Industrial Microbiology: An Introduction

Introduction to industrial microbiologyTraditional fermentation processes, such as thoseinvolved in the production of fermented dairy productsand alcoholic beverages, have been performed forthousands of years. However, it is less than 150 yearsago that the scientific basis of these processes was firstexamined. The birth of industrial microbiology largelybegan with the studies of Pasteur. In 1857 he finallydemonstrated beyond doubt that alcoholic fermentation in beer and wine production was the result ofmicrobial activity, rather than being a chemical process.Prior to this, Cagniard-Latour, Schwann and severalother notable scientists had connected yeast activitieswith fermentation processes, but they had largely beenignored. Pasteur also noted that certain organisms couldspoil beer and wine, and that some fermentations wereaerobic, whereas others were anaerobic. He went on todevise the process of pasteurization, a major contribution to food and beverage preservation, which was originally developed to preserve wine. In fact, many of theearly advances of both pure and applied microbiologywere through studies on beer brewing and wine making.Pasteur’s publications, Études sur le Vin (1866), Étudessur la Bière (1876) and others, were important catalystsfor the progress of industrial fermentation processes.Of the further advances that followed, none weremore important than the development of pure culturetechniques by Hansen at the Carlsberg Brewery inDenmark. Pure strain brewing was carried out here forthe first time in 1883, using a yeast isolated by Hansen,referred to as Carlsberg Yeast No. 1 (Saccharomycescarlsbergensis, now classified as a strain of Saccharomyces cerevisiae).During the early part of the 20th century, furtherprogress in this field was relatively slow. Around theturn of the century there had been major advancements in the large-scale treatment of sewage, enablingsignificant improvement of public health in urbancommunities. However, the first novel industrial-scalefermentation process to be introduced was theacetone–butanol fermentation, developed by Weiz-mann (1913–15) using the bacterium Clostridium acetobutylicum. In the early 1920s an industrial fermentation process was also introduced for the manufacture ofcitric acid, employing a filamentous fungus (mould),Aspergillus niger. Further innovations in fermentationtechnology were greatly accelerated in the 1940sthrough efforts to produce the antibiotic penicillin,stimulated by the vital need for this drug during WorldWar II. Not only did production rapidly move fromsmall-scale surface culture to large-scale submergedfermentations, but it led to great advances in both mediaand microbial strain development. The knowledge acquired had a great impact on the successful developmentof many other fermentation industries.More recent progress includes the ability to producemonoclonal antibodies for analytical, diagnostic, therapeutic and purification purposes, pioneered by Milsteinand Kohler in the early 1970s. However, many of thegreatest advances have followed the massive developments in genetic engineering (recombinant DNA technology) over the last 20 years. This technology has had,and will continue to have, a tremendous influence ontraditional, established and novel fermentation processes and products. It allows genes to be transferred fromone organism to another and allows new approaches tostrain improvement. The basis of gene transfer is the insertion of a specific gene sequence from a donor organism, via an expression vector, into a suitable host. Hostsfor expression vectors can be prokaryotes such asthe bacterium Escherichia coli; alternatively, wherepost-translational processing is required, as with somehuman proteins, a eukaryotic host is usually required,e.g. a yeast.A vast range of important products, many of whichwere formerly manufactured by chemical processes, arenow most economically produced by microbial fermentation and biotransformation processes. Microorganisms also provide valuable services. They have proved tobe particularly useful because of the ease of their masscultivation, speed of growth, use of cheap substrates1

