: possess gene for B-galactosidase. In it’s presence MUG (e-methylumbelliferyl-B-D- galactopyranoside), it metabolizes to produce a fluorescent product visible under UV light. - E. coli: posses gene for B-glucuronidase. Metabolizes indoxyl B-D-glucuronide (IBDG) to a blue compound. MUG turns into fluorescent product and IBDG turns into a insoluble blue compound. - These tests use the MF method and media containing both MUG and IBDG (MI Media). Results are examined after 24 hours. - Diseases: - Cholera (Vibrio cholera): caused by ingestion of contaminated water or raw shellfish/veggies o Gram-negative, curved rod-shaped and free living. o Attaches to epithelial cells in the small intestine where they grow and release cholera enterotoxin. Stomach acidity is responsible for the large inoculum needed to initiate cholera. o Causes severe diarrhea that can result in dehydration and death. Patients can loose up to 20L + water per day. If untreated mortality can reach 60%. Detected by rice water stools. o Treatment: Intravenous or oral liquid and electrolyte replacement therapy. 20g of glucose, 4.2 g of sodium chloride, 4.0g of sodium bicarbonate, and 1.8g of potassium chloride. o V. cholerae is an aquatic bacterium that often lives in association with other aquatic microbiota. - Typhoid Fever (Salmonella tyhpi): caused by contaminated water, can also be foodborne or by direct contact with infected individuals. o Carrier State: individual that harbours organism but shows no disease symptoms. o S. typhi: gram negative rod, one of the enterobacteria o Systemic infection with sustained bacteremia (bacteria in blood). High fever for several weeks. Headache, constipation, diarrhea. Perforation of the intestinal wall. - Giardiasis and Cryptosporidiosis: waterborne disease in areas with regulated water supplies o Has cysts/oocytes that are found in most water surfaces. o Giardia Intestinalis – flagellated protest. Has a cyst (trophozoites) that is environmentally resistant. The cyst germinates, attaches to the intestinal wall, and causes an explosive, foul- smelling and watery diarrhea, intestinal cramps, and nausea, and weight loss, flatulence. o Cryptosporidium pravum: parasites in warm-blooded animals. Small, round cells, that invades and grows intracellularly in mucosal epithelial cells of the stomach an intestine. Protist produces thick-walled, chlorine-resistant, infective cells – oocytes. Mild, self-limiting in healthy individuals. Chronic diarrhea in individuals with impaired immunity. - Legionellosis: may be asymptomatic. Can cause Pontiac Fever – mild, cough, sore throat, headache, self-limiting. Or Legionellosis – type of pneumonia, more likely to affect the elderly, immune-impaired. Intestinal disorder, high fever, chills, muscle aches, dry cough, abdominal pain. o Present in small numbers in lakes, streams, and soil. Relatively resistant to heating and chlorination. o Human infection via airborne droplets through air conditioning or heating systems that use water. Therefore, no person-person transmission. Grows when there are not lot nutrients available. - Waterborne viral diseases – can cause gastroenteritis, eye/throat infections, hepatitis or polio. Most are neutralized by chlorination. - Amebiasis: o Entamoeba histolytica – Transmitted primarily by water, cysts grow in mucosal cells. Amebic Dysentery – invasive version of liver, lung or brain – death. o Naegleria fowleri – free-living amoeba, may right through nose, burrow into brain – multiplies, meningoencephalitis (brain damage) Topic 8 – Food and Industrial - Food spoilage – any change in the appearance, smell, or taste of a food product that makes it unacceptable to the consumer. - Spoilage usually occurs because of nonpathogenic microorganisms but it makes it unacceptable to consume. - Foods = organic materials  they are suitable as microbial media - Perishable food – fresh food items; Semiperishable – potatoes and nuts; Nonperishable – flour and sugar - Water activity – a measure of available water for metabolic process. Differ greatly with regards to their moisture content. How much water is available for the microbes to exploit (some will be tied up, complexed with molecules or in a crystalline/gel form). - Nonperishable foods have low water activity, perishable and semi-perishable have higher water activities - Staph can cause foodborne diseases; it is relatively resistant to low water activity. - For a microbe to spoil a food it must have access to