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Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology ' basis of cell function, today’s classification is based on the sequence of ribosomal RNA (rRNA)in the 16S of the small sub-unit (SSU) of the procaryotic ribosome, and the 18S ribosomal unit of eucaryo...

Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology ' basis of cell function, today’s classification is based on the sequence of ribosomal RNA (rRNA)in the 16S of the small sub-unit (SSU) of the procaryotic ribosome, and the 18S ribosomal unit of eucaryotes. The logical question to ask is, why do we use the rRNA sequence? It is used for the following reasons: (i) 16S (or 18S) rRNA is essential to the ribosome, an important organelle found in all living things (i.e. it is universally distributed); (ii) its function is identical in all ribosomes; (iii) its sequence changes very slowly with evolutionary time, and it contains variable and stable sequences which enable the comparison of closely related as well as distantly related species. The classification is evolutionary and attempts to link all livings things with evolution from a common ancestor. For this approach, an evolutionary time-keeper is necessary. Such a time-keeper must be available to, or used by components of the system, and yet be able to reflect differences and changes with time in other regions appropriate to the assigned evolutionary distances. The 16S ribosomal RNAs meet these criteria as ribosomes are involved in protein synthesis in all living things. They are also highly conserved (remain the same) in many groups and some minor changes observed are commensurate with expected evolutionary distances (Fig. 2.2). Fig. 2.2 Diagram Illustrating Evolutionary Relationship between Organisms with Time According to the currently accepted classification living things are placed into three groups: Archae, Bacteria, and Eukarya. A diagram depicting the evolutionary relationships among various groups of living things is giving in Fig. 2.3, while the properties of the various groups are summarized in Table 2.1. Archae and Bacteria are procaryotic while Eucarya are eucaryotic. 2.3 TAXONOMIC GROUPING OF MICRO-ORGANISMS IMPORTANT IN INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY The microorganisms currently used in industrial microbiology and biotechnology are found mainly among the bacteria and eukarya; the Archae are not used. However, as discussed in Chapter 1, the processes used in industrial microbiology and biotechnology are dynamic. Consequently, out-dated procedures are discarded as new and more effi- cient ones are discovered. At present organisms from Archae are not used for industrial processes, but that may change in future. This idea need not be as far fetched as it may   Modern Industrial Microbiology and Biotechnology Fig. 2.3 The Three Domains of Living Things Based on Woese’s Work seem now. For as will be seen below, one of the criteria supporting the use of a microor- ganism for industrial purposes is the possession of properties which will enable the organism to survive and be productive in the face of competition from contaminants. Many organisms in Archae are able to grow under extreme conditions of temperature or salinity and these conditions may be exploited in industrial processes where such physi- ological properties may put a member of the Archae at an advantage over contaminants. Plants and animals as well as their cell cultures are also used in biotechnology, and will be discussed in the appropriate sections below. Microorganisms have the following advantages over plants or animals as inputs in biotechnology: i. Microorganisms grow rapidly in comparison with plants and animals. The generation time (the time for an organism to mature and reproduce) is about 12 years in man, about 24 months in cattle, 18 months in pigs, 6 months in chicken, but only 15 minutes in the bacterium, E coli. The consequence is that biotechnological products which can be obtained from microorganisms in a matter of days may take many months in animals or plants. ii. The space requirement for growth microorganisms is small. A 100,000 litre fermentor can be housed in about 100 square yards of space, whereas the plants or animals needed to generate the equivalent of products in the 100,000 fermentor would require many acres of land. iii. Microorganisms are not subject to the problems of the vicissitudes of weather which may affect agricultural production especially among plants. iv. Microorganisms are not affected by diseases of plants and animals, although they do have their peculiar scourges in the form phages and contaminants, but there are procedure to contain them. Despite these advantages there are occasions when it is best to use either plants or animals; in general however microorganisms are preferred for the reasons given above.  