Preface In the new millennium, extensive application of bioprocesses has created an environment for many engineers to expand knowledge of and interest in biotechnology. Microorganisms produce alcohols and acetone, which are used in industrial processes. Knowledge related to industrial microbiology has been revolutionised by the ability of genetically engineered cells to make many new products. Genetic engineering and gene mounting has been devel- oped in the enhancement of industrial fermentation. Finally, application of biochemical engineering in biotechnology has become a new way of making commercial products. This book demonstrates the application of biological sciences in engineering with theo- retical and practical aspects. The seventeen chapters give more understanding of the know- ledge related to the specified field, with more practical approaches and related case studies with original research data. It is a book for students to follow the sequential lectures with detailed explanations, and solves the actual problems in the related chapters. There are many graphs that present actual experimental data, and figures and tables, along with sufficient explanations. It is a good book for those who are interested in more advanced research in the field of biotechnology, and a true guide for beginners to practise and establish advanced research in this field. The book is specifically targeted to serve as a useful text for college and university students; it is mostly recommended for undergraduate courses in one or two semesters. It will also prove very useful for research institutes and postgraduates involved in practical research in biochemical engineering and biotechnology. This book has suitable biological science applications in biochemical engineering and the knowledge related to those biological processes. The book is unique, with practical approaches in the industrial field. I have tried to prepare a suitable textbook by using a direct approach that should be very useful for students in following the many case studies. It is unique in having solved problems, examples and demonstrations of detailed experi- ments, with simple design equations and required calculations. Several authors have con- tributed to enrich the case studies. During the years of my graduate studies in the USA at the University of Oklahoma and the University of Arkansas, the late Professor Mark Townsend gave me much knowledge and assisted me in my academic achievements. I have also had the opportunity to learn many things from different people, including Professor Starling, Professor C.M. Sliepcevich and Professor S. Ellaison at the University of Oklahoma. Also, it is a privilege to acknowledge Professor J.L. Gaddy and Professor Ed Clausen, who assisted me at the University of Arkansas. I am very thankful for their courage and the guidance they have given me. My vision in research and my success are due to these two great scholars at the University of Arkansas: they are always remembered. ECAFERP This book was prepared with the encouragement of distinguished Professor Gaddy, who made me proud to be his student. I also acknowledge my Ph.D. students at the University of Science Malaysia: Habibouallah Younesi and Aliakbar Zinatizadeh, who have assisted me in drawing most of the figures. I am very thankfial to my colleagues who have contributed to some parts of the chapters: Dr M. Jahanshahi, from the University of Mazandaran, Iran, and Dr Nidal Hilal from the University of Nottingham, UK. Also special thanks go to Dr H. Younesi, Dr W.S. Long, Associate Professor A.H. Kamaruddin, Professor S. Bhatia, Professor A.R. Mohamed and Associate Professor A.L. Ahmad for their contribution of case studies. I acknowledge my friends in Malaysia: Dr Long Wei Sing, Associate Professor Azlina Harun Kamaruddin and Professor Omar Kadiar, School of Chemical Engineering and School of Industrial Technology, the Universiti Sains Malaysia, for editing part of this book. I also acknowledge my colleague Dr Mohammad Ali Rupani, who has edited part of the book. Nor should I forget the person who has accelerated this work and given lots of encouragement: Deirdre Clark at Elsevier. G. D. RUOPFAJAN Professor of Chemical Engineering University of Mazandaran, Babol, Iran Ch001.qxd 8/24/2006 9:34 AM Page 1 CHAPTER 1 Industrial Microbiology 1.1 INTRODUCTION Microorganisms have been identified and exploited for more than a century. The Babylonians and Sumerians used yeast to prepare alcohol. There is a great history beyond fermenta- tion processes, which explains the applications of microbial processes that resulted in the production of food and beverages. In the mid-nineteenth century,Louis Pasteur understood the role of microorganisms in fermented food, wine, alcohols, beverages, cheese, milk, yoghurt and other dairy products, fuels, and fine chemical industries. He identified many microbial processes and discovered the first principal role of fermentation, which was that microbes required substrate to produce primary and secondary metabolites, and end products. In the new millennium, extensive application of bioprocesses has created an environ- ment for many engineers to expand the field of