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BIOL 241 – Post-Midterm Notes

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University of Waterloo
BIOL 241
Barbara Butler

BIOL  241  –  Introduction  to  Applied  Microbiology     Lecture  8:  Wastewater  Treatment     Septic  Tank   • Small-­‐scale,  not  meant  to  handle  a  lot  of  waste   • Influent  goes  into  septic  tank  found  below  ground  surface,  where  the  solids  settle  and   limited  anaerobic  digestion  occurs   • Fluid  flows  from  the  distribution  box  into  leaching  field/tile  bed  then  gravity  causes  it  to   trickle  down  through  the  soil   • Sieving  effect  as  material  moves  through  soil  naturally  purifies  the  system   o Works  best  with  medium -­‐porosity  soils  such  as  loa ms   o If  sandy  soil,  flows  too  fast  and  is  discharged  into  receiving  body  before  naturally   cleaned   o In  clay,  there  is  too  little  porosity,  so  the  fluid  pools  in  the  soil   • No  disinfection  system  so  intestinal  pathogens  are  not  reliably  destroyed     Water  that  is  released  from  treatment  is  not  100%  free  of  organisms   • Natural  bodies  of  water  are  contaminated  by  microbes  from  wastewater  treatment  effluents,   land  runoff,  wildlife     Lecture  9:  Drinking  Water  Treatment     The  Water  Cycle   • 70%  of  the  Earth’s  surface  is  covered  by  water   o 3%  is  usable;  ~70%  is  locked  away  in  glaciers,  permafrost,  etc.   • Accessible  water  is  a  precious  resource  that  we  can’t  live  without     Purification  of  Drinking  Water   • Goals:   o Remove  potentially  pathogenic  microorganisms  in  the  interest  of  public  healt h   o Decrease  turbidity  and  remove  particulates  to  give  clear  water  appearance   o Eliminate  taste  and  odour  due  to  organic  compounds  present  in  water   o Reduce  nuisance  chemicals  (ex.  Fe  stains  clothing,  Mn  is  also  problematic)   • Water  from  raw  source  comes  into  plan t  and  enters  sedimentation  tank,  where  the  solids   settle   • Flows  into  coagulation  tank   o Floculation:  alum  (aluminum  sulfate),  anionic  polymers  interact  with  small,   suspended  particles  and  colloids  (coagulation)  to  form  larger  flocs  containing   insoluble  material   o Flocs  settle  by  gravity,  trapping  and  removing  microbes,  adsorbing  organic  matter   and  sediment   • Filtration  removes  all  remaining  particulates,  most  organic  and  inorganic  compounds,   microorganisms   o Critical  to  removal  of  disinfectant -­‐resistant  microbes  like  Giardia  and   Cryptosporidium   o Slow  sand  filtration:  water  flows  into  a  tank  containing  sand  and  trickles  through   due  to  gravity,  where  it  is  cleaned  by  microbes;  results  in  a  layer  of  biofilm  and   precipitated  material  on  surface   o Rapid  filtration:  mixed  media  (sand,  activated  charcoal)  acts  as  a  good  absorbent   and  removes  biofilm/biological  material   • Disinfection  kills  remaining  microorganisms   o Chlorination  kills  most  microorganisms  within  30  minutes  and  oxidizes  organic   compounds  to  improve  taste  and  odour   o Chloramination  ensures  that  residual  chlorine  levels  are  maintained  throughout   the  distribution  system     HOCl  +  3H  à2  NH C2  +  H O   o Ozonation  and  UV  irra diation  are  alternate  technologies  to  avoid  the  production  of   disinfection  byproducts  that  are  weakly  carcin ogenic  (ex.  trihalomethanes)   • Water  goes  into  storage,  which  is  typically  a  clear  water  reservoir    a  water  tower • Distribution  system  distributes  finished  water  throughout  town     Monitoring  of  Water  Quality   • Water  courses:  sources  of  raw  water  for  drinking  (ex.  lakes,  rivers,  finished  water  from   drinking  plant)   • Necessary  to  monitor  for  presence  of  potential  disease -­‐causing  agents   • Many  waterborne  pathogens  cause  gastrointestinal  upset  (gastritis,  diarrhea,  c  is) o Transmitted  by  the  fecal-­‐oral  route/cycle   o Acquired  from  physical  contact  or  ingestion  of  water  contaminated  by  the  disease -­‐ causing  agent   o Usually  causes  problems  where  there  is  no  wastewater/drinking  water  treatment   plants  or  where  they’re  not  working  properly   • Potential  pathogens  include  bacteria,  vir uses,  protozoa     Indicator  organisms  are  used  to  monitor  for  pathogens   • Choice  of  indicator  may  depend  on  goal   o Occurrence  of  fecal  contamination   o Treatment  efficiency  (water  purification,  wastewater)  typically  looks  at   E.  coli   though  it  may  also  look  at  total  coliforms/coliphages   o Post-­‐treatment  contamination  in  water  distribution  systems  use  total  coliforms  as   opposed  to  E.  coli   o Food  industry  typically  looks  at   Staphylococci   • Ideal  indicator  should  be:   o Member  of  intestinal  microbiota  of  warm -­‐blooded  animals,  so  it  will  be  present  if   feces  are  in  water   o Equally  resistant  as  the  pathogen  to  varying  insults  (ex.  