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology  Table 2.1 Summary of differences among the three domains of living things, (from Madigan and Martimko, 2006) S/No Characteristic Bacteria Archae Eukarya Morphology and Genetics 1 Prokaryotic cell structure + + - 2 DNA present in closed circular form + + - 1 3 Histone proteins present - + + 4 Nuclear membrane - - + 5 Muramic acid in cell wall + - - 6 Membrane lipids: Fatty acids or Branched hydrocarbons Fatty acids Branched Fatty acids hydrocarbons 7 Ribosome size 70S 70S 80S 8 Initiator tRNA Formyl- Methionine Methionine methionine 2 9 Introns in most genes - - + 3 10 Operons + + - 11 Plasmids + + Rare 12 Ribosome sensitive to diphtheria toxin - + + 13 Sensitivity to streptomycin, chloramphenicol, and kanamycin + - - 4 14 Transcription factors required - + + Physiological/Special Structures 15 Methanogenesis + - - 16 Nitrification + -? - 17 Denitrification + + - 18 Nitrogen fixation + + - 19 Chlorophyll based photosynthesis + - + (plants) 20 Gas vesicles + + - 21 Chemolithotrphy + + - 22 Storage granules of poly-b- hydroxyalkanoates + + - 23 Growth above 80oC + + - 24 Growth above 100oC - + - 1 Histone proteins are present in eucaryotic chromosomes; histones and DNA give structure to chromosomes in eucaryotes. 2Non-coding sequences within genes; 3Operons: Typically present in prokaryotes, these are clusters of genes controlled by a single operator; 4Transcription factor is a protein that binds DNA at a specific promoter or enhancer region or site, where it regulates transcription. 2.3.1 Bacteria Bacteria are described in two compendia, Bergey’s Manual of Determinative Bacteriology and Bergey’s Manual of Systematic Bacteriology. The first manual (on Determinative Bacteriology) is designed to facilitate the identification of a bacterium whose identity is  Modern Industrial Microbiology and Biotechnology unknown. It was first published in 1923 and the current edition, published in 1994 is the ninth. The companion volume (on Systematic Bacteriology) records the accepted published descriptions of bacteria, and classifies them into taxonomic groups. The first edition was produced in four volumes and published between 1984 and 1989. The bacterial classification in the latest (second) edition of Bergey’s Manualof Sytematic Bacteriology is based on 16S RNA sequences, following the work of Carl Woese, and organizes the Domain Bacteria into 18 groups (or phyla; singular, phylum) It is to be published in five volumes. Volume 1 which deals with the Archae and the deeply branching and phototrophic bacteria was published in 2001; Volume 2 published in 2005, deals with the Proteobacteria and has three parts while Volume 3 was published in 2006 and deals with the low G+C Gram-positive bacteria. The last two volumes, Volume 4 (the high C + C Gram-positive bacteria) and Volume 5 (The Plenctomyces, Spirochaetes, Fibrobacteres, Bacteriodetes and Fusobacteria) will be published in 2007. The manuals are named after Dr D H Bergey who was the first Chairman of the Board set up by the then Society of American Bacteriologists (now American Society for Microbiology) to publish the books. The publication of Bergey Manuals is now managed by the Bergey’s Manual Trust. Of the 18 phyla in the bacteria, (see Fig. 2.4) the Aquiflex is evolutionarily the most primitive, while the most advanced is the Proteobacteria. The bacterial phyla used in industrial microbiology and biotechnology are found in the Proteobacteria, the Firmicutes and the Actinobacteria. Green sulfur bacteria Deferribacter Spirochetes Deinococci Flavobacteria Plectomyces/ Pirella Green non-sulfur Cytophaga bacteria Verucomicrobia Thermotoga Chlamydia Cyanobacteria Thermodesulfo bacterium Actinobacteria Aquifex Gram-positive bacteria Nitrospira e – Proteobacteria d –Proteobacteria a – Proteobacteria b – Proteobacteria g – Proteobacteria Fig. 2.4 The 18 Phyla of Bacteria Based on 16S RNA Sequences (After Madigan and Matinko, 2006)  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology ! 