Industrial Microbiology Chapter 2 PDF (Nile University)

BACHELOR OF BIOTECHNOLOGY Major: Applied Biotechnology Course: Industrial Microbiology (MICR404) Academic year 2023/ 2024 Fall 2023 Chapter 2 Handouts Prof. Abdelrahim H. A. Hassa...

BACHELOR OF BIOTECHNOLOGY Major: Applied Biotechnology Course: Industrial Microbiology (MICR404) Academic year 2023/ 2024 Fall 2023 Chapter 2 Handouts Prof. Abdelrahim H. A. Hassan Professor of Food Safety and Technology School of Biotechnology, Nile University INDUSTRIAL MICROBIOLOGY (MICR404) 1  Chapter 2 Microorganisms in Industrial Microbiology Contents: 1. Classification of living organisms into three domains. 2. Taxonomic grouping of industrial microorganisms. 3. Important characteristics of industrial microbes. 1. CLASSIFICATION OF LIVING ORGANISMS INTO THREE DOMAINS The earliest classification placed living organisms into two simple categories, plants and animals. When the microscope was discovered in the middle of the 16th century, it enabled the observation of microorganisms for the first time. Living organisms were then divided into plants, animals, and Protista (microorganisms). From the 1960s and the 1970s, Whittaker’s division of living organisms into five groups was the accepted grouping. The classification was based on cell type: prokaryotic or eukaryotic, organizational level: single-celled or multi-cellular, and nutritional type: heterotrophy and autotrophy. On the basis of these characteristics, living organisms were divided into five groups: Monera (bacteria), Protista (algae and protozoa), Plants, Fungi, and Animals. According to the currently accepted classification living things are placed into three groups: Archaea, Bacteria, and Eukarya. Archaea and Bacteria are prokaryotes, while Eukarya are eukaryotes. The current classification of living organisms is based on the work of Carl R. Woese of the University of Illinois (Fig. 1). INDUSTRIAL MICROBIOLOGY (MICR404) 2  Fig. 1. The Three Domains of Living Things Based on Woese’s Work 2. TAXONOMIC GROUPING OF INDUSTRIAL MICROORGANISMS The microorganisms currently used in industrial microbiology and biotechnology are found mainly among bacteria and Eukarya. However, the processes used in industrial microbiology and biotechnology are dynamic. Consequently, outdated procedures are discarded, as new and more efficient ones are discovered. At present, organisms from Archaea are not used for industrial processes but that may change in the future. One of the criteria supporting the use of a microorganism for industrial purposes is the possession of properties that will enable the organism to survive and be productive in the face of competition from contaminants. Many organisms in Archaea are able to grow under extreme conditions of temperature or salinity. These conditions may be exploited in industrial processes where such physiological properties may put a member of the Archaea at an advantage over contaminants. Plants and animals as well as their cell cultures are also used in biotechnology. Microorganisms have the following advantages over plants or animals as inputs in biotechnology: INDUSTRIAL MICROBIOLOGY (MICR404) 3  i. Microorganisms grow rapidly in comparison with plants and animals. The generation time (the time for an organism to mature and reproduce) is about 24 months in cattle, 18 months in pigs, and 6 months in chickens, but only 15 minutes in the bacterium, E. coli. The consequence is that biotechnological products that 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-liter fermenter can be housed in about 100 square yards (83.6 m2) of space, whereas the plants or animals needed to generate the equivalent of products in the 100,000-liter fermenter would require many acres of land. iii. Microorganisms are not affected by the changes in weather conditions 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 of phages and contaminants, but there are procedures to contain them. 2.1. Bacteria Bergey’s Manual of Systematic Bacteriology classifies Bacteria into taxonomic groups. The bacterial classification in Bergey’s Manual of Systematic 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). The bacterial phyla used in industrial microbiology and biotechnology are found in the Proteobacteria, the Firmicutes, and the Actinobacteria. 2.1.1. The Proteobacteria The Proteobacteria are a major group of bacteria 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 spp. 