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Food 3260 Industrial Micro Exam Material

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University of Guelph
Food Science
FOOD 3260
Keith Warriner

Micro Notes Brewing History • Reinheitsgebot – Bavarian purity law o Hops o Water o Malted barley o Malted wheat o Yeast not yet discovered • 1762 - Thermometer introduced in brewing o M Cumbrune • 1769 – Baverstock introduced hydrometer • 1843 – Balling introduced method for measuring and controlling conversion of wort sugar to ethanol in brewery • 1837 – yeast are living organisms • 1855-1875 - Pasteur laid foundation for brewing by establishing role of yeast in alcoholic fermentation – aerobic and anaerobic metabolism • 1883 – Hansen isolated 3 yeast at Carlsberg o Original o Wild yeasts o Developed pure yeast culture brewing Raw Materials Malt • Dried cereal grain (barley) after germination in water • Provides o Complex carbs and sugars o Gives colour, flavour, aroma, yeast nutrients, body, head, and general beer characteristics • Malted barley because o Malting converts large, insoluble starch chains of endosperm to water soluble starches o Activates proteolytic and diastatic enzymes for the mash • Barley requirements include o Resistance towards disease, stability, efficient nutrient use, high yield, homogenous grain shape, good water soaking, low protein, fast germination, good enzyme formation potential and high extract yield o Starch endosperm = food reserve for plant embryo and source of ferm sugars o Aleurone layer = degenerates enzymes that degrade starch endosperm o 6 row vs 2 row Barley  2 row has lower yield, 6 row higher yield  6 row has lower endosperm to husk ratio  2 row has higher extract  2 row has lower protein and enzymatic power • FHB infected / DON + o Fusarium head blight and presence of deoxynivalenol impacts quality of malt and therefore the quality of the beer • Dimethyl Sulphide (DMS) o Adds cooked corn flavour in beer o Produced during grain germination o Reduced in kilning of malt so high in pale malt o Boiled off in wort kettle (volatile)  Ales 10-20ppb  Lager 40-175 ppb o Control  2 row for pale malt (low SMM, DMS precursor)  Avoid corn adjunct  Boil wort 90 min + in open vessel  Rapidly cool wort  Yeast selection and ferm. temp  Ensure beer is aged sufficiently • Various profiles of different malts o Mod protein (11-12.5%)/ mod enzymes = high demand o Mod protein / high enzymes = high demand o Mod protein / low enzymes = mod demand o Low protein (>11%)/ low enzymes = limited demand Malting • Yeast cant degrade starch in barley • Malting induces enzymes to degrade starch to fermentable sugars • Seed is heated, kilned to point where seed is killed so it won’t germinate but not denature to starch degrading enzymes • Kilning also gives toasty flavours • Diastatic Power (DP) o Ability of malt to hydrolyze starch to ferm sugars o Measured in degrees Lintner o Result of the alpha and beta amylase activity o 6 row = DP or 140-160L and 2 row = DP of 125-135L • Process o Steeping – soaking in water for 48-60hrs  Increase gibberellins (hormone that stimulates hydrolytic enzyme production) o Germination – 3-8 days  Amylotic enzymes break down starch  Proteolytic enzymes break down proteins o Screening and blending o Drying/kilning  Germ halted by drying  Provides protection for enzymes and prevents malt spoilage  Drying = flavour and colour Hops • Humulus lupulus – flower contains lupulin with bitter substances and aromatic oils • Components o Alpha acids (90% of bitterness)  Humulones – bitter, antiseptic  Cohumulone – harsh  Adumulone – complements humulones o Beta Acids  Lupulone, colupulone, adlupulone  Adjunct to yeast washing o Oils  Responsible for odour  Volatile, care when boiling  Humulene component • Production o Use of hop cones is rare o Pellets – hammer milled and compressed = more stable o Extract  Extracted with liquid or super critical CO2  Can be separated into hop resin and oil  Pre isomerized hop can be used to add hop character to finished beers o Amount determines style of beer Adjuncts • Provide extract at lower cost than malt o Corn, rice, barley • Enhance physical stability, superior chill proof properties, and brilliancy • Adjust fermentability of wort o Sugar or syrups • Corn o Corn grits extensively used • Rice o Rice grits give better yield than corn o Requires additional cooking process • Syrup o Extend brew house capacity o High gravity brewing o Improve beer stability o Easy to handle o Invert sugar  Sucrose split into gluc and fruc  Can be added to the kettle  HFCS Water • 