Microbiology and Biotechnology

Lessons

Senior 4 · Biology

Microbiology and Biotechnology

5 lessons

Lesson 5 of 5

Biotechnology: enzymes, fermenters, antibiotics and biogas

25 min

Cooking with cow dung

Since 2007 Rwanda has run a National Domestic Biogas Programme to help households, schools and prisons cook with gas made from dung and other wastes instead of firewood and charcoal. According to Rwanda Energy Group (REG), about 10 200 household digesters and 86 large institutional digesters in secondary schools and prisons have been built, and the potential market is about 150 000 households. Inside each digester, no person does any work: communities of bacteria and archaea turn waste into a fuel and a fertiliser.

Biogas is one example of biotechnology, the use of living organisms or their enzymes to make useful products. This lesson looks at four: enzymes used outside cells, fermenters, antibiotics and biogas.

Definition

Biotechnology: the use of living organisms, cells or their enzymes to make products or carry out processes useful to people.

Immobilised enzyme: an enzyme fixed to, or trapped inside, an insoluble material (such as calcium alginate beads), so that it can be separated from the product and reused.

Fermenter (bioreactor): a vessel in which microorganisms are grown under controlled conditions to make a product. In industry, "fermentation" means any such large-scale growth of microbes, aerobic or anaerobic.

Primary metabolite: a substance made during normal growth (amino acids, ethanol). Secondary metabolite: a substance not needed for growth, made mainly in the stationary phase (most antibiotics).

Antibiotic: a substance made by a microorganism (or a modified version of it) that kills or inhibits the growth of bacteria.

Biogas: a fuel gas, mainly methane and carbon dioxide, produced by the anaerobic digestion of organic matter.

Enzyme technology and immobilised enzymes

Enzymes extracted from microbes work outside cells as catalysts in industry and at home:

Proteases and lipases in biological washing powders digest protein and fat stains at low temperatures.
Pectinases break down pectin in fruit cell walls, so more juice is pressed out and it is clearer.
Amylases break starch into sugars in brewing, baking and syrup making.
Lactase breaks lactose into glucose and galactose to make lactose-free milk for people who cannot digest lactose.
Chymosin (rennet) made by genetically modified microbes clots milk in cheese making.

Enzyme in solution is lost in the product after one use. Immobilising it solves this. Methods include adsorption onto a surface (glass, clay), covalent bonding to a support, entrapment in a gel (calcium alginate beads) and membrane enclosure. A common school method: mix the enzyme with sodium alginate solution and drip it into calcium chloride solution, where each drop sets into a jelly bead holding the enzyme. Packed into a column, the beads convert substrate poured in at the top into product collected at the bottom.

Advantages of immobilised enzymes	Disadvantages
Reused many times, so cheaper over time	More expensive to set up
Product is not contaminated with enzyme, so less purification	Slightly slower: substrate must diffuse into the bead to reach the enzyme
Often more stable to changes of temperature and pH	Some activity is lost when the enzyme is fixed
Allows continuous production in a column	A contaminating microbe can block or spoil a column

Worked example 1: is immobilisation worth it?

A dairy makes lactose-free milk (illustrative costs). Free lactase costs 12 000 Frw per batch and is lost in the milk. The same amount of lactase immobilised in alginate beads costs 180 000 Frw and can be used for 30 batches. (a) Calculate the enzyme cost per batch with immobilised lactase. (b) Calculate the percentage saving. (c) Give one further advantage and one disadvantage.

(a) $\frac{180\,000\ \text{Frw}}{30} = 6000\ \text{Frw}$ per batch.
(b) $\frac{12\,000 - 6000}{12\,000} \times 100\,\% = 50\,\%$ saving.
(c) Advantage: the milk contains no enzyme, so it needs no extra purification. Disadvantage: the reaction is slower because lactose must diffuse into the beads, so the milk must flow through the column more slowly, or the column must be longer.

Fermenters

To make a product on a large scale, microbes are grown in fermenters holding up to hundreds of thousands of litres. Every condition from Lesson 3 is controlled automatically:

Everything is sterilised with steam before use, and air and nutrients enter through sterile filters, so that no contaminating microbe competes with the culture or spoils the product.
Temperature: respiration releases heat, so cold water in the jacket removes it and keeps the optimum temperature for the microbe's enzymes.
pH: wastes change the pH; the probe triggers additions of acid or alkali.
Oxygen and mixing: for aerobic microbes, sterile air is bubbled in and the impellers break bubbles up and keep cells, nutrients and heat evenly spread.

