Commercial biotechnology Learning objectives: To be able to explain the advantage of using microorganisms for industrial processes.To be able to explain the advantage of using microorganisms for industrial processes. To be able to describe the screening procedures carried out to identify the most suitable microorganisms for a particular process.To be able to describe the screening procedures carried out to identify the most suitable microorganisms for a particular process.

Microbes are particularly useful for industrial purposes because They have fast growth rates. They have fast growth rates. They have simple nutritional requirements and can often be fed on cheap or even waste substrates such as molasses, whey, wood pulp, etc. They have simple nutritional requirements and can often be fed on cheap or even waste substrates such as molasses, whey, wood pulp, etc. They can be grown indoors and their growth does not depend on seasons, climate, latitude, etc. They can be grown indoors and their growth does not depend on seasons, climate, latitude, etc. They are often tolerant of a wide range of temperatures and pH. They are often tolerant of a wide range of temperatures and pH. There are fewer ethical problems, when compared to animals. There are fewer ethical problems, when compared to animals. Prokaryotes can be more easily genetically modified than eukaryotic cells, since they don't have a nucleus. Genes from other species can easily be inserted into the bacterial DNA to produce a range of gene products by fermentation, or the microbe can be altered to produce far more of the product than normal. Prokaryotes can be more easily genetically modified than eukaryotic cells, since they don't have a nucleus. Genes from other species can easily be inserted into the bacterial DNA to produce a range of gene products by fermentation, or the microbe can be altered to produce far more of the product than normal.

Screening This is selecting a suitable species and strain of bacteria for a particular job.This is selecting a suitable species and strain of bacteria for a particular job. This involves testing every bacterium you can find to see if it makes the product you want in the right quantities and at the right speed.This involves testing every bacterium you can find to see if it makes the product you want in the right quantities and at the right speed. This can account for most of the cost of development as it can take years.This can account for most of the cost of development as it can take years.

Extracellular enzymes (ones that are secreted by the microorganism) have three main advantages over those that are intracellular: a. The enzyme is already outside the cell, so the cells do not need to be broken open. b. Only a limited number of enzymes are secreted, so isolation is much easier. c. Extracellular enzymes are less likely to be broken down by heat/chemicals (more robust).

Screening can be used to detect the presence of extracellular enzymes: a. Amylases are detected by plating on agar containing starch. After incubating, iodine is added, so that the microbes that break down the starch have clear zones around them.

b. Proteases are detected by plating on agar containing casein (makes the plates look cloudy). The microbes that break down the casein have clear zones surrounding them.

Antibiotic production can be detected by plating the microbes with a test organism such as Staphylococcus aureus. The presence of areas in which the test organism doesn’t grow indicates the presence of antibiotics.

Substances produced by microorganisms are classified into two groups: Primary metabolites – produced during the growth of the organism (normal growth products). These include amino acids, nucleotides, acids, ethanol and enzymes:Primary metabolites – produced during the growth of the organism (normal growth products). These include amino acids, nucleotides, acids, ethanol and enzymes: –i. Acetobacter sp. – production of ethanoic acid. –ii. Aspergillus niger – production of pectinases. –iii. Bacillus subtilis – production of proteases (tenderising meat).

Secondary metabolites – chemicals that are not directly involved in normal growth, e.g. antibiotics. These are produced after the main growth period is completed or under extreme or unusual conditionsSecondary metabolites – chemicals that are not directly involved in normal growth, e.g. antibiotics. These are produced after the main growth period is completed or under extreme or unusual conditions

Microorganisms make some enzymes continually, whilst others maybe made only when neededMicroorganisms make some enzymes continually, whilst others maybe made only when needed For example, some microorganisms may only make a particular antibiotic or enzymes in overcrowded conditions to kill competing microbes.For example, some microorganisms may only make a particular antibiotic or enzymes in overcrowded conditions to kill competing microbes. Primary and secondary metabolites must be harvested at different stages of cell growth, so it is important to be able to make a distinction between themPrimary and secondary metabolites must be harvested at different stages of cell growth, so it is important to be able to make a distinction between them

In order to maximise production of a particular metabolite, different methods are used:

The first method is Continuous Process: –the nutrients are continually inputted into the fermentation vessel, and the material is continually removed and processed. –This may be more economical, as it doesn’t have to be shut down on a regular basis. This method maximises primary metabolite production as the microbes are keep in the exponential phase.This method maximises primary metabolite production as the microbes are keep in the exponential phase.

