Microbiology of Wastewater Treatment Lecture 4 PDF

Microbiology of Wastewater Treatment Lecture 4 Other Sources: http://www.splammo.net/JLbactsite.html Black, Microbiology Principles and Exploration 7th Ed Madigan, Brock Biology of Microorganisms, 11th Ed.  What is wastewater? Industrial sources: –Petrochemical, dairy,...

Microbiology of Wastewater Treatment Lecture 4 Other Sources: http://www.splammo.net/JLbactsite.html Black, Microbiology Principles and Exploration 7th Ed Madigan, Brock Biology of Microorganisms, 11th Ed.  What is wastewater? Industrial sources: –Petrochemical, dairy, food, pharmaceutical, metallurgical, etc. Domestic sources: –Form households and non-industrial businesses Domestic sewage: –Sinks, toilets, and showers –US domestic sewage varies little from community to community across the country –Our waste is not unique. We even flush our toilets at  Lecture Topics Why treat wastewater The wastewater treatment plant Important microbial processes Microbial monitoring  Also called sewage: it looks What’s in domestic like spent dishwater The chemists only care wastewater? about the organic content, specifically carbohydrates, fats, and proteins. http://nsm1.nsm.iup.edu/tsi Sewage is 99.9% water mmons and 0.02-0.04% solids Example: Washington DC, 200 tons of solids per day is produced – 40-50% is proteins, 40-50% carbohydrates, 5-10% fats What would happen if this was released into the surrounding environment?  Environmental consequences of not treating sewage: People can get sick from pathogen contaminated water. Big problem in developing nations. Releasing wastewater directly into a stream leads to oxygen depletion. –This is caused by aerobic respiration linked to high organic carbon loads in the waste. –Low oxygen in water can cause fish kills. http://ian.umces.edu The degree of oxygen consumption in wastewater can be quantified. –This is called the Biological Oxygen Demand (BOD)  How to measure BOD Five day bioassay for oxygen consumption, BOD5 300 ml bioassay in special bottles. Uses an oxygen meter to measure dissolved oxygen (DO) consumption in a five day period The BOD5 is calculated by: BOD5 (mg/L) = D1-D2/P Domestic sewage: 150-200 mg/L – D1= initial DO (mg/L) of the sample Milk processing/cannery waste: – D2= sample DO (mg/L) after 5 days 5000-6000 – P= decimal volumetric fraction of Pulping operations: 10,000-15,000 sample used. mg/L Why the different numbers?  The need for wastewater treatment plants Goal of wastewater treatment: –Protect health –Preserve natural resources –Prevent ecological damage How to accomplishes these goals: –Use wastewater treatment plants (WWTP) –The WWTP removes energy-rich organic matter before discharge into the environment. –And uses technology to prevent/lower the occurrence of water borne diseases. Sewage Treatment Primary treatment: Non-biological treatment Removes solids Waste has high nutrient load (eg C, N, S, and P) Secondary treatment: Decreases dissolved organic carbon (DOC) Uses biological treatment Aerobic and anaerobic secondary treatment Microbiology of anoxic secondary treatment This is used for breaking down solid waste. Done in an anoxic sludge digester. Solids are complex polymers e.g. cellulose and fiber. – Microbes secret lipases, proteases, amylases, etc. Fermentation is the major metabolism in this treatment Lots of methane is produced by methanogenic archaea. The methane is collected and used to generate electricity. Overview of sewage treatment plant Anoxic secondary waste treatment To the landfill Aerobic secondary waste treatment: trickle filter Trickling filter is a bed of crushed rocks—the 1º- treated sewage is trickled over it. Lots of surfaces for microorganisms to attach to. Complete mineralization of waste to CO2, ammonia, nitrate, sulfate, and phosphate Same process occurring in a fish aquarium. www.unclestu.com Much of the organic material binds to the floc and is eventually taken up by the microbial biofilm. 