Microbiology and Organisms in Industry Essential Idea: Microorganisms can be used and modified to perform industrial processes. 1

TOK Alexander Fleming discovered penicillin in England in 1928 on a discarded petri dish. To what extent was Dr. Fleming’s discovery a lucky observation, or do we only perceive what we are open to? 2

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Metabolic Diversity of Microbes Microorganisms have a very diverse set of metabolisms. Photoautotrophs Chemoautotrophs Photoheterotrophs Chemoheterotrophs 4

Metabolic Diversity of Microbes Microbes also show diversity in the way they use oxygen. Aerobes Anaerobes Facultative Obligate 5

Metabolic Diversity of Microbes Photoautotrophs are photosynthetic microorganisms. They use light as an energy source and inorganic carbon (CO2) as their carbon source. 6

Metabolic Diversity of Microbes Photoheterotrophs use light as their energy source and any of a variety of organic compounds as a carbon source, such as: Carbohydrates Lipids Amino acids http://textbookofbacteriology.net/procaryotes_2.html Purple, non-sulfur Heliobacteria 7

Metabolic Diversity of Microbes Chemotrophs obtain energy by oxidizing electron donors from within the environment. These donors can be organic or inorganic molecules that become oxidized when they deliver their electrons. 8

Metabolic Diversity of Microbes These electrons then flow through various transport chains and synthesize compounds needed for survival. 9

Metabolic Diversity of Microbes Most chemotrophs are bacteria that live in hostile environments such as deep sea hydrothermal vents. http://www.photolib.noaa.gov/htmls/nur04506.htm 10

Metabolic Diversity of Microbes Chemoautotrophs use inorganic chemicals as their source of energy and CO2 as their carbon source. They derive their energy from the redox reactions that transfer electrons to the various electron transport chains within these organisms. 11

Metabolic Diversity of Microbes Chemoautotrophs use the energy from inorganic compounds involving sulfur, iron, manganese or nitrogen to provide electrons for redox reactions. They also use CO2 to synthesize organic compounds they need for survival. 12

Most chemoautotrophs live in hostile environments. 13

Metabolic Diversity of Microbes Chemoheterotrophs use organic compounds as an energy source and as a carbon source. The oxidation-reduction reactions of the organic compounds provide both the energy and building blocks needed for survival. 14 http://classes.midlandstech.edu/carterp/Courses/bio225/chap12/lecture1.htm

Common Microbes: Summary 15

Microorganisms in Industry Microorganisms are used in industry because they are diverse, small, and have a very fast growth rate. They are used industrially to produce metabolites of interest. Citric acid Insulin Penicillin 16

Pathway Engineering Pathway engineering is used in industry to optimize genetic and regulatory processes within microorganisms to maximize the production of metabolites of interest. 17

Pathway Engineering Pathway engineering seeks to redesign microorganisms (bacteria and fungi) to efficiently produce metabolites of interest. 18

Fermenters (Bioreactors) Once organisms of interest have been identified or redesigned, the process of culturing them has to be worked out. Largely due to the microorganisms becoming limited by their own waste products. Fermenters (bioreactors) are used to allow for large-scale production of these metabolites. 19

Fermenters (Bioreactors) People using fermenters have to monitor the conditions within them using various probes. These probes measure things like pH, temperature, salinity, waste build-up, etc. Optimal conditions need to be maintained to ensure optimal growth and maximal production of product. 20

Optimal Conditions Most microorganisms grow optimally between a pH of 6 and 8, and a temperature of 20-40°C. Extremophiles grow in conditions much different from these. 21

Batch or Continuous Culture Fermentation/growth can be carried out by Batch Culture or Continuous Culture. Batch Culture is where microorganisms are grown within a nutrient medium in a closed fermenter (bioreactor). At the end of the growth cycle, the desired product is separated out. 22

Batch or Continuous Culture In Continuous Culture, a sterile, nutrient solution is continuously added to the fermenter at the same time as an equivalent amount of nutrient solution with microorganism is removed. Steady growth is maintained by ensuring enough nutrient medium is added to maintain a maximum growth rate. This ensures maximum yield of product. 23

Deep-Tank Batch Fermentation Deep tank fermentation isn’t really fermentation. Yeast and bacteria ferment. Aspergillus and Penicillium are fungi, and like nearly all funguses, they require oxygen. Many of these reactions produce gas as a biproduct, and this biogas must be bled off to prevent explosions. Some gases can be used as an energy source (CH4). 24