the food and the food must be a suitable environment for them to grow. - Bacterial growth in batch culture: o Last few doublings of exponential growth are the ones that cause noticeable changes to the food. o Oxygen, temp, pH, water activity, nutritive value of food, natural antimicrobial substances, makeup of food’s microbial community affect growth of the microorganisms. o Extent of lag phase depends on microbial contaminants, their previous growth history o The aim is to preserve food, and essentially extend the lag phase indefinitely. - Food Preservation: o COLD TEMP – lower storage temp results in less microbial growth a slower spoilage. It slows down growth but does not stop it completely. Psychrotolerant microorganisms can spoil refrigerated food. Freezing as well doesn’t kill microbes, physical damage or slow chemical reactions, very slow microbial growth sometimes. Ideal temps – refrigerator (3-5) and freezer (- 20 normally but -80 in some applications). o HEAT PROCESSING: used to reduce the bacterial load or even sterilize a food product; especially useful for the preservation of liquids and wet foods.  Cooking – kills most of the microbes (sometimes all depending on temp and time), but as soon as you tale it out of the oven inoculation happens. Reduces microbial load; must avoid reinoculation.  Canning - expose cans to a high temperature, kill any microbes present and kill endospores (very high temp), produce an essentially (commercially) sterile food. Produces food that is stable indefinitely even without refrigeration. Temp-time relationships depend on the type of food, it’s pH, size of container, consistency, and density of food. Acidic food can be canned effectively by heating just to boiling; non- acidic foods must be heated to autoclave temps. Retort canning – autoclave under pressure.  Pasteurization – try to prevent wine going sour; use a very low hot temperature for small amt of time. Reduce microbial growth, kill pathogens. LTH – 62ºC for 30 min. HTST - 71ºC for 15 min  Aseptic food processing – uses flash heating – rapid, short heating cycle or sterilization by cooking. Off the shelf products like juice boxes. o PICKLING – Vinegar, a dilute acetic acid fermentation product of the acetic acid bacteria, usually added in the process. Weak acids are used for food preservation. Mix vinegar with large amounts of salt or sugar to decrease water availability and further inhibit microbial growth. o FERMENTATION – natural community or added inoculum.  Lactic acid bacteria (Lactobacillus) – milk  yogurt, cheeses, sour cream; Cabbage  sauerkraut.  Acetic acid bacteria (acetobactor)  Propionic acid bacteria (propionibacteria) – flavor and holes characteristic of swiss cheese o Drying – natural sunlight. Spray drying – spraying, atomizing, liquids such as milk in a heated atmosphere. Reduces droplets, increases surface to volume ratio of the liquid promoting rapid drying without destroying the food.  Lyophilization (freeze-drying) – camping rations or military food. Foods are frozen and water is then removed under vacuum.  Addition of sugar or salt – molecules of water flow freely. Microbial cells must compete with solute for the reduced amount of water. Bacteria are poor competitors. So you do not have to take away water molecules, just add sugar or salt and reduce water activity for bacterial growth. o Irradiation – effective method for reducing contamination by bacteria, fungi and even insects. Treatment for herbs, potatoes, dehydrated seasonings. Works very well, underutilized because worried about food becoming radioactive. o MAP – impermeable films, evacuation, elevation of carbon dioxide content. Vacuum packing anaerobic environment. Shift potential for growth in microbial community towards harmless organisms and away from pathogens. Treats raw meats. Organisms which is readily killed by cooking, but in case people do not cook their food properly. - Use of microorganisms in food production: - Various food fermentations to run a particular reaction in an industrial process. - Beverage alcohol: ethanolic fermentation: glucose  2 ethanol. - Vinegar production. Conversion of sugar to different end products - Biotransformation – Biocatalysis. Cortisone production o Rhizopus nigricans (fungus) will catalyze the biotransformation of progesterone  11 alpha- hydroxyprogesterone. Difficult to do via chemical reaction. o To get cortisone, need O on that specific carbon  takes many chemical steps to get it