90% composition of most beers • Salts in water impact enzyme and chemical processes • ~3-20 hL / hL beer (3.4-3.8 opt) • Biggest consumption from mashing, blending, filling (pastuer), CIP • Must de-chlorinate o Carbon filtration • Resterilizing of water after dechlorination • Deionization, reverse osmosis Minerals • Calcium o Hardness o Increases mash acidity and inverts malt phosphate o Stimulates enz activity and improves protein digestion and stabilizes a-amylase o Essential in yeast-cell composition – small amounts neutralize substances toxic to yeast • Magnesium o Second in hardness o Essential cofactor for some enzymes and as yeast nutrient o Excess can interfere with rxns of Ca b/c phosphates are more soluble • Sodium o Adds fullness and sweetness o High conc cause sour or salty taste … eww • Sulphur o Weakly basic, but overcome by acids o Fairly soluble o Dry, fuller flavour • Chloride o Basic, readily neutralized o Accentuates bitterness, increases stability, improved clarity • Carbonates o Hop bitterness becomes more harsh o Reddens colour o Hinders protein coagulation o Prevents over acidification of mash o Reacts with Ca to form insoluble precipitates so they can strip out the Ca needed to set the pH level • Potassium o Yeast cofactor and required at traces levels o Works better than sodium for salty without sour taste Yeast • Select strains that are already in use • Some breweries isolate, select and maintain yeast, but others have labs do it • Make backups and store at multiple locations • Qualities o Rapid initiation of fermentation o High ferm efficiency o High ethanol tolerance o Desired flavour characteristics o High genetic stability o Range of alcohol production • Ale yeast o S. cerevisiae o Top fermenter  Forms thick layer on surface  Higher temp (10-25oC)  More ferm by products: higher alcohols, esters  Differences may disappear with recycled yeast o Unable to ferment melibiose • Lager yeast o S. pastorianus o Bottom fermenter  Lower temp (5-15oC)  Slower growing, less head formation  Aggregate  Less by products  90% of worlds beer production o Can ferment melibiose (MEL gene) • Yeast condition o Viability  Measure of alive yeasts • Methylene blue • Eosin Y  Microscopic techniques o Vitality  How healthy  Sterol measurement  Acidification power  Glycogen  O2 consumption o Yeast storage  Sterile tanks  Cold 0-4oC  Periodic/gentle agitation to keep uniform temp  Can press yeast into cakes The Brewing Process Milling  • Grain processed through roller mill • Leaves husk intact • Brewery specific and measured on standard sieve (ASBC) Mashing • Crushed grain mixed with water in mash tun o Protein degradation  Reduction in of high MW proteins to AAs • Proteinase – reduce large protein to medium protein • Peptidase – degrade med size to AAs for yeast growth o Starch degradation  Conversion of starch to ferm sugar • A-amylase = rapidly reduces starch to shorter chains • B-amylase = reduces starch and short chains to maltose  Gelatinization – formation of long chain aq starch  Liquefication – reduction of long chains to shorter – reduce viscosity  Saccharification – reduction to ferm sugar • Factors affecting mashing o Temp – rise in temp controls amount of ferm extract o Time – influence yield and fermentability o Mash pH – 5.1-5.6 opt o Mash water – Ca req o Mash viscosity – thin mash favours conversion to sugars Lautering  • Separation of wort from converted mash • Latuer tun is primary separation device o Produces clear wort o Obtain good extract recovery • Mash pumped on false bottom of slits to filter • Rakes assist in leveling grain bed • Sparging with water removes all extract • Spent grains  animal feed Wort Boiling • Inactivation of enzymes • Sterilization of wort o Destruction of residual microbes • Isomerization of hop components o Adding hops too early = very bitter and loss of wanted volatiles, unwanted cause haze o Production of iso-a-acid – hop flavour • Removal of unwanted volatiles o DMS • Precipitation of proteins (trub) o Removal of HMW protein • Some concentration of the wort by evaporation • Colour formation by maillard rxn • Additions o Hops  Bitter and aroma based on time of addition  Multiple additions  Can be added as liquid o Adjuncts (syrup)  Added to increase extract o Salts  Ca to reduce oxalate haze  Copper – remove S compounds Wort Cooling  • Hot wort tank o Whirlpool to clear protein o Tangential entry o Trub spun into center of tank o Clear wort collected and sent to cool • Wort cooler o Paraflow, plate heat exchanger o Reduces to initial ferm temp o Additional sterile oxygen added for yeast – ceramic