Biogas Biogas is generally a mixture of CH4 and CO2, and may have some H2S. Thus, biogas is often used as a fuel. http://highacreslandfill.wm.com/facility/landfill-gas.jsp 25

Typical Composition of Biogas Compound Molecular Formula Percentage Methane CH4 50-75% Carbon Dioxide CO2 25-50% Nitrogen N2 0-10% Hydrogen H2 0-1% Hydrogen Sulfide H2S 0-3% Oxygen O2 0% Biogases are produced by the anaerobic breakdown of organic matter. Common sources of biogas are farming and land fills. 26 http://www.sierrainstruments.com/blog/?biogas-flow-meter-measurementthe-problem

Biogas Capturing biogas is beneficial to both society and to the environment. Methane and other compounds are 20- 300 times more potent greenhouse gases than CO2. Capturing the gas for sale allows it to be used for generating electricity, heat, and powering vehicles. It is a very important renewable energy. 27

Biogas Biogas can also be produced using fermenters. Using anaerobic digesters, biodegradable material can be fed into the tanks. Microorganisms (bacteria and archeans) transform the waste into biogas and the gases are collected. 28 http://eng3060.pbworks.com/w/page/18918910/Insert%20a%20landfill%20gas%20extraction%20system

29 http://en.wikipedia.org/wiki/Anaerobic_digestion#/media/File:Anaerobic_digesters_overhead_view.jpg

http://en.wikipedia.org/wiki/Anaerobic_digestion#/media/File:Haase_Lubeck_MBT.JPG

Biogas Leftover digestate can be spread on fields to condition the soil, providing nutrients and moisture holding properties. http://en.wikipedia.org/wiki/Digestate#/media/File:Anaerobic_digestate.JPG 31

Deep-Tank Batch Fermentation Deep tank fermentation is performed in a bioreactor. These can be HUGE! 40K liter! They started off as small, one-liter tanks that were modified and then were scaled up. Just like with individual cells, tanks need to take surface area to volume into account. 32

Deep-Tank Batch Fermentation These tanks are deep and include a lot of fluid. The fluid is continuously mixed at all levels within the tank and aerated with sterile air to prevent contamination--one of the main obstacles in deep-tank fermentation. http://en.wikipedia.org/wiki/Bioreactor#/media/File:Bioreactor_principle.svg 33

Deep-Tank Batch Fermentation Here’s an example of a small-scale bioreactor. http://en.wikipedia.org/wiki/Bioreactor#/media/File:Autoclavable_bench-top_laboratory_bioreactor_%26_fermenter,_Lambda_MINIFOR.jpg 34

Aspergillus niger A. niger is used to produce citric acid. Citric acid is used as a preservative and flavoring in the food industry. It is also used as an acid to descale coffee machines! 35

Aspergillus niger The production of citric acid by A. niger is achieved in a continuous fermenter. In this process, the growth of A. niger is held in the stationary phase (as the citric acid cycle produces the desired product) and while nutrients are added and an equivalent amount of liquid and micoorganisms are taken out. 36

Citric Acid As the liquid is draw out of the fermenter, the A. niger is filtered out and the citric acid is precipitated with Ca(OH)2 to yield calcium citrate salt. Treatment of this salt with sulfuric acid yields citric acid. 37

Penicillium chrysogenum/notatum Penicillin production has a very storied history. A lot of time elapsed from when Alexander Flemming discovered its antibiotic properties to when it was produced on an industrial scale. It was used exclusively during wartime (WWII) before being used publicly. 38

http://www. nationalww2museum http://www.nationalww2museum.org/learn/education/for-teachers/lesson-plans/pdfs/penicillin-fact-sheet.pdf

Penicillium chrysogenum/notatum P. chrysogenum is the fungus used to produce mass quantities of penicillin. Like A. niger, the production of penicillin makes use of a continuous fermenter. 40

Penicillium chrysogenum/notatum During the production of the penicillin, P. chrysogenum/notatum is cultured with sugar, nutrients and a nitrogen source. Penicillin is a secondary metabolite, and is usually produced when the organisms enter the stationary phase of growth. Secondary metabolites help organisms, but are not immediately necessary for growth. 41

Penicillium chrysogenum/notatum Here you can see the stationary phase of growth as well as the production of secondary metabolite. http://nopr.niscair.res.in/bitstream/123456789/4242/1/IJMS%2038(1)%2038-44.pdf 42

Penicillium chrysogenum/notatum As the culture is in the stationary phase, waste is removed and nutrients are added. The desired product is also removed and purified using a variety of techniques. The product is then ready for use. 43