2.3.1.1 The Proteobacteria The Proteobacteria are a major group of bacteria. Due to the diversity of types of bacteria in the group, it is named after Proteus, the Greek god, who could change his shape. Proteobacteria include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio and Helicobacter, as well as free-living bacteria some of which can fix nitrogen. The group also includes the purple bacteria, so-called because of their reddish pigmentation, and which use energy from sun light in photosynthesis. All Proteobacteria are Gram-negative, with an outer membrane mainly composed of lipopolysaccharides. Many move about using flagella, but some are non-motile or rely on bacterial gliding. There is also a wide variety in the types of metabolism. Most members are facultatively or obligately anaerobic and heterotrophic, but there are numerous exceptions. Proteobacteria are divided into five groups: a (alpha), b (beta), g (gamma), d (delta), e (epsilon). The only organisms of current industrial importance in the Proteobacteria are Acetobacter and Gluconobacter, which are acetic acid bacteria and belong to the Alphaproteobacteria. An organism also belonging to the Alphaproteobacteria, and which has the potential to become important industrially is Zymomonas. It produces copious amounts of alcohol, but its use industrially is not yet widespread. 2.3.1.1.1 The Acetic Acid Bacteria The acetic acid bacteria are Acetobacter (peritrichously flagellated) and Gluconobacter (polarly flagellated). They have the following properties: i. They carry out incomplete oxidation of alcohol leading to the production of acetic acid, and are used in the manufacture of vinegar (Chapter 14). ii. Gluconobacter lacks the complete citric acid cycle and can not oxidize acetic acid; Acetobacter on the on the other hand, has all the citric acid enzymes and can oxidize acetic acid further to CO2. iii. They stand acid conditions of pH 5.0 or lower. iv. Their property of ‘under-oxidizing’ sugars is exploited in the following: a. The production of glucoronic acid from glucose, galactonic aicd from galactose and arabonic acid from arabinose; b. The production of sorbose from sorbitol by acetic acid bacteria (Fig. 2.4), an important stage in the manufacture of ascorbic acid (also known as Vitamin C) v. Acetic acid bacteria are able to produce pure cellulose when grown in an unshaken culture. This is yet to be exploited industrially, but the need for cellulose of the purity of the bacterial product may arise one day. 2.3.1.2 The Firmicutes The Firmicutes are a division of bacteria, all of which are Gram-positive, in contrast to the Proteobacteria which are all Gram-negative. A few, the mycoplasmas, lack cell walls altogether and so do not respond to Gram staining, but still lack the second membrane found in other Gram-negative forms; consequently they are regarded as Gram-positive.  " Modern Industrial Microbiology and Biotechnology CH2OH CH2OH CO Acetobacter suboxydans CH2OH CH2OH D-sorbitol L-sorbose Fig. 2.5 Conversion of Sorbitol to Sorbose Originally the Firmicutes were taken to include all Gram-positive bacteria, but more recently they tend to be restricted to a core group of related forms, called the low G+C group in contrast to the Actinobacteria, which have high G+C ratios. The G+C ratio is an important taxonomic characteristic used in classifying bacteria. It is the ratio of Guanine and Cytosine to Guanine, Cytosine, Adenine, and Thymine in the cell. Thus the GC ratio = G+C divided by G+C+A+T x 100. It is used to classify Gram-positive bacteria: low G+C Gram-positive bacteria (ie those with G+C less than 50%) are placed in the Fermicutes, while those with 50% or more are in Actinobacteria. Fermicutes contain many bacteria of industrial importance and are divided into three major groups: i. spore-forming, ii. non- spore forming, and iii) wall-less (this group contains pathogens and no industrial organisms.) 2.3.1.2.1 Spore forming firmicutes Spore-forming Firmicutes form internal spores, unlike the Actinobacteria where the spore-forming members produce external ones. The group is divided into two: Bacillus spp, which are aerobic and Clostridium spp which are anaerobic. Bacillus spp are sometimes used in enzyme production. Some species are well liked by mankind because of their ability to kill insects. Bacillus papilliae infects and kills the larvae of the beetles in the family Scarabaeidae while B. thuringiensis is used against mosquitoes (Chapter 17). The genes for the toxin produced by B. thuringiensis are also being engineered into plants to make them resistant to insect pests (Chapter 7). Clostridia on the other hand are mainly pathogens of humans and animals. 