or sintered metal diffusers Fermentation • Dependent on o Wort composition – nutrients, FAN, oxygenation, oP o Yeast – proper pitch rate, viability o Process conditions – temp • Yeast pitching o Large inoculum of cells used (7-18mil/mL wort) o Cell density increases 3-4 fold per ferm o 1/3 – ¼ of yeast crop is used for inoc in next batch o Excess dried and used in animal feed o Pitching is economic o Disadvantages  Mutants  Contaminants  Off flavours or retarded ferm  Wash in 7.5% P acid • Yeast washing o Improves yeast quality by red contam bacteria o Methods  Sterile water wash  Acid wash  Acid wash with 0.75% ammonium phosphate  Chlorine dioxide st • 1 day = aerobic cycle o Conversion of sugar to CO2 and water (no alcohol) o Yeast derive energy for reproductive and ferm cycle • Day 1-4 = anaerobic cycle o Ferm and reproduction o Yeast suspended and optimally dispersed o Sugar to alcohol and CO2 • Day 4-5.5 = sedimentation o Growth ceases, flocculation begins o Near end, beer appears clear • Crabtree effect- at >1% glucose, ethanol ferm even if O2 present because it metabolizes glucose quickly not efficiently. But only glucose, so not in beer • Diacetyl formation o o End of ferm = diacetyl rest  Fermenter held at end temp to allow yeast to reduce the diacetyl  11oC for 2-10 days towards end of ferm o High ferm temp = diacetyl formation Storage • Temp lowered to 0oC • Residual yeast/solids removed by centrifuge o Collects and transfers beer in one step • Filtration o Temp still around 0 o Chillproofing agents added prior  Silica gels, PVPP  Stabilizes, removes haze forming elements like protein o Depth filtration  Powder • Diatomaceous earth • Various grades  Filters • Plate and frame – cellulose pad holds DE • Leaf - stainless steel screen holds DE • Candle – perforated stainless steel tubes • Filtered beer stored in bright beer vessels Packaging • Bottled, canned or kegged • Pasteurized, sterile filtration or bottle fermentation • Warehouse then customer Quality • Based on brewery and legislation o Alcohol content (excise) o Package fills (consumer affairs) o Food safety (CFIA) • Sampling and specs based on o Microbiology o Sensory o Analytical o Packaging Microbiology • Metabolic pathways o Fermentation – sugar to CO2 and ethanol • Diacetyl o By product of yeast metabolism o Excess taken up by yeast at end of ferm o Can be prod by bacterial contamination  Beyond yeast absorption capability  P. damnous, other lactics  Produce high levels and outcompete yeast • Remedies include o Ensure purity of pitching yeast o Sanitation o Adequate preventative maintenance o Adequate testing o Add acetolotate decarboxylase to reduce formation o Buttery flavour o Control parameters  Increase primary fermentation (PF) temp = increase diacetyls  Control of temp = decrease diacetyls  Insufficient O2 Primary Ferm= increase diacetyls  Increase temp at end of ferm to reabsorb = decrease diacetyls  Insufficient FAN = increase diacetyls • Esters, aldehydes and higher alcohol production o Fusel oils = higher alcohols  1-propanol, 2-propanol, butanol, amyl alcohol, furfural  Derived from AA, FA and carb fermentation  Prevent by • Avoiding high wort gravity • High ferm temp • Avoid N limitation • Pitch selected yeast strain at sufficient lvl o Esters  Produced from equiv alcohol through catalysis by the enzyme alcohol acetyl transferase and acetyl coenzyme A  Acyl CoA esters with alcohols  Acetyl coA + ethanol = ethyl acetate (nail polish remover)  Controls • Control temp • Yeast strain purity, avoid wild yeasts • Basic testing o Pour plate o Spread plate o Membrane filtration o Medias  Universal beer agar  Cycloheximide to inhibit yeast  Raka ray, MRS, LMDA • Acetic Acid production o Oxidation of ethanol o Intermediate in acetate formation or incomplete ethanol ferm o Causes  Introduction of oxygen  Low Zn, ADH cofactor  Contam by acetic acid bacteria  Contam by wild yeast or mutated yeast o Solution  Yeast selection, purity  Avoid oxygenation during bottling • Real time PCR o Detection of spoilage bacteria Sanitation • Hotspots include o Heat exchangers o Sample valves, viewing windows, faucets o Gaskets o Pipe and hose lines o Gas lines o Kegs and filling heads • Drains o Sufficient number and construction o Floors slope to them • Walls o Hard, smooth, can be cleaned • Food contact surfaces o Non absorbent o Free of pitting, crevices and loose scale o Can withstand repeated cleaning • Sanitation Std Operating Procedure (SSOP) Plans o Sanitation schedule o Routine monitoring o Id trends to prevent recurring problems • Cleaning methods o Manual  Dismantle  Pads