Batch culture	Continuous culture
Nutrients added once; the culture runs through the growth curve; the vessel is emptied, cleaned and restarted	Fresh nutrients added and culture removed at the same steady rate, keeping cells in the log phase
Suits secondary metabolites made in the stationary phase (penicillin)	Suits primary metabolites and cell products made during growth (single-cell protein, some enzymes)
Contamination affects only one batch	Higher output, but contamination can ruin a long run

Penicillin and other antibiotics

In 1928 Alexander Fleming noticed that a contaminating Penicillium mould had killed the staphylococci around it on an old culture plate. In the early 1940s Howard Florey and Ernst Chain worked out how to purify penicillin and produce it in quantity, and showed that it cured infections. Penicillin stops bacteria building their peptidoglycan cell walls, so growing cells burst; it does not harm human cells, which have no such wall.

Penicillin is made today by high-yielding strains of Penicillium chrysogenum in aerated, stirred fermenters at about 25 °C, as a fed-batch culture: after the mould has grown, sugar is fed in slowly, keeping the fungus alive but short of food, because penicillin is a secondary metabolite made mainly when growth slows. After several days the mould is filtered off and the penicillin is extracted from the liquid, purified and crystallised (downstream processing). Many modern penicillins are made by chemically modifying this natural product. Other antibiotics come from soil bacteria: in the 1940s Selman Waksman's team found streptomycin, made by Streptomyces, the first drug effective against tuberculosis.

Worked example 2: when is penicillin made?

In a batch fermenter (illustrative data), the mass of mould rose quickly from 0 to 40 hours and then levelled off at about 30 g per dm³. Penicillin was hardly detectable before 40 hours, then rose steadily to 160 hours. (a) Explain the timing of penicillin production. (b) Suggest why the manufacturer keeps the culture short of sugar after 40 hours.

(a) Penicillin is a secondary metabolite, not needed for growth, so it is made mainly after the log phase, when growth has slowed. In the wild it may help the mould by killing competing bacteria when food is scarce.
(b) Plenty of sugar would make the mould keep growing and making biomass rather than penicillin (high sugar also switches off the genes for penicillin synthesis). Slow feeding keeps the cells alive and productive but not growing fast: this is fed-batch culture.

Biogas

In a sealed digester with no oxygen, a community of microbes breaks down organic matter in four overlapping stages:

Hydrolysis: bacteria secrete enzymes that break carbohydrates, proteins and fats into sugars, amino acids and fatty acids.
Acidogenesis: fermenting bacteria turn these into short-chain organic acids, alcohols, hydrogen and carbon dioxide.
Acetogenesis: other bacteria convert the acids and alcohols into ethanoic acid (acetic acid), hydrogen and carbon dioxide.
Methanogenesis: methanogenic archaea (Lesson 1) produce methane: $\mathrm{CH_3COOH \rightarrow CH_4 + CO_2} \qquad \mathrm{CO_2 + 4H_2 \rightarrow CH_4 + 2H_2O}$

Biogas is typically 50–80 % methane and 15–50 % carbon dioxide, with traces of hydrogen sulfide. Digesters work best without oxygen, at about 30–40 °C, at a pH near neutral, and with a steady daily feed of dung mixed with water. The digested slurry left behind is a valuable fertiliser, and because the waste is broken down, flies and smells are reduced.

Worked example 3: energy from a household digester

A household digester fed with the dung of two cows produces 1.2 m³ of biogas a day containing 60 % methane (illustrative). Burning 1 m³ of methane releases about 36 MJ, and 1 kg of charcoal about 29 MJ. (a) Calculate the energy available each day. (b) How much charcoal would release the same energy? (c) Give two further benefits of the digester.

(a) Volume of methane: $1.2 \times 0.60 = 0.72\ \text{m}^3$ . Energy: $0.72 \times 36 = 25.9\ \text{MJ}$ per day.
(b) $\frac{25.9\ \text{MJ}}{29\ \text{MJ kg}^{-1}} = 0.89\ \text{kg}$ of charcoal per day, or about 325 kg a year, saved from being made from trees. (A gas stove also wastes less heat than a charcoal stove, so the real saving is larger.)
(c) The slurry is a fertiliser; no smoke in the kitchen (fewer lung infections); less time spent collecting firewood; dung no longer attracts flies; less deforestation.

FuseSchool: "Penicillin". Watch for how Fleming noticed the effect of the mould, and how penicillin is produced on a large scale today.

Warning

Safety and common mistakes

Making alginate beads: wear eye protection, since calcium chloride solution irritates the eyes; wipe up spills, which make floors slippery.
Biogas is flammable and its hydrogen sulfide is poisonous: keep flames away from leaking pipes, never enter a digester or its pit, and let only trained masons repair one.
Saying penicillin kills viruses. It acts on bacterial cell walls; viruses have no cell wall, so antibiotics do not cure colds or influenza.
Thinking industrial "fermentation" always means anaerobic respiration. Penicillin production is strongly aerobic.
Saying bacteria make the methane in biogas. The final step is carried out by methanogenic archaea.