biotechnology. One of the useful applica- tions of biotechnology is the use of microorganisms to produce alcohols and acetone,which are used in the industrial processes. The knowledge related to industrial microbiology has been revolutionised by the ability of genetically engineered cells to make many new prod- ucts. Genetic engineering and gene mounting have been developed in the enhancement of industrial fermentation. Consequently, biotechnology is a new approach to making com- mercial products by using living organisms. Furthermore, knowledge of bioprocesses has been developed to deliver fine-quality products. Application of biological sciences in industrial processes is known as bioprocessing. Nowadays most biological and pharmaceutical products are produced in well-defined industrial bioprocesses. For instance,bacteria are able to produce most amino acids that can be used in food and medicine. There are hundreds of microbial and fungal products purely available in the biotechnology market. Microbial production of amino acids can be used to produce L-isomers; chemical production results in both D- and L-isomers. Lysine and glu- tamic acid are produced by Corynebacterium glutamicum. Another food additive is citric acid, which is produced by Aspergillus niger. Table 1.1 summarises several widespread applications of industrial microbiology to deliver a variety of products in applied industries. The growth of cells on a large scale is called industrial fermentation. Industrial fermen- tation is normally performed in a bioreactor,which controls aeration,pH and temperature. Microorganisms utilise an organic source and produce primary metabolites such as ethanol, 1 Ch001.qxd 8/24/2006 9:34 AM Page 2 2 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY TABLE1.1. Industrial products produced by biological processes12 Fermentation product Microorganism Application Ethanol (non-beverage) Saccharomyces cerevisiae Fine chemicals 2-Ketogluconic acid Pseudomonas sp. Intermediate for D-araboascorbic acid Pectinase,protease Aspergillus niger,A. aureus Clarifying agents in fruit juice Bacterial amylase Bacillus subtilis Modified starch,sizing paper Bacterial protease B. subtilis Desizing fibres,spot remover Dextran Leuconostoc mesenteroides Food stabilizer Sorbose Gluconobacter suboxydans Manufacturing of ascorbic acid Cobalamin (vitamin B ) Streptomyces olivaceus Food supplements 12 Glutamic acid Brevibacterium sp. Food additive Gluconic acid Aspergillus niger Pharmaceutical products Lactic acid Rhizopus oryzae Foods and pharmaceuticals Citric acid Aspergillus niger orA. wentii Food products,medicine Acetone-butanol Clostridium acetobutylicum Solvents,chemical intermediate Insulin,interferon Recombinant E. coli Human therapy Baker’s yeast Yeast and culture starter Lactobacillus bulgaricus Cheese and yoghurt production Lactic acid bacteria Microbial protein (SCP) Candida utilis Food supplements Pseudomonas methylotroph Penicillin Penicillium chrysogenum Antibiotics Cephalosporins Cephalosparium acremonium Antibiotics Erythromycin Streptomyces erythreus Antibiotics which are formed during the cells’exponential growth phase. In some bioprocesses,yeast or fungi are used to produce advanced valuable products. Those products are considered as secondary metabolites, such as penicillin, which is produced during the stationary phase. Yeasts are grown for wine- and bread-making. There are other microbes, such as Rhizobium,Bradyrhizobiumand Bacillus thuringiensis,which are able to grow and utilise carbohydrates and organic sources originating from agricultural wastes. Vaccines, anti- biotics and steroids are also products of microbial growth. 1.2 PROCESS FERMENTATION The term ‘fermentation’was obtained from the Latin verb ‘fervere’, which describes the action of yeast or malt on sugar or fruit extracts and grain. The ‘boiling’is due to the pro- duction of carbon dioxide bubbles from the aqueous phase under the anaerobic catabolism of carbohydrates in the fermentation media. The art of fermentation is defined as the chem- ical transformation of organic compounds with the aid of enzymes. The ability of yeast to make alcohol was known to the Babylonians and Sumerians before 6000 BC. The Egyptians discovered the generation of carbon dioxide by brewer’s yeast in the preparation Ch001.qxd 8/24/2006 9:34 AM Page 3 INDUSTRIAL MICROBIOLOGY 3 of bread. The degradation of carbohydrates by microorganisms is followed by glycolytic or Embden–Myerhof–Parnas pathways.1,2 Therefore the overall biochemical reaction mechanisms to extract energy and form products under anaerobic conditions are called fer- mentation processes. In the process of ethanol production, carbohydrates are reduced to pyruvate with the aid of nicotinamide adenine dinucleotide (NADH); ethanol is the end product. Other fermentation processes include the cultivation of acetic acid bacteria for the production of vinegar. Lactic acid bacteria preserve milk; the products are yoghurt and cheese. Various bacteria and mold are involved in the production of cheese. Louis Pasteur, who is known as the father of the fermentation process,in early nineteenth century defined fermentation as life without air. He