and brushes  Physical force  Cheaper  Variable performance  Scratches and cross contamination? o CIP  In situ with controlled cycles  Consistent and fast  Reduced water and chemical use  Expensive  Minimal physical action  Hidden spots? • 5 steps o Dry clean o Pre rinse o Apply detergent o Post rinse o Sanitize • Detergents o Brewing soils include beer, carbs, lipids, protein o Alkali – caustic soda for protein and organic removal o Acid wash – Phosphoric acid for scale removal • Sanitizers o Chlorine  Cheap, easy to apply, bleach, electrolyzed water, foams  pH dependent, sequestered in presence of organics, disinfection byproducts, corrosive  chlorine dioxide • stable in presence of organics, minimal byproducts • expensive, poor water solubility, degraded at high temp o Quaternary ammonium salts  Compat with alkali, stable, residual activity/no rinse, potent against gram + and yeast, non-corrosive, non-oxidizing  Inactivated by surfactants, gram – resistance, resistant mutants, foaming, residual can impact fermentation, $$$$ o Hydrogen peroxide o Peroxyacetic acid  Stable with organics, no disinfection by prod, non-volatile, no foam in CIP  $$$, hazardous to handle, reactive with metals, corrosive to Cu, brass and mild steel o Anionic acids/surfactants  Mix of acids and surfactants, dislodge biofilms, enhance antimicrobial action, no disinf byprod, stable at high temp  $$$, pH sensitive, high conc in hard water, foaming in CIP, corrosive to non st steel o Iodophores  Insensitive to pH, broad activity, effective at low conc  $$$, flavour taints, heat sensitive, staining, excessive foam in CIP o Ozone  Powerful AOX, generated on site, no residual byprod  $$$, poor water solubility, off gassing, rapidly seq by organics, limited against virus and spores o Need to consider  Concentration  Contact time  Temperature Types of Testing • Validation o Quantitive evidence sanitations procedures are adequate to reduce microbe pop’n by a designated lvl without equipment corrosion o Challenge tests o Microbial analysis prior to sanitation plan • Verification o Observations and test to ensure plant sanitation plan is implemented as written and procedures are effective at ensuring sanitary conditions o Observations o Rapid testing o Microbiological and routine testing • Sanitation testing o Rapid  Indirect measure of organic material and microbes • Visual inspection o Sensory evaluation of environment o Easy, low cost o Unreliable because can’t see microbes • Black light o More sensitive than visual but non-specific • ATP luminometer o Increase in organism = increase in ATP = increase in RLU o As little as 30 seconds • Protein residue o Detects protein left behind from poor sanitation • Rapid but poor sensitivity and selectivity o Culture based  Direct microbial counts  Swab/sponge • Sponge for wet environment and large SA • Swab for bacteria and hard to reach sites  Contact RODAC plates • Pre-prepared plates, press onto surface • Incubate and count colonies • Plates can be expensive and limited shelf life • Hard with irregular surface  Agar overlay  Sticky tape  Rinse • For pipes following CIP • Pass solution of known vol through pipes and collect samples • Concentrate by filtration or plate directly  Sensitive and selective but long analysis time Analytical • Lab testing o Alcohol content  All stages of production  Specific gravity and refractive index or GC or alcohol sensors or distillation o Colour o Bitterness o Diacetyl  Colorimetric testing • Napthol- KOH- creatine method (in lab)  GC method • Measures 2,3-butanedione (BD) and 2,3-pentanedione (PD) • BD/PD>5 = possible bacteria contamination o Yeast produce both, LAB produce only BD o pH o packaging  haze  dissolved oxygen • oxidation • DO meter  Fill • 341mL bottle, 355mL can govt requirement Sensory • Can’t get full characterization from instrumental and chemical testing • Get objective measures of beer attributes (trained panel) • Subjective assessments (liking) determined by consumer evaluation • Can have panel measure attributes such as o Bitterness o Sweetness o Maltiness o Hoppiness o Astringency o Defects  Skunky, Chlorine, musty/moldy, diacetyl, oxidation, Sulfur, or other • Appearance, drinkability and flavour important to consumers o Colour- as it gets darker, presumed bitterness increases o Head  High = dirty glass perception  Moderate = good  Low = associated with low alcohol o Thirst quenchability  Thirst potential index: bitter>strong lager>mild>weak lager  Quenching characteristics • High carbonation and high bubble density • Low foam, flavour, malty, hoppy, burnt, bitter, acidic, metallic, astringent