2.3.1.2.2 Non-spore forming firmicutes The Lactic Acid Bacteria: The non-spore forming low G+C members of the firmicutes group are very important in industry as they contain the lactic acid bacteria. The lactic acid bacteria are rods or cocci placed in the following genera: Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus and are among some of the most widely studied bacteria because of their important in the production of some foods, and industrial and pharmaceutical products. They lack porphyrins and cytochromes, do not carry out electron transport phosphorylation and hence obtain  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology # energy by substrate level phosphorylation. They grow anaerobically but are not killed by oxygen as is the case with many anaerobes: they will grow with or without oxygen. They obtain their energy from sugars and are found in environments where sugar is present. They have limited synthetic ability and hence are fastidious, requiring, when cultivated, the addition of amino acids, vitamins and nucleotides. Lactic acid bacteria are divided into two major groups: The homofermentative group, which produce lactic acid as the sole product of the fermentation of sugars, and the heterofermentative, which besides lactic acid also produce ethanol, as well as CO2. The difference between the two is as a result of the absence of the enzyme aldolase in the heterofermenters. Aldolase is a key enzyme in the E-M-P pathway and spits hexose glucose into three-sugar moieties. Homofermentative lactic acid bacteria convert the D- glyceraldehyde 3-phosphate to lactic acid. Heterofermentative lactic acid bacteria receive five-carbon xylulose 5 phosphate from the Pentose pathway. The five carbon xylulose is split into glyceraldehyde 3-phosphate (3-carbon), which leads to lactic acid, and the two- carbon acetyl phosphate which leads to ethanol (Fig. 2.6). Fig. 2.6 Splitting of 6-carbon Glucose into Three-carbon Compounds by the Enzyme Fructose Diphposphate Aldolase Use of Lactic Acid Bacteria for Industrial Purposes: The desirable characteristics of lactic acid bacteria as industrial microorganisms include a. their ability to rapidly and completely ferment cheap raw materials, b. their minimal requirement of nitrogenous substances, c. they produce high yields of the much preferred stereo specific lactic acid d. ability to grow under conditions of low pH and high temperature, and e. ability to produce low amounts of cell mass as well as negligible amounts of other byproducts.  $ Modern Industrial Microbiology and Biotechnology The choice of a particular lactic acid bacterium for production primarily depends on the carbohydrate to be fermented. Lactobacillus delbreuckii subspecies delbreuckii is able to ferment sucrose. Lactobacillus delbreuckii subspecies bulgaricus is able to use lactose while Lactobacillus helveticus is able to use both lactose and galactose. Lactobacillus amylophylus and Lactobacillus amylovirus are able to ferment starch. Lactobacillus lactis can ferment glucose, sucrose and galactose and Lactobacillus pentosus has been used to ferment sulfite waste liquor. 2.3.1.3 The Actinobacteria The Acinobacteria are the Firmicutes with G+C content of 50% or higher. They derive their name from the fact that many members of the group have the tendency to form filaments or hyphae (actinis, Greek for ray or beam). The industrially important members Table 2.2 Characteristics of the lactic acid bacteria S/No Group Description Habit Importance 1 Streptococcus Cocci in pairs or Some in respiratory Some cause sore short chains tract, mouth, intestine; throat; non- others found in pathogenic strains fermenting used in yoghurt vegetable and silage manufacture 2 Enterococcus Cocco-bacilli Found as commensals Can be used to usually in pairs; in the human alimentary monitor water previously classified canal; sometimes cause quality, (like E. coli) Streptococcus urinary tract infections Lancefield Group D 3 Lactococcus Coccoid, usually occuring Plant material and Used as starter in in pairs; hardly form alimentary canals of yoghurt manufacture; chains animals Used as probiotic for intestinal health; Produces copious amounts of lactic acid. 4 Pediococcus Growth in tetrads Found on plant materials Spoils beer; but required in special beers such as lambic beer drunk in parts of Belgium 5 Leuconostoc Cocco-bacili Associated with plant Tolerates high con- materials centrations of salt and sugar and involved in the pickling of vegetables; produce dextrans from sucrose  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology % Fig. 2.7 Formation of lacttic acid by homofermentative bacteria Table 2.3 Distinguishing characteristics of lactic acid bacteria Character Lactobacillus Enterococcus Lactocococcus Leuconostoc Pediococcus Streptococcus Tetrad formation – – – – + – CO2 from glucose ± – – + – – Growth at 10°C ± + + + ± – Growth at 45°C ± + – – ± ± Growth at 6.5% NaCl ± + – ± ± – Growth at pH 4.4 ± + ± ± + – Growth at pH 9.6 – + – – – – Lactic acid (optical orientation) D, L, DL L L D L, DL L of the group are the Actinomycetes and Corynebacterium. Corynebacterium spp are important industrially as secreters of amino acids (Chapter 21). The rest of this section will be devoted to Actinomycetes.  & Modern Industrial Microbiology and Biotechnology Enzymes involved: 1, Hexokinase; 2, Glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydrogenase; 4, Ribulose-5-phosphate 3-epimerase; Phosphoketolase; 6, Phosphotransacetylase; 7, Acetaldehyde dehydrogenase; 8, Alcohol dehydrogense; 9, Enzymes of the homofermentative pathway Fig. 2.8 Fermentation of Glucose by Heterofermentative Bacteria  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology ' Lactobacillus bulgaricus Lactococcus lactis Colour Fig. 2.9 Photomicrographs of Lactic Acid Bacteria 2.3.1.3.1 The Actinomycetes They have branching filamentous hyphae, which somewhat resemble the mycelia of the fungi, among which they were originally classified. In fact they are unrelated to fungi, but are regarded as bacteria for the following reasons. First they have petidoglycan in their cell walls, and second they are about 1.0m in diameter (never more than 1.5m), whereas fungi are at least twice that size in diameter. As a group the actinomycetes are unsurpassed in their ability to produce secondary metabolites which are of industrial importance, especially as pharmaceuticals. The best known genus is Streptomyces, from which many antibiotics as well as non-anti-microbial drugs have been obtained. The actinomycetes are primarily soil dwellers hence the temptation to begin the search for any bioactive microbial metabolite from soil. 2.3.2 Eucarya: Fungi Although plants and animals or their cell cultures are used in biotechnology, microorganisms are used more often for reason which have been discussed. Fungi are members of the Eucarya which are commonly used in industrial production. The fungi are traditionally classified into the four groups given in Table 2.4, namely Phycomycetes, Ascomycetes, Fungi Imprfecti, and Basidiomycetes. Among these the following are those currently used in industrial microbiology Phycomycetes (Zygomycetes) Rhizopus and Mucor are used for producing various enzymes Ascomycetes Yeasts are used for the production of ethanol and alcoholic beverages Claviceps purperea is used for the production of the ergot alkaloids ! Modern Industrial Microbiology and Biotechnology Actinomyces Actinoplanales Micromonospora Nocardia Streptomyces Saccharomonospora Thermoactinomyces Thermomonospora Fig. 2.10 Different Actinomycetes Fungi Imperfecti Aspergillus is important because it produces the food toxin, aflatoxin, while Penicillium is well-known for the antibiotic penicillin which it produces.  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology ! Basidiomycetes Agaricus produces the edible fruiting body or mushroom Numerous useful products are made through the activity of fungi, but the above are only a selection. Table 2.4 Description of the various groups of fungi Group Ordinary Septation Sexual Spores Representative Name of hyphae Zygomycetes Bread molds Non-septate Zygospre Rhizopus, Mucor (Phycomycetes) Ascomycetes Sac fungi Septate Ascospore Neurospora, (in Perithecia) Saccharomyces (Yeasts) Basidiomycetes Mushrooms Septate Basidiomycetes Agaricus (Mushrooms) Deuteromycetes Fungi imperfecti Septate None Penicillium, Aspergillus 2.4 CHARACTERISTICS IMPORTANT IN MICROBES USED IN INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLGY Microorganisms which are used for industrial production must meet certain requirements including those to be discussed below. It is important that these characteristics be borne in mind when considering the candidacy of any microorganism as an input in an industrial process. i. The organism must be able to grow in a simple medium and should preferably not require growth factors (i.e. pre-formed vitamins, nucleotides, and acids) outside those which may be present in the industrial medium in which it is grown. It is obvious that extraneous additional growth factors may increase the cost of the fermentation and hence that of the finished product. ii. The organism should be able to grow vigorously and rapidly in the medium in use. A slow growing organism no matter how efficient it is, in terms of the production of the target material, could be a liability. In the first place the slow rate of growth exposes it, in comparison to other equally effective producers which are faster growers, to a greater risk of contamination. Second, the rate of the turnover of the