Industrial biotechnology and fermentation

Why industry cultures microorganisms

Microorganisms are used in industrial biotechnology because they grow rapidly, can be cultured at high density, use relatively simple feedstocks and can be selected or genetically modified for a high product yield. Production in a fermenter allows temperature, pH, aeration, mixing, nutrients and contamination risk to be monitored and controlled. It is less dependent on season than agriculture, although energy, water, feedstock and infrastructure still affect cost and performance.

Generation time accounts for the rate. A cow doubles its numbers in years, a fungus in hours, and a bacterium such as E. coli, given warmth and nutrients, in about twenty minutes.

The remaining reasons are distinct from speed and from each other. Microorganisms use low-cost substrates: many grow on by-products of other industries, whey from cheese-making, molasses from sugar refining, straw and paper pulp, which are cheaper than purpose-grown feedstocks and in some cases carry a disposal cost for the producer. Culture conditions are set inside the vessel rather than by the weather, so production does not depend on season, though it does depend on local energy, water and infrastructure. The organisms raise no animal-welfare objections of the kind livestock farming does. And their genomes are small and readily altered, so a strain can be improved by selection over a few weeks, or transformed with a human gene, which is how recombinant insulin is manufactured at scale.

Biotechnology

The use of living organisms, usually microorganisms, or of their components such as enzymes, in industrial processes that make useful products.

Fermentation (industrial)

The large-scale culture of microorganisms in a fermenter to make a product. The industrial word covers aerobic processes as well as the anaerobic pathway the respiration unit gave the same name.

The word 'fermentation' has two meanings. In respiration, fermentation is the anaerobic regeneration of NAD. In industry, a fermentation is any large-scale culture with a product at the end, and most of them, penicillin and mycoprotein included, are aerated. The industrial term identifies a vessel of microorganisms and does not indicate whether oxygen is involved.

The products span every unit of this course: yoghurt, cheese and bread from traditional fermentations; ethanol for fuel as well as for drink; enzymes such as the proteases in washing powder and the lactase and glucose isomerase you met immobilised; antibiotics; and human proteins from transformed cells. Two of those categories, food and medicines, are treated in the sections below.

Microorganisms as human food

Most industrial biotechnology sells a product made by a microorganism. The microorganism itself is also sold, dried and flavoured, as protein-rich food, called single-cell protein whether or not the producer organism is single-celled.

The example on British shelves is mycoprotein: the hyphae of the fungus Fusarium venenatum, grown continuously on glucose syrup with ammonia as the nitrogen source, harvested, heat-treated and pressed into a food that has been sold since the 1980s. The fibrous hyphae matter as much as the protein: they give a texture close enough to muscle for the product to stand in for meat, which yeast or bacterial cells, being small and round, cannot do.

Single-cell protein

Protein-rich food produced from cultured microorganisms, eaten by humans or fed to livestock.

Mycoprotein

Single-cell protein made from the hyphae of the fungus Fusarium venenatum, grown in continuous culture and textured to resemble meat.

Set the argument out on both sides, and attach each point to a mechanism. 'It is unnatural' names no process; 'the nucleic acid content must be lowered because its breakdown product uric acid can cause gout' does.

The stirred-tank fermenter: a controlled environment

A stirred-tank fermenter is a stainless steel vessel, from bench size up to hundreds of thousands of litres, designed to hold a large volume of nutrient medium at the optimum conditions for one organism's enzymes while excluding other organisms.

Asepsis first. The vessel is sterilised with pressurised steam between runs, the medium is sterilised before it enters, and the air is passed through filters fine enough to strip bacteria and spores. The reason is competitive: a contaminating organism consumes the substrate, may out-grow the production strain, may degrade the product, and in a food or medicine makes the batch unsellable in any case. Aseptic technique at industrial scale uses the same principles as laboratory practice, with steam sterilisation in place of a flame.

The conditions controlled are those affecting enzyme rate. A dense culture respires rapidly and respiration releases heat, so an uncooled fermenter warms past its own optimum and the enzymes denature: water circulating through the cooling jacket carries that heat away, holding the culture at its optimum temperature. Metabolism also shifts pH, since carbon dioxide and organic acids accumulate, so a pH probe monitors the broth and alkali or acid is metered in to hold the optimum. Aerobic cultures consume oxygen faster than it dissolves, so sterile air is forced in through a sparger, a ring of fine holes near the bottom, and the impeller above it breaks the rising bubbles up and keeps organisms, nutrients, heat and oxygen evenly mixed, so no part of the tank becomes a stagnant corner running anaerobic. Nutrients are added, the exhaust gas escapes through a filter, and probes report continuously so that each variable is corrected as it departs from its set point, which is negative feedback.

Fermenter

A vessel in which microorganisms are cultured at large scale under controlled conditions of temperature, pH, oxygen and nutrient supply.

Aseptic conditions

Conditions from which unwanted microorganisms are excluded, achieved in a fermenter by steam-sterilising the vessel and medium and filtering the incoming air.

Batch and continuous fermentation

Prerequisite: the closed-culture growth curve, covered in populations and their limits. It has a lag phase while the population adjusts and synthesises the enzymes its new medium requires, an exponential (log) phase of doubling while nothing limits growth, a stationary phase when nutrients run short and wastes accumulate so deaths balance divisions, and a death phase when deaths exceed divisions. The two ways of running a fermenter operate at different points on that curve.

Batch fermentation is a closed culture. The vessel is filled with sterile medium, inoculated, sealed, and left to run through the curve; at the end, usually early in the stationary phase, the whole tank is harvested, emptied, sterilised and set up again. Nothing is added during the run except air and pH correction, so the culture visits every phase the growth curve has.

Continuous fermentation is an open one. In a chemostat, fresh medium enters and culture leaves at the same rate. A limiting nutrient and the dilution rate control growth, allowing a steady state to be maintained without passing through a complete batch growth curve. Nutrient supply and waste removal are balanced against consumption and production rather than being unlimited, and the population grows at a constant rate set by the dilution rate for weeks or months.

Which to choose turns on when the product is made, and the pair of terms that captures it is examined by name. A primary metabolite is made during normal growth, as part of it: ethanol, biomass itself, most enzymes. A secondary metabolite is made when growth slows, typically as the culture enters the stationary phase; penicillin is the standard example, an antibiotic that inhibits the mould's competitors under crowded, nutrient-limited conditions. A product made during growth suits a continuous process that maintains growth; a product made when growth slows is produced in batch, because a continuous culture in steady state does not reach the stationary phase.