and aftertaste o Throat sensations Industrial Enzymes • History o 1883 – Payen - diastase o 1874 – Hansen – rennet and first high purity enzyme prep for industrial use (cheese) o 1876 – Kuhne – “enzyme” to describe ferments from viable organisms o 1880-1895 – Pasteur – live cells necessary for ferm, Liebig disagrees o 1894 – Takamine – digestive aid – diastase o 1897 – Buchner – proves Liebig right – cell free extract to ferment sugar to ethanol o 1913 – pancreatic enzymes patent o 1950 – commercialized alpha-amylase for desizing and brewing o 1967-1971 – DNA restriction, ligase and reverse transcriptase, DNA recombination o 1974 – immobilised glucose isomerase • GMOs o 1980- Human insulin o 1983 -Tomatoes first GM plant approved o 1983 - Tobacco glyphosphate resistance GMO o 1988 - First GMO lipolase in Japanese detergent o 1990 - First GMO food enzyme approved (chymosin) • Golden age of enzymes o Post 90s – enzymes in personal care, detergents, CIP, textiles, feed, biopolymers, fuel, food o 1995 – GMO corn, cotton, potatoes, soy, rice, squash approved Arguments Against GMOs • Big corporations • Farmers cant grown own GMO seed • Forcing small farmers out of existence • GMOs producing new allergens • Resistance genes in weeds • Dependence on GMO varieties < genetic diversity Arguments For GMOs • Resistance to insects, fungi, bacterial pests • Spray requirements lowered • Increased shelf life • Drought and freeze tolerance • Increased yield and waste reduction • Reducing processing costs • Lower reliance on food preservatives • There’s been an increase in life expectancy since we’ve introduced them GMO considerations • Religion • Environmentalists • Food intolerances/allergies • Vegans/vegetarians Why are most enzymes fungal? • Can grow just about anywhere o Temp extremes o Water extremes o Salt ex o pH ex o highly complex nutrient sources o pathogen assaults Exophelia dermatidis • breakout of meningitis from steroid shots contam with black mold • fungal contam increasing public concern • high temp, moist, alkaline environment ideal for opportunistic fungal species harmful to us How can molds survive hostile environments? • Evolved an impressive array of rugged enzymes that degrade, detoxify and absorb nutrients or modify the growth environment o Fungi excrete enzymes which break down polymers into monomers and are then transferred to the hyphae o Standalone enzymes to create a better environment Why use enzymes? • Most rxns require o High heat, pressure, acid or alkali treatments • Can be expensive and create bad by-products otherwise Enzyme Rxns • Mild conditions • Highly specific • High rxn rates • Small amount of enzymes used • Reduce manufacturing impact on environment o Less chemicals, water and energy Enzyme producing fungi • Most are ascomycetes • Zygomycetes • Basidiomycetes Enzyme Classification • EC 1 – Oxidoreductase o Dehydrogenase, oxidases, laccases  Laccase bleach – need phenol or parahydroxybenzoate mediator for redox • EC 2 – Transferase • EC 3 – Hydrolases o Splits molecules by adding water to covalent bonds o Most commonly used in industrial enzymes o Amylase, cellulase, lipase, mannanase, pectinase, phytase, protease, pullulanase, xylanase o Depilling and biopolishers = cellulase, hemicellulose (xylanase), pectinase (endopectin lyase) • EC 4 – Lyases o Addition of groups to double or removal to create double bonds o Pyruvate acetaldehyde o Lager rest – yeast produce byproducts in fermentation  Phenols, esters, diacetyls, DMS • Fungal A-acetolactate decarboxylase reduces conditioning of diacetyl to acetoin • EC 5 – Isomerases o Rearrange atoms within same molecule  Change structure by one molecule so same chemical formula  Nothing added/removed or redoxed o Isomerase, epimerase, racemase o Glucose isomerase glucose  fructose • EC 6 – Ligases o Construction of GMO strains Exoenzymes – produce monomers, dimers Endoenzymes – produce oligomers, and LMW polymers Proteases • Attack both internally and externally • Most don’t need cofactors • Rugged – don’t denature readily under adverse pH, salt, lipid or temperature conditions • Expression can be modified genetically • Cheese making and chymosin • Solubilize soils o Soaps and detergents solubilize soils by interactions with hydrophobic regions o Some may be part protein, carb or lipid, so normal soaps cant solubilize o Enzymes digest soils to soluble fragments Keritinase • Many fungal trychophytons can degrade keratin • Athletes foot, jock itch, ringworm, nail rot • P. griseofulvum to produce anti-fungal agent Pectic Methyl Esterase • Undesirable component