proved that existing microbial life came from pre- existing life. There was a strong belief that fermentation was strictly a biochemical reac- tion. Pasteur disproved the chemical hypothesis. In 1876, he had been called by distillers of Lille in France to investigate why the content of their fermentation product turned sour.3 Pasteur found under his microscope the microbial contamination of yeast broth. He discovered organic acid formation such as lactic acid before ethanol fermentation. His greatest contribution was to establish different types of fermentation by specific microor- ganisms,enabling work on pure cultures to obtain pure product. In other words,fermenta- tion is known as a process with the existence of strictly anaerobic life: that is, life in the absence of oxygen. The process is summarised in the following steps: (cid:127) Action of yeast on extracts of fruit juice or,malted grain. The biochemical reactions are related to generation of energy by catabolism of organic compounds. (cid:127) Biomass or mass of living matter,living cells in a liquid solution with essential nutrients at suitable temperature and pH leads to cell growth. As a result, the content of biomass increases with time. In World War I, Germany was desperate to manufacture explosives, and glycerol was needed for this. They had identified glycerol in alcohol fermentation. Neuberg discovered that the addition of sodium bisulphate in the fermentation broth favored glycerol production with the utilization of ethanol. Germany quickly developed industrial-scale fermentation, with production capacity of about 35 tons per day.3 In Great Britain, acetone was in great demand; it was obtained by anaerobic fermentation of acetone–butanol using Clostridium acetobutylicum. In large-scale fermentation production,contamination was major problem. Microorganisms are capable of a wide range of metabolic reactions,using various sources of nutrients. That makes fermentation processes suitable for industrial applications with inexpensive nutri- ents. Molasses,corn syrup,waste products from crystallisation of sugar industries and the wet milling of corn are valuable broth for production of antibiotics and fine chemicals. We will discuss many industrial fermentation processes in the coming chapters. It is best to focus first on the fundamental concepts of biochemical engineering rather than the applications. There are various industries using biological processes to produce new products,such as antibiotics,chemicals,alcohols,lipid,fatty acids and proteins. Deep understanding of bio- processing may require actual knowledge of biology and microbiology in the applications of the above processes. It is very interesting to demonstrate bench-scale experiments and Ch001.qxd 8/24/2006 9:34 AM Page 4 4 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY make use of large-scale advanced technology. However, application of the bioprocess in large-scale control of microorganisms in 100,000 litres of media may not be quite so simple to manage. Therefore trained engineers are essential and highly in demand; this can be achieved by knowledge enhancement in the sheathe bioprocesses. To achieve such objec- tives we may need to explain the whole process to the skilled labour and trained staff to implement bioprocess knowhow in biotechnology. 1.3 APPLICATION OF FERMENTATION PROCESSES Man has been using the fermentative abilities of microorganisms in various forms for many centuries. Yeasts were first used to make bread; later,use expanded to the fermentation of dairy products to make cheese and yoghurt. Nowadays more than 200 types of fermented food product are available in the market. There are several biological processes actively used in the industry,with high-quality products such as various antibiotics,organic acids, glutamic acid,citric acid,acetic acid,butyric and propionic acids. Synthesis of proteins and amino acids,lipids and fatty acids,simple sugar and polysaccharides such as xanthan gum, glycerol,many more fine chemicals and alcohols are produced by bioprocesses with suit- able industrial applications. The knowledge of bioprocessing is an integration of biochem- istry,microbiology and engineering science applied in industrial technology. Application of viable microorganisms and cultured tissue cells in an industrial process to produce specific products is known as bioprocessing. Thus fermentation products and the ability to cultivate large amounts of organisms are the focus of bioprocessing, and such achievements may be obtained by using vessels known as fermenters or bioreactors. The cultivation of large amounts of organisms in vessels such as fermenters and bioreactors with related fermenta- tion products is the major focus of bioprocess. A bioreactor is a vessel in which an organism is cultivated and grown in a controlled manner to form the by-product. In some cases specialised organisms are cultivated to pro- duce very specific products such as antibiotics. The laboratory scale of a bioreactor is in the range 2–100 litres,but in commercial processes or in large-scale operation this may be up to 100 m3.4,5 Initially the term ‘fermenter’was used to describe these vessels,but