UV,  temperature   fluctuations,  disinfection)   o Present  in  greater  numbers  than  pathogen   o Detectable  via  easy,  rapid,  inexpensive  methods   o Non-­‐pathogenic   o Shouldn’t  multiply  in  the  environment     Coliforms:  facultative  aerobes,  gram-­‐negative,  non-­‐spore-­‐forming,  rod-­‐shaped  bacteria  that  ferment   lactose  with  gas  formation  within  48h  at  36 C   • Total  coliforms  include  E.  coli,  Enterobacter  aerogenes,  Klebsiella  pneumoniae   • Total  coliform  test  uses  the  most  probable  number  procedure   o Presumptive,  confirmed,  completed  steps   o Membrane  filter  technique :  filter  sample  through  4.5  µm  membrane,  place  on  top   of  medium,  look  for  colonies  with  green  metallic  sheen  ( E.  coli)   • Fecal  coliforms:  all  coliforms  that  can  ferment  lactose  at  44.5 C   o May  not  be  intestinal   o May  grow  under  environmental  conditions   • Fecal  coliform  test:  use  MFC  medium,  membrane  filtration,  pick  out  blue  colonies   o Also  referred  to  as  “thermotolerant  coliform  test”  since  mor e  than  just  fecal   organisms  may  grow       Defined  Substrate  Technology :  based  on  possession  or  expression  of  a  gene  for  enzymatic  activities   rather  than  growth   • Coliforms  possess  the  gene  for   ß-­‐galactosidase,  which  converts  MUGal  into  a  fluorescent   product  within  24h  at  35 C   • E.  coli  possesses  the  gene  for  ß-­‐glucuronidase,  which  converts  MUG  into  a  fluorescent   product  or  IBDG  into  an  insoluble  blue  compound  within  24h   o Other  coliforms  do  not  possess  this  enzyme     E.  coli  is  a  great  indicator  for  bacterial  pathogens,  but  not  for  enteroviruses,  pathogenic  protists   (Giardia,  Cryptosporidium ),  etc.     • Persist  in  the  environment  longer  than   E.  coli  or  are  more  resistant  to  disinfection   • Alternate  indicators:   o Bacteriophages/coliphages  as  indicators  for  enteric  viruses   o Enterococci/fecal  streptococci     Epidemics  of  waterborne  disease  often  show  pattern  of  cases  consistent  with  a   common  source  of   infection   • Treatment  plant  malfunction,  undetected  break  in  distribution  system,  disruption  of   treatment  by  natural  disasters  (earthquake,  flood)     Cholera   • Transmitted  almost  exclusively  via  contaminated  water  and  the  fecal -­‐oral  route,  though  it   may  also  be  associated  with  shellfish  and  water -­‐washed  foods  (ex.  vegetables)   • 7  or  8  worldwide  pandemics  since  1817   • Endemic  in  Africa,  parts  of  As ia,  Indian  subcontinent,  Central  &  South  America   • Controlled  by  applying  appropriate  water  treatment  and  sanitation  methods   • Vibrio  cholerae  is  a  gram-­‐negative,  curved  rod  that  i s  free-­‐living  in  coastal  waters   o Lives  in  association  with  other  aquatic  microbio ta  (ex.  the  alga  Volvox)   • Cholera  is  initiated  when  ingested  bacteria  attach  to  epithelial  cells  of  the  small  intestine,   grow,  and  release  enterotoxin  that  affects  the  GI  tract   o Characterized  by  copious  watery  diarrhea   o Fluid  loss  may  exceed  20L/day   o Skin  starts  to  shrink,  hollow  cheeks  from  water  loss;  may  die  if  untreated   • Treatment:  intravenous  or  oral  liquid  and  electrolyte  replacement  therapy   o If  enough  fluid  comes  through,  toxin  is  washed  out  as  the  i ntestinal  mucosa  is   replaced  frequently,  allowing  new  cells  to  grow  and  get  rid  of  bacteria   o Typically  don’t  treat  with  antibiotics     Typhoid  Fever   • Salmonella  typhi  is  a  gram-­‐negative  rod  that  is  a  member  of  the  Enterobacteriaceae   • Classically  waterborne;  may  also  be  foodborne  or  due  to  direct  contact  with  infected   individuals   • Virtually  eliminated  in  developed  countries  as  a  result  of  water  treatment  practices   • Carrier:  an  individual  that  harbours  the  organism  but  shows  no  disease  symptoms   o Carrier  state:  someone  can  have  an  asymptomatic  situation  but  still  carry  and   transmit  the  pathogen   o ex.  Typhoid  Mary   • Systemic  infection  with  sustained  bacteremia  that  is  characterized  by  high  fever,  initial   headache,  constipation,  then  diarrhea   • Microbes  can  end  up  in  the  kidney  or  intestine,  perforating  the  intestinal ng  l  and  causi the  contents  to  leak  into  the  body  cavity   • Treated  with  antibiotics  (ex.  chloramphenicol,  ampicillin,  cephalosporins)   • Mortality  may  reach  15%  in  untreated  typhoid     Cryptosporidiosis  and  Giardiasis   • Common  waterborne  diseases  in  areas  with  regulated  water   supplies   • Environmentally  resistant   cysts  or  oocysts  of  these  parasitic  protozoa  found  in  most  surface   waters   o Chlorine-­‐resistant;  dose  rates  can  be  low   o Can  be  filtered  out  in  distribution  system   • Excysts  into  vegetative  forms  in  body  (trophozoite,  sporozoite)  that  cause  symptoms  and   cannot  survive  in  the  external  environment     Giardia  intestinalis:  flagellated  protozoa,  infects  animals  and  humans   • Giardiasis  is  also  known  as  “beaver  fever”   • Environmentally  resistant  cyst  is  infective  agent   o Ingested,  move  through  digestive  tract   o Stomach  acid  triggers  excystation,  causing  the  cysts  to  germinate  in  the  small   intestine   o Trophozoites  metabolize  and  grow  on  the  small  intestinal  wall   • Often  acute  symptoms,  though  they  can  be  chronic  (nausea,  weight  loss,  explosive,  watery   diarrhea)     Giardia  lamblia  life  cycle:     cyst  à  moves  through  food/ water  into  host  or  through  some  sexual  contact  (fecal  oral  cycle)   à   excision  à  trophozoite  à  attach  to  intestinal  wall  à  grow,  complete  life  cycle  à  cysts  released  with   feces  à  reingested  by  another  host      Cryptosporidium  parvum:  infects  a  variety  of  warm-­‐blooded  animals,  resistant  oocysts  transmitted  to   new  host  via  feces-­‐contaminated  water   • Oocysts  are  smaller  than   Giardia  and  more  chlorine  resistant   • Ingested  oocysts  germinate  into  sporozoites,  which  become  trophozoites   o Invade  intestinal  wall,  mucosis,  epithelial  cells  to  the  stomach  and  gall  bladder   o Mild,  self-­‐limiting  diarrhea  in  healthy  individuals;  immune  system  eventually  fights   it  off   o Chronic  diarrhea  and  dehydration  in  impaired  immune  systems;  may  lead  to   complications,  since  they  can’t  get  rid  of  it      Cryptosporidium  life  cycle:     excystation  à  trophozoite  invades  tissue  à  sporozoite  à  complete  life  cycle  à  oocyst  released  from   body  à  can  continue  on  infection     Legionellosis   • Waterborne  disease  that  causes  pneumonia  (respiratory  tract  infection)   • Severity  of  infection  varies  depending  on  the  strain  of   Legionella   o Asymptomatic   o Pontiac  fever:  mild  cough,  sore  throat,  mild  headache,  self -­‐limiting   o Legionellosis:  a  type  of  pneumonia,  more  likel y  to  affect  elderly  and   immunocompromised  individuals;  intestinal  disorder,  high  fever,  chills,  muscle   aches,  dry  cough,  chest  and  abdominal  pain   • Legionella  pneumophila  is  present  in  small  numbers  in  natural  waters  and  soils,  may  live   inside  free-­‐living  protozoa;  heat-­‐  and  chlorine-­‐resistant;  non-­‐spore-­‐forming   o Lives  happily  in  cooling  towers,  air  conditioning  systems,  hot  water  tanks,  whirlpool   spas,  etc.   • No  person  to  person  transmission   o Bacteria  is  disseminated  in  humidified  aerosols   o Human  infection  is  via  airborne  droplets     Waterborne  Viral  Diseases   • May  cause  gastroenteritis  (ex.  rotaviruses,  noroviruses)   • May  cause  eye,  throat  infections  (ex.  adenoviruses)   • Hepatitis  (liver  disease)   o Hepatitis  A  and  E  are  waterborne   o B  and  C  are  spread  parentally   o Jaundice  may  be  a  symptom:  liver  malfunction,  skin  and  whites  of  eyes  turn  yellow   o A  may  be  asymptomatic,  have  chronic  or  mild  symptoms   o Can  cause  cirrhosis  of  the  liver,  which  is  fatal  or  seriously  damaging   • Polio:  wild  poliovirus  has  been  eliminated  from  the  western  he misphere,  but  is  still   endemically  spread  in  Afghanistan,  Pakistan,  Nigeria   • Most  of  these  viruses  are  neutralized  by  chlorination     Amebiasis   • Entamoeba  histolytica  is  transmitted  primarily  by  water  (cysts)   o Excysted  trophozoites  grow  on  or  in  intestinal  muco sal  cells   o Amebic  dysentery  is  the  invasive  version  that  leads  to  intestinal  inflammation,   ulceration,  fever,  blood  and  mucus  in  feces;  may  result  in  invasion  of  liver,  lung,   brain,  abscessing/death   o Common  in  tropics,  subtropics   • Naegleria  fowleri  is  a  free-­‐living  amoeba  found  in  soil,  water  runoff   o May  enter  through  nose  and  burrow  into  brain,  where  it  multiplies   o Meningoencephalitis :  swelling  of  the  brain,  extensive  hemorrhaging,  brain  damage   o Unless  quickly  diagnosed  and  treated  with  amphotericin  B,  death  occ urs     Lecture  10:  Food  Microbiology     Food  spoilage:  any  change  in  the  appearance,  smell,  or  taste  of  a  food  product  that  makes  it   unacceptable  to  the  consumer   • Spoilage  organisms  are  not  necessarily  pathogens;  therefore,  spoiled  food  isn’t  necessarily   unsafe,  but  is  considered  to  be  unpalatable     Perishable  foods:  ex.  meat,  eggs,  milk,  most  fruits,  vegetables   Semiperishable  foods:  more  stable;  ex.  potatoes,  nuts,  some  apples   Non-­‐perishable  foods:  very  stable,  won’t  spoil  if  kept  in  cupboard;  ex.  sugar,  rice,  f lour,  dry  beans     Water  Activity  (a ):  a  measure  of  available  water   w • Vapour  pressure  of  air  in  equilibrium  with  substance  or  solution  /  vapour  pressure  of   solvent  (pure  water)   • V p  of  pure  water  is  1   • Water  activity  is  not  the  same  as  moisture  content   o Water  can  be  tied  up  by  solutes  that  are  present  in  the  food  (sugar,  salt)  so  that  it  is   unavailable  to  be  used  by  microbes   • Every  microbe  has  a  mininmal  water  activity  below  which  it  cannot  grow   o Gram  negatives  tend  to  require  high  water  activities   o Staphylococcus  aureus  is  an  exception,  and  is  resistant  to  low  water  activity  of  about   0.86   o Lower  than  that,  specialist  organisms  that  can  deal  with  high  stress,  salty  conditions   (halophiles),  dry  conditions  (molds)   • Lower  aw  =  more  stable  food     Microbial  Spoilage  of  Fresh   Food   • Fruits  and  vegetables  have  sugar,  pectin  (cellular  cement   in  plants,  makes  plant  tissue)     o Spoilage:  fermentation  of  sugars  by  yeasts,  etc.   o Certain  bacteria  are  good  at  attacking  pectin,  break  it  down  causing  “soft  rot”  where   the  food  becomes  watery  and  mushy  because  its  tissue  structure  has  fallen  apart     o ex.  Erwinia  is  pectinolytic,  Pseudomonas,  Corynebacterium   • Meats  have  a  lot  of  protein  which  can  be  broken  down;  typically  by  microbes  associated  with   the  GI  tract  of  animals   o ex.  E.  coli,  Salmonella,  Campylobacter,  Listeria,  Pseudomonas   • The  main  sugar  in  milk  is  lactose,  which  can  be  broken  down  by  lactic  acid  bacteria   o May  be  fermented  to  lactic  acid,  souring  the  milk   o ex.  Streptococcus,  Lactobacillus,  Pseudomonas     Bacterial  Growth  in  Batch  Culture   • Recall:  microbial  growth  curve  from  BIOL  240   • In  terms  of  food  spoilage,  lag  and  exponential  phases  are  important   o By  the  time  it  reaches  the  end  of  the  exponential  phase,  it  is  visibly  spoiled   o Important  to  shelf  life  estimates   • Exponential  growth  affected  by  tempe rature,2  O , w  pH,  a ,  nutritive  value  of  food,  natural   antimicrobial  substances  (ex.  lysozyme  in  eggs),  makeup  of  food’s  microbial  community   • Extent  of  lag  phase  depends  on  microbial  contaminants,  previous  growth  history,   disinfectants,  handling  it  receives   • Aim  of  food  preservation:  extend  lag  phase  indefinitely,  slowing  growth     Food  Preservation:  Temperature  Control     Storage  at  Cold  Temperatures   O O O • Refrigeration  at  5 C;  freeze  to  -­‐20 C,  some  to  -­‐80 C   • Slows  down  microbial  growth  but  does  not  stop  them  from  growin g   o Psychrotolerant  microorganisms  may  spoil  refrigerated  foods   • Freezing  increases  the  shelf  life  of  food   o Perishable  goods  have  a  short  shelf  life  since  psychrotolerant  organisms  will  still  be   able  to  grow   • Freezing  may  damage  food   o Freeze-­‐thaw  sequence  alters  physical  structure  of  foods   o Damaged  food  is  a  better  growth  substrate  for  certain  microbes,  which  is  why  it’s   important  to  use  thawed  food  up  quickly   • Freezing  at  -­‐80 C  is  also  a  good  way  to  keep/store  microbial  colonies     Heat  Processing   • Cooking  markedly  reduces  the  microbial  load   o Must  avoid  reinoculation   • Canning:  vacuum  sealed  can  of  food  cooked  at  high  temperature  (often  121 C  for  15  min)  to   produce  food  that  is  stable  indefinitely  on  the  shelf   o Doesn’t  need  to  be  refrigerated   o Produces  “commercially  steril ized  product”   o Anaerobic  spore  formers  may  survive  in  home -­‐canned  goods   • Pasteurization:  designed  to  reduce  microbial  load,  kill  pathogens   o Low  temperature  hold  (LTH):  batch  process;  63 C  for  30  min  knocks  out  most   microbes,  including   Mycobacterium  bovus   o High  temperature,  short  time  (HTST):  continuous  process;  72 C  for  15  sec   • Aseptic  food  processing :  ex.  juice  boxes,  milk  products   O o Uses  flash  pasteurization/sterilization  (133 C)  and  aseptic  packaging     Flash  Pasteurization:  continuous  flow  system  that  sterilizes  a  small  volume  of  raw  milk  over  a   short,  specified  amount  of  time   • Milk  flows  through  metal  plates/tubing  heated  to  a  very  high  temperature   • Immediate  passage  to  cooling  unit  lowers  temperature  quickly  to  prevent  excessive  heat   denaturation  of  product’s  nut rients  and  flavor     Increase  Acidity  of  Food   • Decreasing  the  pH  of  food  makes  it  more  stable   • Adding  acidulant  (pickling)   o Adding  vinegar  or  other  acids  to  cucumbers,  peppers,  meats,  fish,  etc.   o Often  entails  other  additions  (ex.  sugar,  salt,  spices  for  flavor,  substrate  for  microbial   fermentation)   • Encouraging  fermentation  by  microorganisms   o May  be  natural  community  or  added  inoculum   o Lactic  acid  bacteria  (ex.  Lactococcus,  Lactobacillus,  Leuconostoc )  transform  milk  à   yogurt,  cheese,  sour  cream  and  cabbage   à  sauerkraut   o Acetic  acid  bacteria  (ex.   Acetobacter,  Gluconobacter )   o Propionic  acid  bacteria  ( Propionibacteria)  create  the  flavor  and  holes  characteristic   of  Swiss  cheese  due 2to  CO  and  lactic  acid  production   o Inoculate  raw  milk  with  lactic  acid  bacteria,  incubate  at  38-­‐46 C  for  6-­‐8h;  casein   denatures,  forming  a  curd;  lactic  acid  contributes  to  flavor  and  preserving  qualities;   aldehydes  produced  may  contribute  to  flavour/aroma     Decrease  Water  Activity  of  Food   • Drying:  natural  sunlight,  oven,  may  be  in  conjunction  with  smoking   o Phenolics,  phenaldehydes  in  smoke  have  a  preservative  effect   o ex.  fish,  meats,  fruit   • Lyophilization:  freeze  food,  pull  of  water  by  vacuum,  store  in  controlled  atmosphere   o Can  rehydrate  by  adding  water  back   o ex.  camping  rations   • Addition  of  sugar  or  sal t   o Tie  up  water  and  stabilize  food,  wropping  a  and  making  it  harder  to  spoil   o ex.  sugar  added  to  fruits  (jellies,  jams,  preserves)   o ex.  salt  added  to  meats,  fish  (sausage,  ham)     Chemical  Preservation  of  Food   • GRAS  (“generally  recognized  as  safe”)  are  often  ad ded   • Organic  acids  inhibit  the  growth  of  many  icrobes   • Nitrite  added  to  cure  foods  (ex.  ham,  bacon);  anticlostridial  agent  that  inhibits   Clostridium   botulinum   • Formaldehyde  from  food  smoking  processes  used  on  meat,  fish   • Ethylene/propylene  oxides:  microbial  st atic,  lethal  to  microbes,  alkylating  agents   o Can  only  be  used  in  limited  ways  to  gas  spices,  dried  fruits,  nuts     Irradiation   60 137 • Co  or Cs  sources   • Food  does  not  become  radioactive   • Physiochemical  damage  may  occur  making  food  inedible/unappealing  to  consumers   • Used  to  knock  out  endospores,  insects,  larvae  in  wheat,  flower   • Prevent  potatoes  and  onions  from  sprouting  as  readily   • May  be  used  to  reduce  microbial  load  and  limit  pathogen  contamination   o Campylobacter  in  raw  poultry,  E.  coli  in  hamburger   • Sterilization   o NASA  space  flights,  special  hospital  diets     Modified  Atmospheric  Packaging  (MAP)   • Use  of  impermeable  films,  evacuation,  elevation 2 of  CO  content  to  prevent  growth  of  aerobic   spoilers   o Often  used  in  conjunction  with  refrigeration  temperature,  acidic  pH,  non -­‐optimal   water  activity   • Used  to  increase  the  shelf  life  of  food     Lecture  11:  Industrial  Microbiology     Biotransformation  (Biocatalysis)   • ex.  cortisone  production   • First  step  is  very  difficult  to  accomplish  via  chemical  reactions   • Rhizopus  nigricans  (fungus)  will  catalyze  the  biotransformation  of  progesterone  to  11   α-­‐ hydroxyprogesterone   o After  that,  the  rest  of  the  transformations  are  chemical     Primary  Metabolite:  one  that  is  formed  during  the  growth  phase  (exponential  phase)  of  the   microorganism   • ex.  ethanol  produced  by  fermenting   Saccharomyces  cerevisiae   • As  ethanol  builds  up,  it  is  toxic;  not  always  the  case  with  primary  metabolites     Secondary  Metabolite:  one  that  is  formed  near  the  end  of  exponential  phase,  near/in  stationary   phase  (fairly  late  in  the  cycle)   • Not  essential  for  growth/reproduction   • Often  the  starting  material  to  produce  this  is  a  primary  metabolite   • If  you’re  not  careful  with  the  conditions  under  which  a  microbe  is  grown,  this  production  can   be  inadvertently  inhibited  or  repressed   o Alternatively,  you  can  also  get  a  microbe  to  overproduce  a  secondary  metabo lite;   often  done  in  antibiotic  production   • ex.  penicillin  from  Penicillium  chrysogenum  (fungus)     Making  Vinegar   • Can  make  vinegar  chemically  from  ethanol,  but  it  would  be  harsh  to  use   • Requires  a  source  of  alcohol  (pure  ethanol,  wine,  hard  cider)  plus  certain  strains  of  the   “acetic  acid”  bacteria  (Acetobacter,  Gluconobacter )   • Strains  are  strict  aerobes,  grow  on  alcohol  and  oxidize  it  to  acetic  acid   o Incomplete  oxidizers;  don’t  oxidize  electron  donors  completely  to  CO  and  H O   2 2   Vinegar  Generator   • “Trickle  method”  uses  a  container  made  of  stainless  steel  that  is  filled  with  woodchips,  and   allows  a  biofilm  of  acetic  acid  to  develop  on  woodchips  over  time   o Alcoholic  solution  trickled  in  at  the  top,  makes  its  way  through  woodchips  via   gravity   o Adequate  aeration  is  import ant  for  strict  aerobe  metabolism   o Fluid  recirculated  until  desired  acetic  acid  content  is  reached   o Operates  continuously  with  shaving  lifetime  of  ~5 -­‐30  years   o Other  materials  produced  in  the  microbial  reaction  contribute  to  vinegar  flavour   • Low-­‐tech  method:  vat  of  alcohol  containing  bacterium  eventually  turned  into  acetic  acid   • High-­‐tech  method:  bioreactor  (Fring’s  Acetater)  and  submerged  fermentation  process       Vitamin  Synthesis   • Vitamin  B 12  is  an  important  coenzyme  and  a  necessary  part  of  human  diet   o Deficiency  can  lead  to  pernicious  anemia,  which  reduces  red  blood  cell  numbers   and  can  cause  nervous  system  disorders   o Natural  synthesis  is  exclusively  by  microorganisms   o Pseudomonas  and  Proteonome  bacteria  are  exploited  to  make  this   • Riboflavin  (vitamin  B 2  is  needed  for  flavin  synthesis   o FAD,  FMN  are  coenzymes  that  participate  in  oxidation -­‐reduction  reactions,  involved   in  electron  transport  activity   o Ashbya  gossypii  (fungus)  has  been  manipulated  to  produce  7  g/L     Amino  Acid  Production   • Glutamic  acid/glutamate  used  as  part  of  MSG  salt   • Glycine  used  as  sweetner  to  improve  flavor;  used  to  start  organic  chemical  synthesis   • Aspartame  is  an  artificial  sweetner  in  sugar-­‐free  products  made  from  two  natural  amino   acids  (phenylalanine  and  aspartic  acid)  bound  together  in  an  unnatural  way   • Use  overproducing  strain  of  bacteria  (ex.   Brevibacterium  flavum)  to  produce  a  given  amino   acid   o Mutant,  circumvents  normal  regulatory  control  of  amino  acid  synthesis   • Feedback  inhibition:  enzyme  that  catalyzes  first  reaction  is  shut  off  if  amino  acid  end   product  starts  to  accumulate  in  the  cell   o Binds  to  allosteric,  regulatory  site  and  changes  conformation  so  catalytic  site  isn’t   available,  decreasing  amino  acid  production     Enzyme  Production   • Often  exploit  extracellular  enzymes  ( exoenzymes)   o Excreted  into  supernatant,  easily  recovered   o Found  in  detergents  (ex.  amylase,  lipase  intended  to  break  down  starch/lipid   polymers)   o Can  also  use  intracellular  enzymes   • Catalytic  specificity  of  an  enzyme  is  useful  in  bioconversions   o Some  only  produce  L-­‐amino  acids,  which  are  used  to  make  specific  stereoisomers  in   the  fine  chemical  industry   • Extremophile  enzymes  (extremozymes)  remain  functional  under  harsh  conditions  (acid,   alkaline,  high  temperatures,  high  salinity)   • Detergents:  highly  alkaline,  function  optimally  at  pH  of  9-­‐10   o ex.  amylase,  protease,  lipase,  reductase  from   Bacillus  licheniformis   • Taq  Polymerase:  thermostable  (to  95 C)  DNA  polymerase  from  Thermus  aquaticus   o Used  in  polymerase  chain  reaction  (PCR)  to  amplify  a  certain  piece  of  DNA   • Invertase:  intracellular  enzyme  that  will  break  down  sucrose   • Glucose  Isomerase:  intracellular  enzyme  that  is  used  to  make  high  fructose  sweetener  from   corn,  wheat,  potato  starch   o Produce  glucose  from  starch  using  amylase,  then  use  glucose  isomerase  to  turn  it   into  fructose   o Used  in  soft  drink  production   • Immobilization  of  soluble  enzymes  allows  large -­‐scale  reactions  under  continuous  conditions   o May  stabilize  the  enzyme  so  that  it  lasts  longer  and  can  catalyze  more  reactions     Scale-­‐Up   • Lab  flask  (100  mL)  à  lab-­‐scale  fermenter  (1-­‐10L)  à  pilot  plant  scale  (300-­‐3000L)  à   commercial  scale  (10  000–500  000  L)   • Bioengineers  are  skilled  in  gas  transfer,  fluid  dynamics,  mixing,  thermodynamics   • Oxygen  transfer  is  much  more  difficult  to  obtain  in  large  scale   o Surface  to  volume  decreases,  making  it  harder  to  mix/aer ate   o Sterile  air  flows  in,  uses  an  impeller  to  mix  and  diffuse  out  air  bubbles   • Methods  of  temperature  control  must  be  changed  with  scale -­‐up   o Cooling  jacket  to  help  control  heat  of  system   o Biological  activity  raises  temperature   • Sterilization  protocols  must  cha nge  with  scale-­‐up   o Lab  flask/fermenter  can  be  autoclaved   o With  larger  scale  operations,  live  steam  comes  in  from  the  top,  s  lizing  it  in  place • Commercial  scale  is  all  computerized,  allowing  for  continuous  control  and  decreasing  the   likelihood  of  mistakes     Downstream  Processing     Processing  of  cellular  products  may  entail:   • Removal  of  biomass  from  culture  medium,  concentration  and  purification  of  product  from   spent  medium   • Harvesting  and  fractionation  (centrifugation)  of  biomass  to  recover  desired  product,   separation  from  cellular  debris,  concentration  and  purification  of  product   • If  just  looking  for  enzymes,  just  filter  and  centrifuge  to  get  rid  of  biomass  and  use   ultrafiltration  to  get  rid  of  most  water   • Antibiotics  excreted  into  the  supernatant  are  concentrate d  using  solvent  extraction,  purified   using  chromatography/crystallization,  and  tested  to  ensure  they  aren’t  contaminated   • Cytoplasmic  enzymes  are  harvested  by  centrifugation,  concentrated,  purified     Processing  Yeast  Biomass   • Inoculate  fermenter,  grow  aerobically  on  organic  media   • Collect  cells  using  centrifugation   • Wash  cells  to  get  rid  of  spent  medium   • Dewater  cells  by  centrifugation/filtration  processes   o Commercial  bakeries:  use  compressed  yeast  cakes  that  have  20%  moisture  and  live   yeast;  low  shelf  life,  immed iately  metabolizes  sugar  with  no  lag  phase   o Home:  use  active  dry  yeast  with  8%  moisture;  packaged  in  water -­‐tight  package  that   is  shelf-­‐stable  and  can  last  for  ~1  year   o Nutritional  dry  yeast:  single  cell  protein,  pasteurized  so  yeast  is  dead  and  almost  all   of  the  water  is  removed;  dry  storage   • Package   • Store  until  shipped     Lecture  12:  Biotechnology     Biotechnology:  the  use  of  living  organisms  to  carry  out  defined  chemical  processes  for  industrial  or   commercial  application   • Bioremediation  of  pollutant  spills,  biole aching  of  metals   • Production  foods,  food  additives,  alcohol,  antibiotics,  commodity  chemicals,  biomass,   microbial  plastics   • Bioconversion,  biocatalysis     Molecular  Biotechnology :  implies  the  organism  used  to  carry  out  the  process  has  been  genetically   manipulated  in  vitro   • Molecular  genetic  techniques  provide  an  opportunity  to  isolate,  manipulate,  sequence  DNA,   control  DNA  expression,  genetic  engineering   o Production  of  human  growth  hormone,  insulin,  interferon,  vaccines,  industrial   chemicals,  microbial  plastics,  modified  plants  and  animals   o ex.  Taq  polymerase  now  produced  in   E.  coli  engineered  to  carry  and  express  the   Thermo  aquaticus  gene     Before  the  molecular  age…   • Introduction  of  random  mutations  and  screening  for  mutants  with  desired  traits   • Industrial  strain  development  in  penicillin  production   o Numerous  mutations  achieved  spontaneously  (UV  or  X -­‐ray  irradiation,  chemical   exposure  to  nitrogen  mustard)  led  to  the  development  of  an  overproducing   P.   chrysogenum  strain  that  produced  enough  penicillin  for  efficient  comm ercial   production   • Feedback  inhibition  in  wild -­‐type  cells  abolished  in  mutants   o AEC  is  a  lysine  analog  that  will  bind  to  aspartokinase  and  shut  down  the  pathway   o Mutants  of  Brevibacterium  flavum  (also  called  Corynebacterium  glutamicum)  that   overproduce  lysine  were  obtained  by  looking  for  AEC -­‐resistant  cells  that  had   mutations  in  aspartokinase   Today…   • Site-­‐directed  mutagenesis:  a  technique  whereby  a  gene  with  a  specific  mutation  can  be   constructed  in  vitro   o Modify  regulatory  part,  not  catalytic  part   o Ex.  eliminate  capacity  for  modification  of  aspartokinase  enzyme  by  lysine/AEC   o Still  need  to  test  mutant  to  ensure  it  functions  as  planned     Ways  to  Move  DNA  Around     pUC19:  a  cloning  vector  (synthetic  plasmid)  that  replicates  in   E.  coli   • Origin  of  replication  allows  independent  replication   • Polylinker:  region  with  single  cleavage  sites  for  various  restriction  enzymes  (ex.   BamHI)   o Restriction  enzymes  recognize  specific  sites,  cut  and  make  blunt/sticky  ends  that   allow  for  insertion  of  foreign  DNA   • Selectable  marker  gene  (ex.  antibiotic  resistance)   o E.  coli  is  not  naturally  resistant  to  ampicillin,  but  E.  coli  carrying  this  plasmid  will  be   able  to  grow  and  form  colonies   • Differential  marker  gene  (ex.  lacZ  for  blue-­‐white  screening)   o Enables  easy  detection  of  transformants   o If  insert  is  present,  it  will  disrupt  lacZ;  won’t  be  able  to  produce  ß-­‐galactosidase,   appear  white  on  X-­‐gal   o If  insert  is  not  taken  up,  lacZ  is  still  functional  and  can  produce  ß-­‐galactosidase,   forming  blue  colonies  on  X-­‐gal   • Transformation:  transfer  of  genetic  information  via  free  DNA     Bacteriophage  Lambda:  modified  lysogenic  E.  coli  phage  used  as  a  cloning  vector   • Digest  with  restriction  enzymes,  ligate  with  foreign  DNA   • Hybrid  DNA  can  be  packaged  inside  of  phage  particles  and  injected  into  host  chromosomes   where  it  is  expressed   • Artificial  transduction   o Transduction:  transfer  of  host  genes  from  one  cell  to  another  by  a  virus     Electroporation:  the  use  of  electric  pulses  to  enable  cells  to  take  up  DNA   • Artifical  transformation   • Millisecond  length  pulses  of  electricity  open  sm all  pores  in  cell  membranes   • DNA  can  move  into/out  of  the  ells  via  pores,  creating  transformants   • Don’t  need  to  use  a  plasmid   • Works  with  any  cell  type     Microprojectile  “DNA  Gun” :  microprojectiles  fly,  embed  themselves  in  target  cells   • Pierce  cell  membranes  and  land  somewhere  into  cellular  tissue;  DNA  incorporated  into  cell   • Don’t  know  where  the  piece  of  DNA  will  go  in  the  genome   • Can  use  with  whole  tissues     Commonly  Used  Hosts:   • E.  coli  is  the  most  common  host  in  molecular  biology  as  we  know  more  about  its  geneti cs   than  any  organism  on  the  planet   o Some  strains  are  pathogenic  to  certain  people   o Gram-­‐negative,  proteins  may  get  hung  up  in  periplasm   • Bacillus  subtilis  is  a  soil  organism  that  produces  a  lot  of  extracellular  proteins   o Forms  endospores,  making  it  easy  to  sto re   o Naturally  competent   o Don’t  know  much  about  its  genetics   • Saccharomyces  cerevisae   o Easy  to  handle  compared  to  Drosophila   o Can  process  mRNA  and  remove  introns;  prokaryotes  can’t  do  this   o Used  as  model  organism,  cloning  host  in  eukaryotic  systems   o Can’t  handle  prokaryotic  plasmids/genes     Expression  of  human  genes  in  prokaryotes  requires  a  method  of  dealing  with   introns   • Introns:  intervening  sequences  that  are  removed  and  not  expressed   • Spliceosomes  used  to  produce  mature  mRNA  with  introns  removed  and  exons  stitched   together   • Bacteria  can’t  remove  introns  and  will  try  to  process  them   • Reverse  transcriptase  enzyme  takes  mRNA  that  has  been  processed  from  a  eukaryotic   organism  and  converts  it  to  cDNA  that  a  bacterium  can  understand   o No  introns  present   • Can  also  synthesize  a  synthetic  gene  and  clone  that  into  bacterium     Expression  Vector:  controls  expression  of  a  foreign  gene;  makeup  similar  to  cloning  vector   • Polylinker:  used  to  insert  desired  DNA   • Selectable  marker  (ex.  ampicillin  resistance)   • Promoter:  turns  on  gene  expression,  gets  a  lot  of  transcription;  upstream  of  foreign  gene   • Operator:  regulatory  region  that  works  with  promoter   • Shine-­‐Dalgarno  Sequence:  codes  for  ribosome  binding  site,  immediately  upstream  of   foreign  gene   • Start/stop  codons   • Transcription  terminator  region   • Regulatory  control  (ex.  lacI  the  lac  repressor)   • Peptide  tag  (ex.  His  tag):  used  to  find  foreign  protein  very  easily   o String  of  His  bind  nickel,  creating  nickel  columns;  take  a  solution  of  protein  with   His   tag,  run  through  column,  proteins  bind  and  are  recove red     Producing  Recombinant  Bovine  Somatotrop in   • Somatotropin:  a  growth  hormone  that  interacts  with  two  receptors   o ST  receptor  controls  growth   o Prolactin  receptor  controls  lactation   • Convert  mRNA  for  bovine  somatotropin  into  cDNA  using  reverse  transcriptase  and  clone   into  expression  vector   o Purify,  clean  it  up,  inject  into  cows   o Causes  females  to  lactate  more  and  produce  more  milk   o Doesn’t  maximize  milk  production  though,  since  it’s  hard  on  cow     Producing  Recombinant  Human  Somatotropin   • For  treatment  of  stunted  grow th  in  children   o Doesn’t  work  if  there’s  a  mutation  in  ST  receptor  (ex.  pygmy  tribes)   • Use  site-­‐directed  mutagenesis  to  modify  ST  gene  and  inactivate  prolactin  activity   • Use  host  bacteria  strain  defective  in  protease  activity   o Avoids  protease  digestion  of  recom binant  protein  before  harvesting     Transgenic  plants  may  be  produced  with   binary  vector  system  in  Agrobacterium  tumefaciens   • Get  rid  of  genes  that  will  make  plant  diseased  (ops,  oncogenes)  and  keep  system  that  moves   foreign  DNA  from  bacterium  to  plant   • Binary  vector  system   o Generalized  plant  cloning  vector  that  foreign  DNA  is  inserted  into   o Mutant  TI  plasmid  capable  of  moving  single -­‐stranded  DNA   • Two  origins  of  replication   o One  in  E.  coli,  one  in  Agrobacterium   • Marker  genes:  plant  naturally  sensitive  to  kanamycin,   can  easily  find  transformed  cells  this   way   • Clone  in  E.  coli,  transfer  to  A.  tumefaciens  by  conjugation   • TI  plasmid  mobilizes  vector,  producing  plant  cells  grown  in  tissue  culture   • Whole  transgenic  plants  can  be  generated  from  the  recombinant  cell     Lecture  13:  Foodborne  Illnesses     Gastroenteritis:  acute  inflammation  of  the  gastrointestinal  tract,  especially  the  small  intestine  or   large  intestine  (enterocolitis)   • Characteristic  symptoms:  diarrhea,  vomiting   o Diarrhea  is  a  result  of
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