of pectinase preparations • Cleaves C6 methyl to produce methanol • Methyl problematic to distilled wines and most volatile fraction is discarded Water and Water Treatment • Ground water (GW) preferred over lake/river water  More nutrients, less microbes • Use surface water (SW) if GW is insufficient • SW Supply o Rivers and lakes  Capacity and temp fluctuations, colour problems, microbes  Lake>river b/c of natural settling  Storage in reservoirs before purification  Cdn SW quality is poor o Rain water  Areas with limited fresh water  Low cost of collection  Good quality  Storage in reservoir  Dependent on weather o Spring water  Mainly for bottled water and industrial fermentations • Steam whistle uses spring water from Caledon  Composition and supply variable (summer droughts)  Susceptible to contamination o Salt/Sea water  Areas of limited fresh water  Desalination • High pressure membrane filtration (reverse osmosis) • Pros: o Renewable, predictable o Diversification of supply o Water supply independence for community o No drawdown in upland streams or headwaters • Cons: o Construction/siting of plant impacts o Entrainment and impingement o Brine discharge toxicity o Growth inducement  Susceptible to contamination Water Treatment and Purification  Physical o Undissolved material rendering water turbid (high NTU)  Biological o Viruses, bacteria, algae, other small organisms  Chemical o Dissolved substances from natural and man-made processes  Sedimentations and Floc Settling o Settling basin for sedimentation o Aerations – precipitation of ions, entrainment of odors and tastes o Flocculation and sedimentation aids (coagulants) may be added  Alum or aluminum sulfate  Lime  Polymeric ferric sulfate (PFS)  Coagulants include many Al and Fe based compounds  Coagulation Process: • dispersion(-)adsorption(-/+)compressed(+/-)collisionflocculation • possible entrapment of metals and other organics o solids can easily sediment  LMW are more difficult o How to know what coagulant?  Jar test • Add coagulant to sample, maybe at diff concentrations • Adjust pH • Rapid stir then settling phase • Observe results o Visible clearing, BOD, DOC, TSS, turbidity  Variables: coagulant type and conc, aeration, pH, temp, mixing rate, post filtration method, supplements  Filtration o Passive sand filtration (biofilm, sand, gravel)  Slow, expensive, needs a lot of maintenance o Rapid sand filtration (by pumping, no biofilm)  Higher bacterial counts o Anthracite/sand filters o Pumping through pad filters - $$$ o Reverse osmosis, ultrafiltration, dialysis, tangential flow treatment  Chlorination o Adverse taste from chlorophenols o Cl reacts with amines (protein) to form chloroamine Hazards Associated with Water  Physical o Sticks and stones  Chemical o pollutants  Biological o Bacteria: campylobacter, clostridia, E coli, Listeria, Salmonella, Shigella, Vibrio, Yersinia o Viruses: NLV, Hep A, Rotavirus o Protozoans: cryptosporidium, giardia Water Analysis  Solids content  Colloids  Hardness  Microbiological quality o Screening directly for pathogens  $$$  Sporadic and low levels  Protozoan and viruses hard to cultivate in lab o More common to screen for fecal contamination  Fecal indicators • Coliforms • Fecal coliforms • E coli • Fecal streptococci • Clostridium perfringens • F+ coliphage Analysis of Water Quality  Coliforms including generic E. coli o Total coliforms – gram -, facultatively anaerobic, non sporing, rod shaped bacteria that ferment lactose with gas formation at 35oC o Fecal coliforms – coliforms that can grow at 44.5oC  E coli, enterobacter, klebsiella, citrobacter o Analysis performed on 100mL samples  Methods for enumerating coliforms o Most probable number (MPN) test  Multiple dilutions of lactose or lauryl tryptose broth inoculated with 10, 1, and 0.1 mL water sample and incubated at 35oC for 24hr  Tubes + for gas used to inoc brilliant green lactose bile tubes incubated for 48h • Further confirmed by streaking onto EMB or endo agar  Est value of MPN determined from MPN tables o Membrane filtration technique  Samples filtered on .45um filters and placed on the surface of selective media  Total coliforms = endo medium, 35oC, 24h  Fecal coliforms= MFC med, 44.5oC, 24h  Fecal streptococci = KFS med, 35oC, 48h  Methods for detecting coliforms o Presence/absence (PA) test  100ml sample cultured in single bottle of lactose broth, lauryl tryptose broth and bromocresol purple indicator  Yellow = + presumptive test and requires further testing  Methods for detecting generic E coli o Colilert MUG test  100mL sample to MUG med with ONPG and MUG  Incubated 