there. There is a microbe (fungus) that does this as part of its metabolism. - Primary Metabolite – forms during the exponential growth phase of the microorganism. - Secondary metabolite – forms near the end of growth phase. Non-essential for growth and reproduction. Formation is highly dependent on growth conditions. Often produced as a group of closely related compounds. Often overproduced; products of spore-forming microorganisms and production is linked to the sporulation process itself. - In these sorts of reactions, the end products of the microbes metabolism is what the industry is after  where it can be produced in the growth of a microbe is important - Formation of secondary metabolites is late because it is formed using products of primary metabolism  long, complex, highly regulated. Have to be careful that you do not accidently repress the pathway you are trying to encourage. - Often play around with these organisms to get them to produce a lot of secondary metabolite - Making Vinegar – Must have a source of alcohol and certain strains of acetic acid bacteria (Acetobacter, Gluconobacter). Strict aerobes, the strains of use do not oxidize their organic electron donors completely to carbon dioxide and water. Acetic acid bacteria are acid-tolerant and are not killed by the low pH products that they generate. The aerobic process demands oxygen during growth in high amounts. Microbe’s energy metabolism; partial ethanol oxidation. o Open-vat Method – wine placed in shallow vats to facilitate exposure to the air. Surface of the liquid, acetic acid bacteria develops as a slimy layer. o Trickle Method – alcoholic liquid is trickled over loosely packed beechwood twigs or shavings. Bacteria grow on the surface of wood shavings. Vinegar Generator – Wood shavings in a vinegar generator are the support on which the bacteria grow to produce a biofilm. Operates continuously for 5-30 years. Not much else will grow due to low pH (not much contamination). Aeration is important, fluid circulation is important, can keep this going for years (lifetime depends on when wood starts to fall apart). o Bubble Method – industrial incubation techniques to introduce and mix air into a fermenter containing the alcoholic substrate and inoculated with acetic acid bacteria. - Vitamin Synthesis: o Vitamin B12 – synthesized in nature exclusively by microorganisms but is required as a growth factor by all animals. Plays an important role in methyl transfers. It’s deficiency can lead to debilitating condition called pernicious anemia (low production of RBCs). Microbial strains Propionibacterium and Pseudomonas are used. o Riboflavin – parent compound of the flavins FAD and FMN. Play an important role in oxidation-reduction reactions. Synthesized by fungus Ashbya gossypii. - Amino Acid Production: o There are methods to make every amino acid (biologically, harvested from animal tissue, synthetically via chemical synthesis, depends on what is economically efficient) o Glutamate – produced highest world wide (MSG). Overproduction is done through biotin as the cell is starved. o Lysine – food additive. Is controlled at the level of the enzyme aspartokinase; excess lysine feedback inhibits the activity of this enzyme. Sometimes it can be obtained by isolating mutants of the bacteria and then aspartokinase is no longer needed. Have to get rid of regulatory pathway in overproducing strains – do not have that feedback inhibition, so as long as have starting material they will keep making lysine  metabolic cripple - Production of enzymes: o Often exploit extracellular enzymes (exoenzymes). Specificity of enzyme’s catalytic reaction useful in bioconversions o Detergents – amylase, protease, lipases, reductases from Bacillus. Removed by degrading the polymers into water soluble components. o Intervase – sucrose-digesting enzyme o Glucose isomerase – high fructose sweetener from corn, wheat, and potato starch. o Extremophile enzymes (extremozymes) remain functional under harsh conditions (acid, alkaline, high temperature, high salinity) o Taq Polymerase – thermostable DNA polymerase from Thermus used in PCR – can amplify a certain piece of DNA to very high levels by doing continuous cycles of DNA repliation primers, DNA polymerase. Go through cycles where you need to split dsDNA to ssDNA (via heating it up), annealing of primers to strands, synthesis, join to dsDNA, back to start again. o Immobilization – soluble enzymes allows large-scale reactions