in strict terms fermentation is an anaerobic process whereas the major proportion of fermenter uses aerobic conditions. The term ‘bioreactor’ has been introduced to describe fermentation vessels for growing the microorganisms under aerobic or anaerobic conditions. Bioprocess plants are an essential part of food,fine chemical and pharmaceutical indus- tries. Use of microorganisms to transform biological materials for production of fermented foods, cheese and chemicals has its antiquity. Bioprocesses have been developed for an enormous range of commercial products,as listed in Table 1.1. Most of the products orig- inate from relatively cheap raw materials. Production of industrial alcohols and organic solvents is mostly originated from cheap feed stocks. The more expensive and special bio- processes are in the production of antibiotics, monoclonal antibodies and vaccines. Industrial enzymes and living cells such as baker’s yeast and brewer’s yeast are also com- mercial products obtained from bioprocess plants. Ch001.qxd 8/24/2006 9:34 AM Page 5 INDUSTRIAL MICROBIOLOGY 5 TABLE1.2. Products and services by biological processes Sector Product and service Remark Chemicals Ethanol,acetone,butanol Bulk Organic acids (acetic,butyric,propionic and citric acids) Enzymes Fine Perfumeries Polymers Pharmaceuticals Antibiotics Enzymes Enzyme inhibitors Monoclonal antibodies Steroids Vaccines Energy Ethanol (gasohol) Non-sterile Methane (biogas) Food Diary products (cheese,yoghurts,etc.) Non-sterile Baker’s yeast Beverages (beer,wine) Food additives Amino acids Vitamin B Proteins (SCP) Agriculture Animal feeds (SCP) Non-sterile Waste treatment Vaccines Microbial pesticides Mycorrhizal inoculants 1.4 BIOPROCESS PRODUCTS Major bioprocess products are in the area of chemicals,pharmaceuticals,energy,food and agriculture, as depicted in Table 1.2. The table shows the general aspects, benefits and application of biological processes in these fields. Most fermented products are formed into three types. The main categories are now discussed. 1.4.1 Biomass The aim is to produce biomass or a mass of cells such as microbes, yeast and fungi. The commercial production of biomass has been seen in the production of baker’s yeast,which is used in the baking industry. Production of single cell protein (SCP) is used as biomass enriched in protein.6An algae called Spirulinahas been used for animal food in some coun- tries. SCP is used as a food source from renewable sources such as whey,cellulose,starch, molasses and a wide range of plant waste. Ch001.qxd 8/24/2006 9:34 AM Page 6 6 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY 1.4.2 Cell Products Products are produced by cells, with the aid of enzymes and metabolites known as cell products. These products are categorised as either extracellular or intracellular. Enzymes are one of the major cell products used in industry. Enzymes are extracted from plants and animals. Microbial enzymes,on the other hand,can be produced in large quantities by con- ventional techniques. Enzyme productivity can be improved by mutation,selection and per- haps by genetic manipulation. The use of enzymes in industry is very extensive in baking, cereal making, coffee, candy, chocolate, corn syrup, dairy product, fruit juice and bever- ages. The most common enzymes used in the food industries are amylase in baking, pro- tease and amylase in beef product,pectinase and hemicellulase in coffee,catalase,lactase and protease in dairy products,and glucose oxidase in fruit juice. 1.4.3 Modified Compounds (Biotransformation) Almost all types of cell can be used to convert an added compound into another compound, involving many forms of enzymatic reaction including dehydration,oxidation,hydroxyla- tion,amination,isomerisation,etc. These types of conversion have advantages over chem- ical processes in that the reaction can be very specific, and produced at moderate temperatures. Examples of transformations using enzymes include the production of steroids, conversion of antibiotics and prostaglandins. Industrial transformation requires the production of large quantities of enzyme,but the half-life of enzymes can be improved by immobilisation and extraction simplified by the use of whole cells. In any bioprocess, the bioreactor is not an isolated unit, but is as part of an integrated process with upstream and downstream components. The upstream consists of storage tanks,growth and media preparation,followed by sterilisation. Also,seed culture for inoc- ulation is required upstream, with sterilised raw material, mainly sugar and nutrients, required for the bioreactor to operate. The sterilisation of the bioreactor can be done by steam at 15 pounds per square inch guage (psig), 121 °C or any disinfectant chemical reagent such as ethylene oxide. The downstream processing involves extraction of the product and purification as normal chemical units of operation.7 The solids are separated from the liquid, and the solution and supernatant from separation unit may go further for purification after the product has been concentrated. 