Continuous Fermenters This takes place in an open or flow-through fermenter.This takes place in an open or flow-through fermenter. The culture is continuously run off to make the product, while nutrients are continuously added at the same rate.The culture is continuously run off to make the product, while nutrients are continuously added at the same rate. The rate is set to match the growth rate so the number of cells in the fermenter stays constant.The rate is set to match the growth rate so the number of cells in the fermenter stays constant. This is very efficient, since the microbesbare kept at their exponential growth phase.This is very efficient, since the microbesbare kept at their exponential growth phase.

Batch process – –raw materials and microbes are placed together in a container vessel. –The microbes are then allowed to grow to their maximum population size, then the fermenter is emptied and the products are extracted and purified. This method maximises secondary metabolite production as the microbes are allowed to enter the stationary phaseThis method maximises secondary metabolite production as the microbes are allowed to enter the stationary phase

Batch Fermenters The fermenter is sterilised using high pressure steam to kill any other microbes that may infect the batch.The fermenter is sterilised using high pressure steam to kill any other microbes that may infect the batch. In batch fermentation the cells show a typical growth curve pattern. If left long enough the nutrients will be used up and the cells will die.In batch fermentation the cells show a typical growth curve pattern. If left long enough the nutrients will be used up and the cells will die. This would be waste time and so products are drained off at the end of the stationary phase.This would be waste time and so products are drained off at the end of the stationary phase. Once the product has reached its maximum concentration, the fermentation is stopped and the medium is run off.Once the product has reached its maximum concentration, the fermentation is stopped and the medium is run off. The fermenter is sterilised and a new batch is started.The fermenter is sterilised and a new batch is started.

Advantages and disadvantages: Faster production rateFaster production rate Automated so cheaper to runAutomated so cheaper to run Less labour intensive due to automationLess labour intensive due to automation More continuity of productMore continuity of product Continuous supplyContinuous supply Can use smaller vesselCan use smaller vessel Difficult to get rightDifficult to get right Fermenter only suitable for one processFermenter only suitable for one process High initial cost of complex equipmentHigh initial cost of complex equipment Hard to achieve constant sterilityHard to achieve constant sterility If contaminated more is lost/wastedIf contaminated more is lost/wasted CONTINUOUS

Advantages and disadvantages: Simple to set upSimple to set up Only one batch lost if contaminatedOnly one batch lost if contaminated Can be sterilised between batchesCan be sterilised between batches For fed batch process nutrients can be added at intervals to prolong the stationary phaseFor fed batch process nutrients can be added at intervals to prolong the stationary phase Fermenters are versatile and can be used for many processesFermenters are versatile and can be used for many processes Time lost between batchesTime lost between batches Time to get going – lag phase – slow productionTime to get going – lag phase – slow production Need to maintain consistencyNeed to maintain consistency Need to be sterilisedNeed to be sterilised Nutrient wasted if not all used upNutrient wasted if not all used up Amount of product limited by initial amountAmount of product limited by initial amount Labour intensive due to start/stop cycleLabour intensive due to start/stop cycle Large vessels needed to be cost effectiveLarge vessels needed to be cost effective BATCH

Fermenter requirements: StrongStrong SmoothSmooth Non corrosiveNon corrosive Easily sterilised (metal)Easily sterilised (metal) Non reactiveNon reactive

Fermentation may be: Surface or submergedSurface or submerged Aerobic or anaerobicAerobic or anaerobic