under continuous conditions. - Scale-Up: o Going from a very small scale reaction that we are used to in the lab (test tubes) up to an industrial sized process (hundreds of thousands of litres) = scale up o Involves step wise process, lots of biomass, low oxygen solubility in large volume of liquid, oxygen transfer has to change as you scale up o Oxygen Transfer – difficult to obtain in large-scale o Mixing and Aeration – easily accomplished in small volumes o Temp control – must change with scale-up o Sterilization protocols – must change with scale-up o Bioengineers – skilled in gas transfer, fluid dynamics, mixing, thermodynamics. o Continuous flow type of reactors  quite often get a lot of foam getting produced, put in antifoam to reduce (tubes going to sides are trying to collect the foam) o Huge bioreactors in b, go up a few stories - Downstream Processing – Recover and process product from bioreactor. Processing yeast biomass: collect cells, wash cells, dewater cells, achieve desired consistency, package, store until shipped - Processing cellular products: removal of biomass from culture, concentration, and purification of product. Harvesting + fractionation of biomass to recover desired product, separation from debris, concentration and purification of product. Topic 9 – Biotech - Biotechnology – use of living organisms to carry out defined chemical processes for industrial or commercial application. - Bioremediation of pollutants, bioleaching of metals, bioconversion, biocatalysis - Current usage – implies organism used to carry out the process has been genetically manipulated in vitro – molecular biotechnology. Provide opportunity to isolate, manipulate, sequence DNA, control expression of DNA: “genetic engineering”  GMOs - Production of human growth hormone, insulin, interferon, vaccines, industrial chemicals, microbial plastics, modified plants, animals. - Taq polymerase – nowadays produced in an e. coli strain engineered to carry and express T. aquaticus gene. - Before Molecular Age: o Introduction of random mutations + screening for mutants with desired traits o Looking for a particular sort of mutant (specific phenotype), but lots of other random mutations may be present that you do not detect/ do not see  bad things can happen o Industrial strain development – many mutations achieved spontaneously, by irradiation, chemical exposure  led to development of an overproducing P. chryogenum strain that produced enough penicillin for efficient commercial production. - The Old way: o AEC = lysine analog, will bind to aspartokinase and shut down the pathway  no feedback inhibition. o Mutants of the bacteria that overproduce lysine obtained by looking for AEC-resistant cells. The mutants possess a modified aspartokinase that no longer recognizes AEC or lysine. These overproduce lysing because the feedback inhibition is abolished. o Started with wildtype cells and grew them in a no lysine medium, but had AEC in it. Just put a ton of cells in, looked for cells that reno wildtype cells will be able to grow under these conditions, only mutants. - Site Direction Mutagenesis  a technique whereby a gene with a specific mutation can be constructed in vitro. Would still need to test the mutant to ensure it functions as planned. - Modify cytokinase gene specifically, need to know the sequence of gene and what part did what, but if you did you could use this method. Aim: eliminate capacity for modification of enzyme by lysine (or AEC); leave catalytic function of aspartokinase unaffected o Clone gene in ssDNA phage/vector o Synthesize a synthetic oligonucleotide that is complementary to the strain but will abolish regulatory site o Add polymerase to get dsDNA o Clone it into a whole cell. If this undergoes replication and the daughter cell inherits it, the daughter cell will have the desired phenotype - Ways to move DNA around: o Cloning vector (pUC19) that replicates E coli o Origin of replication will allow independent replication. It is the polylinker with single cleavage site for various restriction enzymes. Ampicillin resistance gene. Insertion into polylinker inactivates LacZ’ gene  enables easy detection of transformants by “blue-white screening”. Insertion of foreign DNA into polylinker region. LacZ/ gene is thus inactivated. Transformants carrying foreign DNA can be selected on ampicillin plates containing Xgal. o Transformation – transfer of genetic information via free DNA. o Marker gene – allows identification