1.5 PRODUCTION OF LACTIC ACID Several carbohydrates such as corn and potato starch, molasses and whey can be used to produce lactic acid. Starch must first be hydrolysed to glucose by enzymatic hydrolysis; then fermentation is performed in the second stage. The choice of carbohydrate material depends upon its availability, and pretreatment is required before fermentation. We shall describe the bioprocess for the production of lactic acid from whey. Large quantities of whey constitute a waste product in the manufacture of dairy products such as cheese. From the standpoint of environmental pollution it is considered a major problem,and disposal of untreated wastes may create environmental disasters. It is desirable Ch001.qxd 8/24/2006 9:34 AM Page 7 INDUSTRIAL MICROBIOLOGY 7 Lactic acid recovery Whey Seed culture 5000 gallon inoculum bioreactor 150 gallons Preparation of Stock inocula Fermentation of culture lactose using Lactobacillus bulgaricus FIG. 1.1. Production of lactic acid from whey. to use whey to make some more useful product. Whey can be converted from being a waste product to something more desirable that can be used for the growth of certain bacteria, because it contains lactose, nitrogenous substances, vitamins and salts. Organisms can utilise lactose and grow on cheese wastes; the most suitable of them are Lactobacillus species such as Lactobacillus bulgaricus,which is the most suitable species for whey. This organism grows rapidly,is homofermentative and thus capable of converting lactose to the single end-product of lactic acid. Stock cultures of the organism are maintained in skimmed milk medium. The 3–5% of inoculum is prepared and transferred to the main bioreactor, and the culture is stored in pasteurised, skimmed milk at an incubation temperature of 43 °C. During fermentation,pH is controlled by the addition of slurry of lime to neutralise the product to prevent any product inhibition. The accumulation of lactic acid would retard the fermentation process because of the formation of calcium lactate. After 2 days of com- plete incubation,the material is boiled to coagulate the protein,and then filtered. The solid filter cake is a useful,enriched protein product,which may be used as an animal feed sup- plement. The filtrate containing calcium lactate is then concentrated by removing water under vacuum, followed by purification of the final product. The flow diagram for this process is shown in Figure 1.1. 1.6 PRODUCTION OF VINEGAR The sugars in fruits such as grapes are fermented by yeasts to produce wines. In wine- making, lactic acid bacteria convert malic acid into lactic acid in malolactic fermentation in fruits with high acidity. Acetobacterand Gluconobacteroxidise ethanol in wine to acetic acid (vinegar). Ch001.qxd 8/24/2006 9:34 AM Page 8 8 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY The word ‘wine’is derived from the French term ‘vinaigre’meaning ‘sour wine’. It is prepared by allowing a wine to get sour under controlled conditions. The production of vinegar involves two steps of biochemical changes: (1) Alcoholic fermentation in fermentation of a carbohydrate. (2) Oxidation of the alcohol to acetic acid. There are several kinds of vinegar. The differences between them are primarily associated with the kind of material used in the alcoholic fermentation, e.g. fruit juices, sugar and hydrolysed starchy materials. Based on US Department of Agriculture (USDA) definitions, there are a few types of vinegar: vinegar, cider vinegar, apple vinegar. The products are made by the alcoholic and subsequent acetous fermentations of the apple juice. The acetic acid content is about 5%. Yeast fermentation is used for the production of alcohol. The alco- hol is adjusted to 10–13%,then it is exposed to acetic acid bacteria (Acetobacterspecies), whereby oxygen is required for the oxidation of alcohol to acetic acid. The desired tem- perature for Acetobacteris 15–34 °C. The reaction is: C H O æZæymoæmoænasæmobæilisæÆ2CH CH OH(cid:1)2CO (1.6.1) 6 12 6 3 2 2 2CH CH OH(cid:1)2O æAæcetæobacæteræspæ.Æ2CH COOH(cid:1)2H O (1.6.2) 3 2 2 3 2 1.7 PRODUCTION OF AMINO ACIDS (LYSINE AND GLUTAMIC ACID) AND INSULIN Many microorganisms can synthesise amino acids from inorganic nitrogen compounds. The rate and amount of some amino acids may exceed the cells’need for protein synthesis, where the excess amino acids are excreted into the media. Some microorganisms are capa- ble of producing certain amino acids such as lysine,glutamic acid and tryptophan. 1.7.1 Stepwise Amino Acid Production One of the commercial methods for production of lysine consists of a two-stage process using two species of bacteria. The carbon sources for production of amino acids are corn, potato starch, molasses, and whey. If starch is used, it must be hydrolysed to glucose to achieve higher yield. Escherichia coli is grown in a medium consisting of glycerol, corn- steep liquor and di-ammonium phosphate under aerobic conditions, with temperature and pH controlled. (cid:127) Step 1:Formation of diaminopimelic acid (DAP) by E. coli. (cid:127) Step 2:Decarboxylation of DAP by Enterobacter aerogenes. E. colican easily grow on corn steep liquor with phosphate buffer for an incubation period of 3 days. Lysine is an essential amino acid for the nutrition of humans,which is used as a