24h at 35oC  Yellow colour = coliforms  Examined under long wave UV lamp for fluorescence • Indicates presence of e coli Parameters of Importance  Carbon content o Indicative of pollution or inadequate treatment o TCC = total carbon content o COD = chemical oxygen demand  Instead of using O2, the org C in the sample is oxidized with strong chemical oxidizer (potassium dichromate) under acidic conditions  After ox complete, amount of Cr3+ determined as an indirect measure of C o BOD = biological oxygen demand  Estimates org C by measuring O2 req by microorganisms in the sample to degrade the organic matter under a set of stdized conditions  Sample seeded with small amount of microbe  Sample diluted with water saturated with O2, initial O2 conc determined with electrode and bottle is sealed and placed in the dark  After 5 days at 20oC, final O2 conc determined… final value subtracted from the initial value and divided by the dilution factor to give BOD o TOC = total organic carbon  Org C in sample is oxidized at high temperature with an oxygen stream  Resulting CO2 quantified by infrared or poentiometric methods Chemical Analysis  Colour  Turbidity – nephelometric turbidity units (NTU)  Chemicals of concern o VOCs o Halogenated disinfectant by products o Nitroso disinf byprod o Semivolatile org compounds o Alkyl phenol ethoxylates o Phenols o Hormones o Metals o Pharmaceuticals o Chelating agents o Pesticides  Hardness o Caused by Ca/Mg in water o Measure in degrees of hardness o Total hardness (TH) – carbonate/temp hardness = noncarbonated/residual hardness  Carbonate hardness- carbonate and bicarbonate  Non carb hardness – Cl and Sulfate ions o Reducing hardness – softening  Ion exchange • H exchange – decarbonisation • Cation/Na exchange • Complete desalination with H exchangers (deionisaztion/demineralization) • Softening by precipitation • Softening by desalination by distillation • Reverse osmosis • Addition of threshold effect substances – polyphosphates Types of Wastewater • Gray water • Domestic – relatively easily treated • Storm – low in pollutants, overload treatment plants • Industry – depends, low pollutants  high toxic constituents Waste Treatment • Waste water o Chemical pollutants o Microbial contamination • Fermentations generate high volumes of waste water requiring treatment • Problems caused by effluents o Pollution of land, water o Toxic effects on living things • Water pollutants o Microbial loading, C, N, P, heavy metals, halogenated organics • Dead zones o Excess nutrients in water o Stimulate algae growth o Oxygen depletion • General effluent characterization o Volume o BOD  Oxygen needed to degrade org material at set conditions o COD  Oxygen needed for chemical oxidation of organic compounds o TSS  Total suspended solids • Treatment Classes o Physical – screens, sediment o Chemical – coagulation, flocculation, precipitation o Biological – aerobic and anaerobic processes • Wastewater – Sewage – Treatment  Primary sedimentation o Solids settle to form sludge and primary effluent o Removal of debris and suspended material  Screens, solids incinerated or sent to landfill  Grit chamber, grit used in building or put in landfill o Fragmentation of residual solids in comminutors (blender) – sep or fats/oil o Reduced TSS and BOD  Secondary treatment (biological) o Primary effluent pumped to secondary system, microbes digest bulk of the organic matter  Aeration tanks / activated sludge systems • Flocculated suspension of mixed microbes • Floc formation through exo polysaccharide formation • Continuous aeration/agitation • Partial recovery of activated sludge from secondary settlement/sediment o Act sludge parameters  Sludge loading rate = BOD/(biomass x time)  Hydraulic retention time (HRT) = mean residence time  Trickling filter systems • Microbial biofilm formation in reactor filled with inert support • Spraying of waste water leads to percolation through filter bed • Biofilm growth leads to sloughing b/c of nutrient and O2 limitation • Bacteria, fungi, protozoa, algae, C oxidizers, nitrifiers • Factors: inner SA (large), void space (big for aeration), porosity and density • Impossible to est biomass • Characterization by organic loading rate OLR • Low rate filters o Higher quality effluent – filter blockage possible o Mineral support material (biofilm form 4-24weeks) o HRT ~20-60min, 90-95% BOD removal • High rate filters o Plastic support- high surface, reduced blockage risk o Pretreatment of concentrated industrial o Sludge processed in anaerobic or aerobic sludge digester where microbes reduce organics  Aerobic – 30-70% BOD converted to biomass  Anaerobic biological treatment • Used for sludge, highly concentrated industrial waste • Facultative and obligate anaerobes • Semi continuous – daily removal and replacement • Can be used for energy/heat production • Microbes o Hydrolytic/fermentative bacteria (group 1)  Produce acetic, butyric, propanoic acids, +H2, CO2, methanol  Various genera producing proteases, cellulases, lipases • Eg Clostridium o Acedogenic bacteria  Fermentative  Acid forming, converts sugars, AAs, FAs to organic acids • Eg Clostridium o Acetogenic bacteria (group 2)  Use end products from group 1 and produce acetic acid, CO2, H2  Convert FAs and alcohols to acetate, CO2, H2 • Eg Syntrophomonas o Methanogenic bacteria (group 3)  Acetotrophs – acetic acid  CH4, CO2  Hydrogenotrophs – reduce CO2 (+H2) to form CH4 o Secondary sludge can be used as fertilizer or put in landfills o Use of mixed microbial cultures to degrade pollutants  Carbon oxidizers, nitrifiers, denitrifiers, protozoan, algae, nematoads  Tertiary treatment o Secondary eff clarified by removing finely divided, suspended solids in a clarification tank  Flocculation agents like alum may be used o Clarified water disinfected by chloronation, ozonation, and or UV treatment o Treated water released to the environment/lake/river Anaerobic Vs Aerobic treatment Aerobic Anaerobic  Large energy consumption (aeration) or  Reduced operational costs (no aeration, space less biosolids, treatment and disposal costs)  High biosolids production ~50% of C  Higher loading possible – more compact  Longer start up and harder to operate/ slower  H2S production  Sludge Treatment and Disposal  Primary sludge (non-biological), good settling  Secondary sludge (lower in SS, difficult thicken)  Sludge thickening (sedimentation, flotation)  Sludge stabilization (reduce pathogens, malodorants) o Chemical oxidation (CI), stabilization (lime) o Physically (heatburn)  Sludge dewatering / solidification o Max 50% water … centrifuge, filter Small Scale Recycling  Physical removal of solids o Parabolic screen, rapid filtration, low maintenance, 40-50% BOD removal o Bag filters – inexpensive, low filtration rates, clogging o Centrifugation/cyclone – rapid, efficient, expensive  Coagulation and Flocculation o Remove particulate impurities and colour o Remove soluble (colloidal) solids o Decrease microbial loading  Filtration o Separation of floc from water o Reduction in suspended solids o Reduction in microbial loading o Decrease turbidity o Types  Sand filter  Pressure filter  Inline filter • Addition of coagulant, low org loading  Hollow fibre filter • Low tmp, frequent air purge to reduce fouling  Cross flow filtration  Disinfection o Decontamination of the water  Reduce microbial loading  Sequester chemicals o Decontam methods  Residue – continuous antimicrobial effect • Chlorine, chlorine dioxide, electrolyzed water  Non residue – reduced environmental impact • UV, ozone o Disinfection by products (DPB)  Hypochlorous acid which is reactive • Fulvic and humic acids, AAs, Iodide, bromide  Trihalomethanes  Haloacetic acids  Chlorite  Flavour and odour taints  Carcinogens  Death from pathogens >>> than carcinogenic effects o DBP reduced by  Acidic pH (<4) • pH adjustment o Citric acid (org acid) – some side products o Inorganic acids (phosphoric, hydrochloric) – handling o Excess chlorine will raise pH  Measure by • Ox/red potential (ORP) • Test strips • Titration  Low precursor / low organics  Low temp and contact time  Low chlorine levels Using Chlorine Advantages Disadvantages  Inexpensive  byproducts  Broad range of antimicrobial action  corrosive  Low risk of developing resistance  pH dependant  available  flavour and colour taints  environmental impact Using Electrolyzed Water  electrolysis of NaCl  acidic fraction (hypoclorous acid), alkaline fraction (hydroxide) Advantages Disadvantages  low DBP - acidic form  equipment costs  high oxidizing power  maintenance  low residues  unstable Using Chlorine Dioxide Advantages Disadvantages  high oxidation power  expensive  high solubility in water  chlorine dioxide generator  less DBP  chlorite residues (ASC)  gas phase – independent of pH  explosive risk  high efficacy against biofilms Using Ozone Advantages Disadvantages  no residues  unstable  minor DBP  low solubility  strong oxidation power  monitoring  broad spectrum of antimicrobial activity  corrosive  high SA  concentration adjustment  increased solubility and stability Using Peroxyacetic Acid Advantages Disadvantages  broad spectrum of activities
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