2Introduction to industrial microbiologythat in many cases are wastes, and the diversity of potential products. In addition, their ability to readily undergo genetic manipulation has opened up almost limitlesspossibilities for new products and services from thefermentation industries.Successful development of a fermentation processrequires major contributions from a wide range ofother disciplines, particularly biochemistry, geneticsand molecular biology, chemistry, chemical and processengineering, and mathematics and computer technology. A typical operation involves both upstream processing (USP) and downstream processing (DSP) stages (Fig.i). The USP is associated with all factors and processesleading to and including the fermentation, and consistsof three main areas.1 The producer microorganism. Key factors relating tothis aspect are: the strategy for initially obtaining a suitable industrial microorganism, strain improvement toenhance productivity and yield, maintenance of strainpurity, preparation of a reliable inoculum and the continuing development of selected strains to improve theeconomic efficiency of the process. For example, theproduction of stable mutant strains that vastly overproduce the target compound is often essential.Some microbial products are primary metabolites,produced during active growth (the trophophase),which include amino acids, organic acids, vitamins andindustrial solvents such as alcohols and acetone. However, many of the most important industrial productsare secondary metabolites, which are not essential forgrowth, e.g. alkaloids and antibiotics. These com-Upstream processingFermentation raw materialsProduction microorganismFermentationDownstream processingProduct purificationEffluent wastesProductFig. i Outline of a fermentation process.pounds are produced in the stationary phase of a batchculture, after microbial biomass production has peaked(the idiophase).2 The fermentation medium. The selection of suitablecost-effective carbon and energy sources, and otheressential nutrients, along with overall media optimization are vital aspects of process development to ensuremaximization of yield and profit. In many instances,the basis of industrial media are waste products fromother industrial processes, notably sugar processingwastes, lignocellulosic wastes, cheese whey and cornsteep liquor.3 The fermentation. Industrial microorganisms arenormally cultivated under rigorously controlled conditions developed to optimize the growth of the organismor production of a target microbial product. The synthesis of microbial metabolites is usually tightly regulatedby the microbial cell. Consequently, in order to obtainhigh yields, the environmental conditions that triggerregulatory mechanisms, particularly repression andfeedback inhibition, must be avoided.Fermentations are performed in large fermentersoften with capacities of several thousand litres. Theserange from simple tanks, which may be stirred or unstirred, to complex integrated systems involving varyinglevels of computer control. The fermenter and associated pipework, etc., must be constructed of materials, usually stainless steel, that can be repeatedly sterilized andthat will not react adversely with the microorganismsor with the target products. The mode of fermenteroperation (batch, fed-batch or continuous systems), themethod of its aeration and agitation, where necessary,and the approach taken to process scale-up have majorinfluences on fermentation performance.Conventional DSP includes all unit processes thatfollow fermentation. They involve cell harvesting, celldisruption, product purification from cell extracts or thegrowth medium, and finishing steps. However, attemptsare now being made to integrate fermentation with DSPoperations, which often increases process productivity.Overall, DSP must employ rapid and efficient methodsfor the purification of the product, while maintainingit in a stable form. This is especially important whereproducts are unstable in the impure form or subject toundesirable modifications if not purified rapidly. Forsome products, especially enzymes, retention of theirbiological activity is vital. Finally, there must be safe andinexpensive disposal of all waste products generatedduring the process.

Introduction to industrial microbiology3Fermentation productsHealth-care productsThe overall economics of fermentation processes are influenced by the costs of raw materials and consumables,utilities, labour and maintenance, along with fixedcharges, working capital charges, factory overheads andoperating outlay. Fermentation products can be broadlydivided into two categories: high volume, low valueproducts or low volume, high value products. Examplesof the first category include most food and beveragefermentation products, whereas many fine chemicalsand pharmaceuticals are in the latter category.In terms of providing human benefit, antibiotics areprobably the most important compounds producedby industrial microorganisms. Most are secondarymetabolites synthesized by filamentous fungi and bacteria, particularly the actinomycetes. Well over 4000 antibiotics have now been isolated, but only about 50 areused regularly in antimicrobial chemotherapy. The bestknown and probably the most medically useful antibiotics are the b-lactams, penicillins and cephalosporins,along with aminoglycosides (e.g. streptomycin) and thetetracyclines. New antibiotics are still being sought asresistance to established antibiotics has become a majorproblem in recent years, through the misuse and overuseof these drugs.Other important pharmaceutical products derivedfrom microbial fermentation and/or biotransformations are alkaloids, steroids and vaccines. More recently,therapeutic recombinant human proteins such as insulin, interferons and human growth hormone havebeen produced by a range of

Industrial microbiology is primarily associated with the commercial exploitation of microorganisms, and involves processes and products that are of major economic, environmental and social importance throughout the world. There are two key aspects of in-dustrial microbiology, the first relating to production of