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 sunlight in photosynthesis. All Proteobacteria are Gram-negative with an outer membrane mainly composed of lipopolysaccharides. Many of them move about using flagella, but some are non-motile or INDUSTRIAL MICROBIOLOGY (MICR404) 4 rely on bacterial gliding. Most members are facultatively or obligately anaerobic and heterotrophic but there are numerous exceptions. Proteobacteria are divided into five groups: α (alpha), β (beta), γ (gamma), δ (delta), and ε (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 that also belongs to the Alphaproteobacteria and has the potential to become important industrially is Zymomonas which produces copious amounts of alcohol. Figure 2. Acetobacter aceti 2.1.1.1. The Acetic Acid Bacteria The acetic acid bacteria are Acetobacter and Gluconobacter. They have the following properties: 1. They carry out incomplete oxidation of alcohol leading to the production of acetic acid and are used in the manufacture of vinegar. 2. Gluconobacter lacks the complete citric acid cycle and cannot oxidize acetic acid; Acetobacter, on the other hand, has all the citric acid enzymes and can oxidize acetic acid further to CO2. 3. They can tolerate acid conditions of pH 5.0 or lower. 4. Their property of ‘under-oxidizing’ sugars is exploited in the following: a) The production of glucoronic acid from glucose, galactonic acid from galactose, and arabonic acid from arabinose; INDUSTRIAL MICROBIOLOGY (MICR404) 5  b) The production of sorbose from sorbitol by acetic acid bacteria, an important stage in the manufacture of ascorbic acid (Vitamin C). 5. Acetic acid bacteria are able to produce pure cellulose when grown in an unshaken culture. 2.1.2. The Firmicutes The Firmicutes are a division of Gram-positive bacteria. They do lack the outer membrane found in other Gram-negative forms; consequently, they are regarded as Gram-positive. Originally, the Firmicutes group included 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. Gram-positive bacteria, with G+C less than 50%, are placed in the Firmicutes, while those with 50% or more are in Actinobacteria. Firmicutes 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). 2.1.2.1. Spore-forming firmicutes Spore-forming Firmicutes form internal spores, unlike Actinobacteria where the spore- forming members produce external ones. The group is divided into the aerobic Bacillus spp. and the anaerobic Clostridium spp. Bacillus spp. are sometimes used in enzyme production. Bacillus papilliae infects and kills the larvae of the beetles in the family Scarabaeidae while B. thuringiensis is used against mosquitoes. Clostridia on the other hand are mainly pathogens of humans and animals. INDUSTRIAL MICROBIOLOGY (MICR404) 6  2.1.2.2. 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. They are among some of the most widely studied bacteria because of their importance in the production of several foods, industrial, and pharmaceutical products. They lack porphyrins and cytochromes, do not carry out electron transport phosphorylation, and hence obtain energy by substrate-level phosphorylation. They grow anaerobically but are not killed by oxygen; 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 for successful cultivation, they require the addition of amino acids, vitamins, and nucleotides. Lactic acid bacteria are divided into two major groups: The homofermentative group, which produces lactic acid as the sole product of the fermentation of sugars, and the heterofermentative which besides lactic acid also produces ethanol as well as CO2. The difference between the two results from the absence of the enzyme aldolase in the heterofermenters. Aldolase is a key enzyme in the Embden-Meyerhof-Parnas (EMP) pathway. 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 phosphate pathway. The five-carbon xylulose is split into: 1) glyceraldehyde 3-phosphate (3-carbon), which leads to lactic acid, and 2) the two-carbon acetyl phosphate which leads to ethanol. Table 1. Differences between the homofermentative LAB and heterofermentative LAB. Homofermentative LAB Heterofermentative LAB Ferment hexoses through glycolysis Ferment hexoses through the (EMP pathway) to generate energy pentose phosphate pathway to (ATP). generate energy (ATP). Have aldolase enzyme. Do not have aldolase enzyme. INDUSTRIAL MICROBIOLOGY (MICR404) 7  Produce lactic acid only (2 molecules) Produce lactic acid (1 mol), ethanol/acetic acid, and CO2. usually used as starter cultures in the dairy industry. rarely used as starter cultures in the dairy industry. Aldolase Fig. 3. Differences between a) homofermentation (glycolysis through EMP pathway) and b) heterofermentation (pentose phosphate pathway). Table 2. Characteristics of the lactic acid bacteria Group Description Habitat Importance 1 Streptococcus Cocci in pairs or Some are in the Some cause a sore throat; short chains respiratory tract, mouth, non-pathogenic