95% of the BOD is reduced during this stage. Aerobic secondary waste treatments: activated sludge Air bubbled through waste water. Bacteria form large flocs. – Zoogloea ramigera is one of the key species that forms a slime and is the base of the floc. After the flocs form they are allowed to settle out Filamentous bacteria can cause sludge bulking problems-- sludge thickens  Important microbes in the sewage treatment plant Nitrifying bacteria – Aerobes – Convert nitrogenous waste into nitrate Denitrifying bacteria – Anaerobes – Convert nitrate to N2 Methanogens – Generate methane from acetate – Or use H2 and CO2 to make methane – Mostly archaea  Nitrifying bacteria Ammonia is converted into nitrate Ammonia has a high BOD because NH3 oxidation requires oxygen. Two groups of microbes are involved: –Ammonia oxidizing bacteria (AOB) –Nitrite oxidizing bacteria (NOB) AOB oxidize NH3 to NO2- in two steps: –Ammonia monooxygenase (AMO) –Hydroxylamine oxidoreductase (HAO) NOB oxidize NO2- to NO3- –Uses the Nor enzyme complex Both AOB and NOB respire oxygen Ammonia Oxidizing Bacteria (AOB): (A,B) Nitrsomonas; (C,D) Nitrosolobus Nitrification Enzymes Nitrite Oxidizing Bacteria (NOB): (E, F) Nitrospira; (G, H) Ntirococcus  Sewage treatment and environmental monitoring Monitoring effluents and the surrounding environment is important (Why?): –Asses the efficacy of the treatment process –C, N, P, metals, microbes, effluent toxicity It is often too difficult to directly monitor a specific pathogen or virus/phage (Why?). Instead, monitoring is usually done for indicator organisms.  Common indicator bacteria Coliforms: –Facultatively aerobic, gram-negative, nonspore- forming, rod-shaped bacteria; ferment lactose with gas formation at 35˚C within 48 hrs. –Usually enteric bacterial group (E. coli, Klebsiella, Citrobacter, Enterobacter, Serratia, Yersinia) –Poor indicator: often found outside of the intestinal tract Fecal coliforms –Thermotolerant coliforms (44.5˚C), 20% of total coliforms. What is an indicator organism? An organism that can be readily cultured that indicates the presence of a pathogenic microorganism or correlates to a health problem. Five criteria for an indicator organism: –Consistently present in feces and at higher concentrations than pathogens. –Should not multiply outside the human intestinal tract. –Should be as resistant or more resistant than the pathogen to environmental conditions and to disinfection. –Easy to assay (culture and quantify) and differentiate from other organisms. –Environmental concentrations should correlate with pathogens or measurable health hazards. Monitoring of indicators is done using culturing techniques and selective media. Gram positives Fermentation: Gas Yes Yes No Growth: Yes No Most probable number (MPN) analysis for quantifying coliforms Quantifies total coliform bacteria in water samples by three sub-tests – Presumptive, Confirmed, and Completed The MPN is used to monitor waste effluents, drinking water systems, and recreational waters. High MPN results can lead to beach closures. In a drinking water system, a positive for coliform is a HUGE deal. This would set off a series of events to find the source of coliform contamination. – Would you want to be drinking water with coliforms in it?  MPN: Presumptive Test Determines presence of coliforms (gas producers). Serial dilution of replicate lactose broth tubes MPN index reports data as per 100 ml  MPN: Confirmed Test Part two of the MPN test Positive tubes in the presumptive test are inoculated into brilliant green lactose bile broth (BGLB) –These tubes will confirm acid and gas production (fermentation end products). –Bile will inhibit gram positive organisms  Example MPN problem: Glucose fermenters in lake water: Choose numbers with three consecutive sets of “dilution to extinction” for sugar fermenting organisms (tubes: C, D, E) The MPN table for 3-1-0 is 0.43 organisms (next slide). 0.43 organisms were inoculated into the middle tube (D). Use the dilution to calculate the MPN per ml Tube D had 0.1 ml of a 10-3 dilution of lake water. Now calculate the MPN in the original sample: 0.43 organisms X 103 ÷ 0.1 ml = 4.3 x 103 glucose fermenters per ml The MPN index would be 4.3 x 103 per ml ÷ 100 = 43 organisms per 100 ml  MPN: Completed Low pH: dye turns Test metallic green Positive tubes from the confirmed test are analyzed by streak plating on eosin methylene blue (EMB) plates. –Incubated at 35˚C for 24- 48 hr –Only coliforms will turn dark with a metallic green sheen –Gram + inhibited by eosin/MB Coliform colonies are gram stained and to verify that they are gram negative and http://www.microbelibrary.org/ non-spore forming (see definition of coliforms). https://microbeonline.com/probable-number-mpn-test-principle-procedure-results/ Molecular techniques for monitoring indicators Molecular methods can be very sensitive. – In theory, one gene copy can be detected via PCR. Molecular methods can be rapid: