Biotechnology

Annotations:

• biotechnlogy is the use of living organism to produce food, drugs or other products

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• bacteria, archea, fungi and protoctistia.

1. Rapid life cycles

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   • have rapid life cycles so large populations can be built up quickly, all prokaryotes (bacteria and archea) reproduce asexually so all organisms are indentical and carry out the same metabolic processes
   1. exam q

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      • The generation time for Escherichia coli in a laboratory can be 20 minutes, but in the intestinal tract it can be as much as 24 hours. Suggest three reasons for this difference.  
      • less oxygen / ora; reduced amount of nutrients / ora;ref to pH / ora;competition from other bacteria / interspecific competition / ora;use of antibiotics;AVP; ref to intestinal enzymes or immune system 
2. Specific requirments

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   • have very specific and simple requirements for growth so can easily be produced in fermenter with minimal attention
3. industry waste

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   • they are often grown using waste mateials from industry
4. No ethical concerns
5. Gene Expression

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   • only have single copy of genes so if gene is altered through gene technology there will be no other genes to mask it, express genes simply so are easily modified
6. High Temps

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   • some evolved to function in higher temps
7. Grown 24/7

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   • they can be grown all the time, in any climate, in any countrys, in small spaces
8. Enzymes

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   • uses part of living organism to make product = enzyme,   
   1. intracellular

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      • be contained within cells of organism (intracellular enxymes)
   2. extracellular

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      • can , they also use enzymes that are not contained cells = isolated enzymes. they can be secreted naturally others have to be extracted (extracellular enzymes) extraction makes them more expensive compared to intracellular enzymes
9. exam q

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   •   Other medically important products, such as insulin and growth hormone, are produced on a large scale using microorganisms. Give reasons for using microorganisms in the production of insulin and growth hormone.  
   • can genetically engineer  microorganisms;ref to risk of infection; e.g. CJD with GHavoids problem with, side effects / allergic effects; A ref. toimmune responselarge amount of product;grow microorganisms in small, area / volume; A less space requiredcan be cultured anywhere in world;ethical advantages, qualified;ref to cost qualified; e.g. insulin uses cheaper feedstock (than forrearing pigs)AVP;AVP; e.g. high replication / growth rateextraction of GH from brains slow process max 4 
10. exam q

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    • fast growth, can be genetically engineered, can be processes at low temperatures which makes them easier and safer to maintain, product is pure and easy to seperate, grow on unwanted food, no animal welfare issues 

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• usual growth curve example is using bacteria ecoli that can respire aerobically and anarobically. In a closed culture with a nutrient broth containing glucose, amino acids and vitamins. the bacteria reproduce asexually and cells split in two with a process of binary fission.

1. 1. Lag

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   • adjusting to new condtions, = synthesising enzymes to take up nitrients, switching on and off genes, trasncription and translation, reproduction rate is slow
2. 2. Log

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   • or Expoential phase. Increase rapidly, growing and dividing at maximum rate for particular conditons they are in, plenty of nutrients and space only limitation to their rate of reproduction is their own in built capacity, the population growth is exponential = repeatdly doubles in a particular length of time.plenty of nutrients and space
3. 3. Stationary Phase

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   • nutrients begin to run out, the number of new cells being produced matches the number of cells dieing, changes in broth due to waste products, fluctuates around amean,
4. 4. Death Phase

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   • death rater is geater than the reproductive rate, food is scarse and waste products become very toxic,
5. Sigmoid Growth Curve

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   • lag, log & stationary phase

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• enzymes are expensive if you use immobilised enzymes they can be reused

1. Attatched to an insoluble material
   1. cross linked

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      • cross linking amino groups of the enzyme between enzymes
   2. carrier bound
   3. microcapsule
   4. exam q

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      • State the meaning of the term immobilised enzyme and describe how immobilisation can be achieved.  
      •  ATTATCHED TO INSLUBLE MATERIAL   (micro)encapsulation / (trapped) in alginate beads;adsorption / stuck onto, collagen / clays / resin / (porous) glass;cross linkage / covalent / chemical, bonding to, cellulose / collagen fibres;gel entrapment / trapped inside gel e.g. silica (lattice / matrix);partially permeable membrane (polymer) microspheres; 
   5. stuck onto collagen
2. Gel Entrapment

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   • enzyme solution mixed with sodium alginate solution droplets of this mixture are then added to a solution of calcium chloride
   1. Enzymes + Sodium Alginate = Droplets + calcium chloride = Jelly
      1. React to form Jelly

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         • turns into  bead, the bead contains the enzyme. The enzyme is held in the bead = immobilised. These beads can be packed into a collum and the liquid can trickle over the beads and the enzymes in the bead catlayse the reaction from substrate to product
   2. Lactose

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      • lactase can be immobilised so that milk trickles over immobilised enzymes and lactase hydrolyses the lactose in milk to glucose and galactose = lactose free millk

1. No product contamination

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   • if not immobilised it is hard to extract enzyme from the product, proudct would be contaminated
   1. exam q

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      •  The student expressed concerns that live yeast cells may be present in the product and that these cells would affect the results of the experiment. Explain whether or not you agree with these concerns.  
      • agree not all yeast cells successfully entrapped / AW ; (in product) yeast cells, respiring / metabolising / using sugar as anenergy source ; (so) lower levels of sugar (in product) ; not agree yeast cells, entrapped (in beads) / immobilised, so product not contaminated/ yeast not present to affect product ;yeast cells unable to pass through, glass wool / filter ;only very low numbers of yeast cells (so unlikely to have great effect) 
2. Cheap
3. Reusable

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   • keep and re use the enzymes and product is enzyme free
4. High Temps & PH

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   • immobilised enzymes are more tolerant to high temps and ph changes because they are held firmly in place in the alginate so do not denature easily 
5. X3 EXAM Q

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   •  Suggest three practical advantages of using an immobilised urease bioreactor in a spaceship  
   •  urine can be processed / no problem of removing urine / AW;pure / drinkable / useable, water produced; A water recycled space saving / less water needs to be taken into space;payload limit / weight reduction / AW;no problem in separating enzyme from products / product not contaminated;ref. to longer shelf-life of enzyme;no need to take more enzymes into space / enzymes reusable; A enzymes recoverable AVP; e.g. larger surface area of enzyme exposed, more stable at extremes, ref. to ease of use (of bioreactor)  
   • DEFINE HYDROLSIS  adding water to break bonds
   •  An investigation was carried out to compare lipase in soluble and immobilised forms. Palm oil was hydrolysed to produce fatty acids and glycerol. • The two forms of lipase showed optimal activity at the same pH and temperature(pH 7.5 and 35°C). • At that pH and temperature, 100% of the oil was hydrolysed in two minutes. • If the temperature was increased to 45°C, the immobilised enzyme hydrolysed 100% of the oil but the soluble enzyme hydrolysed only 80% of the oil in two minutes.  
   •   matrix, protects / stabilises, enzyme / lipase; functions, at optimal rate / more efficiently, at higher temperature / 45 °C; A greater activity / AWref. to soluble lipase begins to denature (reducing activity); ora functions, at optimal rate / more efficiently, at lower pH;ref. to presence of fatty acids changing pH;ref. to ionic bonds breaking (in soluble lipase); ora AVP; e.g. ref to industrial uses ref to effect on R groups  

1. Continouse
   1. add nutrients constantly

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      • steady input and steady output, centrifuge to remove product from solution is an open system
   2. Maintained in Log phase
   3. Mycoprotein

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      • means fungus protein, culture medium contains glucose used a respiratory substate, which is obtained from starch that has been hydrolysed by enzymes this provides the growing fungus with a source of energy and carbon that can be used to make proteins and fat molecules. ammino phosphate is addedas  ntrogen source = so fungus can make protein and nucletides ammonia gas may be bubbles & zinc/copper. no stirrer used as would break the fungal hyphea
   4. Monitored

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      • oxygen, ph and temp constantly monitored.= optimum growing condtions. 
   5. High Yeild

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      • Kept in max rate of reproduction so creates a lot of product
   6. Advatages

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      • needs less space then batch to make more yield
   7. Disadvatages

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      • contamination, if conttaminated large amounts have to be thrown away , cells can often clump together and block inlet and outlet pipes
   8. exam q

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      •  Explain how the apparatus shown in the figure above allows a continuous culture of Chlorella  
      •  1 air pressure will push the medium into the culture vessel; 2 medium / nutrients, added to the culture at a constant rate / AW; 3 algae / cells / Chlorella, removed / harvested, from the sample port 4 at the same rate as / to match, the nutrients added; 5 so volume in fermenter remains constant; 6 removal of, waste / toxic products; 7 that could affect, growth / reproduction; 8 (cells kept in) exponential / log / rapid / main, growth phase;9 algae are photosynthetic; 10 light energy required;11 ref to use of fluorescent light to avoid overheating;12 refto monitoring temperature;13 ref to optimum conditions; A ‘conditions for maximum growth’14 air bubbles to mix culture with nutrients / AW;15 air bubbles to allow algae to get sufficient light;16 air bubbles provide oxygen for (aerobic) respiration;17 and CO2 forphotosynthesis;18 air flowing into the culture vessel flows out through an outflow tube;19 preventing build-up of pressure;20 AVP; e.g. sampling to check for mass of Chlorella 
      1. exam q

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         • (c) Describe the major problems of developing this project to enable the large-scale production of Chlorella. (continouse culutre)  
         • difficulty controlling ph/temp, heating/cooling, foaming, blocking of inlet, difficulties mixing, contamination, constant monitoring of condtions, nutrient requirments may change 
2. Batch

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   • where microorganisms are grown in individual batches
   1. Batch Fed

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      • carb source added regularly = keep ferm process going for longer and can produce more penecillin then standard batch culture
      1. exam q

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         •  Suggest why limited amounts of glucose are added at regular intervals to the culture medium during the fed-batch process  
         •  to provide respiratory substrate / energy ; A for respiration to maintain culture / keep culture alive / prevent (premature) death ofculture ; (limited) maintains in stationary phase / prevents rapid growth  AVP;  
   2. Goes through Lag, Log & maintained in Stat
   3. Ferm stopped at Stat
   4. Fixed Vol

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      • set up and left to proceed, nothing is added or taken away, waste gases are allowed to escape it is a close culture
   5. Penecillin

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      • fungus grown in fermenter until max amount of product (penecillin) is made then fermenter stopped and product harvested. cleaned out and new culture begins 
      1. x2 exam q

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         •  The fungus that produces penicillin needs a supply of carbon and nitrogen. Give the form in which these elements are added to the culture.  
         • carbon – glucose / lactose; nitrogen – amino acids / nitrate ions / ammonium ions / yeast extract;  
         •  production of penicillin and the growth of the fungus, Penicillium, in the fermenter  Explain why there is no antibiotic produced during phase lag and log   
         •  X includes, rapid / exponential / main, growth phase; ora when primary products are made / penicillin is a secondarymetabolic product; excess of nutrients in X or penicillin produced when nutrients,limited / depleted;  
      2. down stream processing

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         •   Penicillin is removed from the fermenter for downstream processing. Describe what happens during downstream processing.  
         •   filter (to remove fungus) fungus  washed (to remove penicillin);continuous countercurrent / chemical extraction;concentration;addition of potassium ions;precipitate crystals / (potassium) salts;solvents used to purify penicillin;AVP; e.g. dried, some are chemically modified, 99.5% pure 
   6. Product yeild is low