of the gene. - Bacteriophage Lamba – modified as a cloning vector. - Transduction – transfer of host genes from one cell to another by a virus. o Native phage lambda have right and left arms o Taking out replaceable region allows us to insert a segment of foreign DNA in its place. o Phages can carry the foreign segment into e.coli hosts. o Artificial transduction the phage infective materials are manipulated. o Dna can be moved through transduction as well as conjugation. o However, this method is inefficient, also, plant and animal cells do not go through transduction and conjugation. o This method is not popularily used. - Electroporation – the use of electric pulses to enable cells to take up DNA. Millisecond long pulses open small pores in cell membranes. DNA can more into/out of the cells via pores. o Electroporation is a method of electrical transformation. o Single stranded DNA is taken up by cell under certain conditions. o Electroporation uses pulses of electric activity to create transient pores, DNA can move through the pore o This way the host cell doesn’t have to be permissive or competent. o Cell + plasmid in solution + electric current o Donor (with plasmid) + recipient + electric current. - Hosts commonly used in molecular cloning: o Gram-positive get hung up in the e. coli periplasm. o If trying to create genetic engineered microbe, E coli might not be best because it’s gram negative  It has most well developed genetic. BUT Periplasm traps proteins. Potentially pathogenic. o Bacilllus Subtilis  Is naturally competent and will take up naked DNA  Not pathogenic. Make extracellular enzymes (secrete proteins). Spore former.  Genetically unstable. Genetics less developed. o Saccharomyces Cerevisiae  Has features of a microbe, this makes it easy to grow and in large numbers.  Not pathogenic. Prokaryotic organisms cannot process mRNA and proteins like eukaryotes can.  Most plasmids used on prokaryotes will not replicate in eukaryotic cells. - Reverse Transcriptase: copies information form RNA to DNA. The cDNA derived from the processed human mRNA could be used to clone in prokaryotic systems. o Prokaryotes typically do not have split genes. That is no good because we want to correct the protein. o It copies info from RNA into DNA – reverse transcriptase o Found in viruses as a part of its genome because it is a part of its lifestyle. o Production of human proteins. Expression of human genes on e. coli. o However, eukaryotic genes are split genes that has introns within exons. o When transcribed, there will be an unprocessed message. o Later introns are spliced out. o Prokaryotes do not have split genes. o Recover animal RNA from recovered tissue and make completentary dna from the recovered RNA. o This is done through reverse transcriptase o Retroviruses have reverse transcriptase that can convert RNA into DNA. o cDNA is cloned into E.coli  there is no introns. - Expression Vector pSE420 o Polylinker – insert desired DNA. Amp resistance. o Artificial construct. Grow it, inducer added. Want the culture to grow first to a lacl and then get it to turn on. o Inclusion bodies. o Quiet often we want to control expression. Grow it to very high o Trc promoter  strong promoter in E.coli (promotes transcription) o Shine-dalgarno site encourages ribosome binding to translate cloning site. o Protein made by lacI will bind to promoter region and shuts it down. o Addition of inducer promotes expression of cloned gene. - Producing recombinant bovine somatotropin: o Interacts with ST receptor (growth) and prolactin receptor (lactation) o Recombinant protein – not to make cows grow bigger but to get them to produce more milk. o One way is to use e.coli to grow human somatotropin. o We make bovine somatotropin (BST) to make cows produce more milk. o Treatment of cow with BST will stimulate lactation. o mRNA of BST is translated into cDNA by reverse transcriptase o Insert cDNA into expression vector o Transform E.coli with vector. o Harvest from E. coli  inject cow. o In humans, we don’t want to target the prolactin receptor. - Producing recombinant human somatotropin: o For treatment of stunted growth in children o Similar strategy is used with human somatotropin gene  clone and express in bacteria o Use site-directed mutagenesis to modify ST gene  inactivate prolactin activity o Use host strain defective in protease activity  avoids protease digestion of recom
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