production of the desired material is lower in a slower growing organism and hence capital and personnel are tied up for longer periods, with consequent lower profits. iii. Not only should the organism grow rapidly, but it should also produce the desired materials, whether they be cells or metabolic products, in as short a time as possible, for reasons given above. iv. Its end products should not include toxic and other undesirable materials, especially if these end products are for internal consumption. ! Modern Industrial Microbiology and Biotechnology Rhizopus Aspergillus Perithecium with Asci and ascospores Conidia Sporangium Sporophore Mucor Penicillium Yeast s with 8 Ascospores Mucor Basidiomycete Fruiting Bodies (Showing Zygospore) (Mushrooms) Fig. 2.11 Representative Structures from Different Fungi v. The organism should have a reasonable genetic, and hence physiological stability. An organism which mutates easily is an expensive risk. It could produce undesired products if a mutation occurred unobserved. The result could be reduced yield of the expected material, production of an entirely different product or indeed a toxic material. None of these situations is a help towards achieving the goal of the industry, which is the maximization of profits through the production of goods with predictable properties to which the consumer is accustomed. vi. The organism should lend itself to a suitable method of product harvest at the end of the fermentation. If for example a yeast and a bacterium were equally suitable for manufacturing a certain product, it would be better to use the yeast if the most appropriate recovery method was centrifugation. This is because while the  Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology !! bacterial diameter is approximately 1m, yeasts are approximately 5m. Assuming their densities are the same, yeasts would sediment 25 times more rapidly than bacteria. The faster sedimentation would result in less expenditure in terms of power, personnel supervision etc which could translate to higher profit. vii. Wherever possible, organisms which have physiological requirements which protect them against competition from contaminants should be used. An organism with optimum productivity at high temperatures, low pH values or which is able to elaborate agents inhibitory to competitors has a decided advantage over others. Thus a thermophilic efficient producer would be preferred to a mesophilic one. viii. The organism should be reasonably resistant to predators such as Bdellovibrio spp or bacteriophages. It should therefore be part of the fundamental research of an industrial establishment using a phage-susceptible organism to attempt to produce phage-resistant but high yielding strains of the organism. ix. Where practicable the organism should not be too highly demanding of oxygen as aeration (through greater power demand for agitation of the fermentor impellers, forced air injection etc) contributes about 20% of the cost of the finished product. x. Lastly, the organism should be fairly easily amenable to genetic manipulation to enable the establishment of strains with more acceptable properties. SUGGESTED READINGS Asai, T. 1968. The Acetic Acid Bacteria. Tokyo: The University of Tokyo Press and Baltimore: University Park Press. Axelssson, L., Ahrne, S. 2000. Lactic Acid Bacteria. In: Applied Microbial Systematics, F.G. Priest, M. Goodfellow, (eds) A.H. Dordrecht, the Netherlands, pp. 367-388. Barnett, J.A. , Payne, R.W., Yarrow, D. 2000. Yeasts: Characterization and Identification. 3rd Edition. Cambridge University Press. Cambridge, UK. Garrity, G.M. 2001-2006. Bergey’s Manual of Systematic Bacteriology. 2nd Ed. Springer, New York, USA. Goodfellow, M., Mordaraski, M., Williams, S.T. 1984. The Biology of the Actinomycetes. Academic Press, London, UK. Madigan, M., Martimko, J.M. 2006. Brock Biology of Microorganisms. Upper Saddle River: Pearson Prentice Hall. 11th Edition. Major, A. 1975. Mushrooms Toadstools and Fungi: Arco New York, USA. Narayanan, N., Pradip, K. Roychoudhury, P.K., Srivastava, A. 2004. L (+) lactic acid fermentation and its product polymerization. Electronic Journal of Biotechnology 7, Electronic Journal of Biotechnology [online]. 15 August 2004, 7, (3) [cited 23 March 2006]. Available from: http:// www.ejbiotechnology.info/content/vol2/issue3/full/3/index.html. ISSN 0717-3458. Samson, R., Pitt, J.I. 1989. Modern Concepts in Penicillium and Aspergillus Classification. Plenum Press New York and London. Woese, C.R. 2002. On the evolution of cells Proceedings of the National Academy of Sciences of the United States of America 99, 8742-8747.