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      • no product is being made whilst you are cleaning and resetting up fermenter 
   7. Advantages

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      • in contaminated not large amounts have to be chucked, easy to set up, fermenter can be used for multiple purposes, no blocking of inlet and outlets
   8. Disadvantages

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      • smaller yeild, more labour intensive

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• substance made by the cell through the course of its metabolism

1. Primary CONTINU

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   • made by most of  the cells in the culture most of the time and is involved in normal metabiolic process = necessary for cell growth e.g. respiration  grows best in favourable ondtions
   1. Favourable Condtions
   2. Vital for growth & resp
2. Secondary BATCH

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   • only produced by some cells or at a particular growth phase does not have any involvment with metabolic processes  grows best in unfavourable growing condtions so produces at max rate on a short supply of nutrients
   1. Unfavorable condtions
   2. Not vital for cell growth
   3. Batch Culture

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      • only made at one stage = need new fermnters (pencillin)
   4. exam q

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      •  Explain why a continuous culture method would not be suitable for the manufacture of penicillin.  secondary metabolite ; produced at start of / during stationary phase / end of growth phase ; A logphase ref to production (at maximum) when kept short of nutrients/ nutrients depleting / factors limiting growth ; continuous culture maintains in, log / rapid growth, phase ; 
   5. exam q

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      •  State which method of fermentation would be used to produce a secondary metabolite and explain your answer.  
      • batch / fed batch; 1 nutrients only added at start; short / rapid, growth phase; required product made, during stationary phase / late in lifecycle; ora  R death phase  shortage / depletion of, nutrients / named nutrients;cell division / reproduction, no longer occurring;ref to addition of, glucose / lactose, at intervals(to avoid death of culture); 

1. pH

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   • kept t optimum so enzymes can work efficently so rate of reaction is kept as high as possible 
   1. exam q

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      • why ph is monitored and how it is controlled 
      •  will affect, enzyme action / metabolic rate; A denature enzymes addition of, buffer / acid / alkali / base;  
2. Temp

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   • water jacket = keeps temp as optimum as heat can rise due to it being a waste product of resp. water jacket keeps cool so enzymes can function
   1. exam q

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      •  Explain why it is necessary to pump water into the jacket surrounding the culture.  
      • water is for, cooling / removing excess  heat;maintains, constant / optimum, temperature; respiration produces heat; which would, denature enzymes / kill cells; heat also produced by, stirrer / motor;  
3. Sterile

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   • superheated steam = no competion with other organisms 
4. Paddles

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   • means the microganisms can always access nutrients required for growth. do not settle at the bottom of fermenter
5. Oxygen

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   • need to respiration= energy = growth. sterile air pumped in 
   1. exam q

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      •  Explain why sterile air is pumped into the fermenter.  
      • provides oxygen for aerobic  respiration; any detail, e.g. oxidative phosphorylation;sterile to prevent contamination;mixes fungus with substrate / prevents settling / bubbles help stirring / AW; 

Annotations:

• asepsis means with out microrganisms other microorgansims may metabolise substrate in a different way = unwanted products, expecially importat when the product will bee food/medicine. 

1. workers

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   • workers must wear clean lab coats as microorgs could be on their skin hair up and gloves on 
2. air pressure

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   • mintaine small pressure difference between air inside room containg the fermenter and air outside room so that air flows from inside to outside, 
3. fermenter

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   • make sure fermenter is sterilised before anything aded to it  using super heated steam necks of culture containers heated to kill any unwanted orgs 
4. inputs

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   • make sure all liquid, solid and gases entering fermeneter are steile,  instruments also sterilised
5. exam q

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   • what is meant by the term pathogen; a disease causing organsim

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• what is biotechnology the commercial use of living organisms to produce food/drugs
• involved in making food; to make beer, microbe used is yeast 
• to make drugs pencillium to make pencilin