Tip

Memory hook. A fermenter controls "S-T-O-P-M": Sterility, Temperature, Oxygen, pH, Mixing. Biogas stages: "Hungry Ants Ate Mangoes": Hydrolysis, Acidogenesis, Acetogenesis, Methanogenesis.

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Practice: biotechnology

Describe how to immobilise an enzyme in alginate beads, and explain one advantage of using immobilised enzymes.
Name four features of an industrial fermenter and explain the purpose of each.
Distinguish between primary and secondary metabolites, with an example of each.
Explain why penicillin is made by fed-batch rather than continuous culture.
Explain how penicillin kills bacteria and why it does not harm human cells.
Describe the four stages of anaerobic digestion in a biogas plant.
A school digester makes 8.0 m³ of biogas a day containing 55 % methane. Using 36 MJ per m³ of methane, calculate the energy available per day.
Evaluate the use of household biogas digesters in rural Rwanda, giving two benefits and two problems.

Show answersHide answers

Mix the enzyme with sodium alginate solution and drip it from a syringe into calcium chloride solution; each drop sets into a calcium alginate bead with the enzyme trapped inside; rinse the beads. Advantages (any one): reusable; the product is free of enzyme; more stable to temperature and pH; allows continuous production.
Any four: water jacket (removes heat from respiration to keep the optimum temperature); pH probe with acid/alkali inlets (keeps the optimum pH); sterile air inlet and sparger (supplies oxygen for aerobic respiration); impellers/stirrer (mix cells, nutrients and heat evenly, break up bubbles); steam sterilisation and sterile filters (prevent contamination); temperature and oxygen probes (monitor conditions); harvest valve (removes product).
Primary metabolites are made during normal growth and are needed for it, for example amino acids or ethanol. Secondary metabolites are not needed for growth and are made mainly in the stationary phase, for example penicillin.
Penicillin is a secondary metabolite made mainly when growth slows. Continuous culture keeps the cells in the log phase, growing fast and making little penicillin. In fed-batch culture the mould grows first, then is fed slowly so it survives without fast growth and keeps making penicillin.
Penicillin blocks the enzymes that build cross-links in peptidoglycan, so growing bacteria cannot make strong cell walls and burst by osmosis. Human cells have no cell wall and no peptidoglycan, so they are not affected (though some people are allergic to penicillin).
Hydrolysis: enzymes break large molecules into sugars, amino acids and fatty acids. Acidogenesis: bacteria ferment these to organic acids, alcohols, H₂ and CO₂. Acetogenesis: these are converted to ethanoic acid, H₂ and CO₂. Methanogenesis: archaea convert ethanoic acid, or CO₂ and H₂, into methane.
Methane: $8.0 \times 0.55 = 4.4\ \text{m}^3$ ; energy: $4.4 \times 36 = 158\ \text{MJ}$ per day (to 3 significant figures).
Benefits: clean cooking fuel with no smoke in the kitchen; less firewood and charcoal, so less deforestation and time saved; slurry is a good fertiliser; better sanitation. Problems: building cost (a subsidy is needed); a household needs enough cattle and water to feed it every day; poor construction or maintenance can make plants fail; gas output falls in cold highland areas. Overall, digesters are worthwhile where these conditions are met and owners are trained and supported.

Quick check

Match each part of a fermenter to its job.

Tap one on the left, then its partner on the right.

Key points

Enzymes from microbes are used in washing powders, juice, baking, dairy and cheese making; immobilised in beads or on supports, they can be reused, leave the product pure and allow continuous processing.
Fermenters control sterility, temperature, oxygen, pH and mixing. Batch culture suits secondary metabolites; continuous culture suits primary products.
Penicillin (Fleming 1928; Florey and Chain 1940s) is a secondary metabolite of Penicillium, made by aerobic fed-batch culture; it blocks bacterial wall synthesis. Streptomycin comes from Streptomyces.
Biogas: hydrolysis, acidogenesis, acetogenesis, then methanogenesis by archaea; 50–80 % methane; Rwanda's programme has built about 10 200 household and 86 institutional digesters (REG).

1Cooking with cow dung

2Enzyme technology and immobilised enzymes

3Fermenters

4Penicillin and other antibiotics

5Biogas

Cooking with cow dung

Enzyme technology and immobilised enzymes

Fermenters

Penicillin and other antibiotics

Biogas