strains are and intestine; others are used in yogurt manufacture found in fermenting vegetables and silage INDUSTRIAL MICROBIOLOGY (MICR404) 8 2 Enterococcus Cocco-bacilli Found as commensals Can be used to monitor water usually in pairs; in the human quality (like E. coli) previously alimentary canal; classified sometimes cause Streptococcus urinary tract infections Lancefield Group D 3 Lactococcus Coccoid, usually Plant material and Used as a starter in yoghurt occurring in alimentary canals of manufacture; Used as a pairs; hardly form animals probiotic for intestinal health; chains Produces copious amounts of lactic acid. 4 Pediococcus Growth in tetrads Found on plant Spoils beer; but required in materials special beers such as lambic beer drunk in parts of Belgium. 5 Leuconostoc Cocco-bacilli Associated with plant Tolerates high concentrations materials of salt and sugar and involved in the pickling of vegetables; produce dextrans from sucrose. 6 Lactobacillus Bacilli Aerotolerant anaerobes Used as a starter in yoghurt or microaerophilic. manufacture; Constitute a significant Used as a probiotic for component of the intestinal health human and animal microbiota in digestive system and female genital system. 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. their production of 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 and negligible amounts of other byproducts. The choice of a particular lactic acid bacterial species for production primarily depends INDUSTRIAL MICROBIOLOGY (MICR404) 9  on the carbohydrate to be fermented: Lactobacillus delbrueckii subspecies delbrueckii is able to ferment sucrose. Lactobacillus delbrueckii subspecies bulgaricus is able to use lactose 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, Lactobacillus pentosus has been used to ferment sulfite waste liquor. Lactobacillus bulgaricus Lactococcus lactis Fig. 4. Photomicrographs of Lactic Acid Bacteria Table 3. Distinguishing characteristics of lactic acid bacteria (For reading only) Characters 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 D, L, DL L L D L, DL L INDUSTRIAL MICROBIOLOGY (MICR404) 10  2.1.3. The Actinobacteria The Actinobacteria are Gram-positive bacteria 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 of the group are the Actinomycetes and Corynebacterium. Corynebacterium spp. are important industrially as secreters of amino acids. The Actinomycetes They have branching filamentous hyphae which resemble the mycelia of fungi. They are unrelated to fungi and regarded as bacteria for the following reasons: first, they have peptidoglycan in their cell walls, and second, they are about 1.0 µ in diameter, whereas fungi are at least twice that size in diameter. As a group, the actinomycetes are unsurpassed in their ability to produce secondary metabolites 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 triggering the search for bioactive microbial metabolite from soil organisms. 2.2. Eukarya: Fungi Fungi are members of the Eukarya which are commonly used in industrial production. The fungi are traditionally classified into four groups, namely Phycomycetes, Ascomycetes, Fungi Imperfecti, and Basidiomycetes. The following are currently used in industrial microbiology. 1. Phycomycetes (Zygomycetes) Rhizopus and Mucor are used for producing various enzymes. 2. Ascomycetes Yeasts are used for the production of ethanol and alcoholic beverages. Claviceps purpurea is used for the production of ergot alkaloids (potent α-blockers that cause direct smooth muscle contraction). INDUSTRIAL MICROBIOLOGY (MICR404) 11 3. Fungi Imperfecti Aspergillus is important because it produces the food toxin aflatoxin while Penicillium is well-known for the antibiotic penicillin which it produces. 4. Basidiomycetes Agaricus produces the edible fruiting body or mushroom. 3. IMPORTANT CHARACTERISTICS OF INDUSTRIAL MICROBES Microorganisms used for industrial production must meet certain requirements including those discussed below. It is important to keep these characteristics in mind when considering the candidacy of any microorganism as an input in an industrial process. 1. 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 that 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. 2. The organism should be able to grow vigorously and rapidly in the medium in use. A slow-growing organism, regardless of its efficiency in terms of the production of the target material, could be a liability. In the first place, the slow rate of growth exposes it to a greater risk of contamination in comparison to other faster growers. Second, the rate of the turnover of the production of the desired material is lower in a slower- growing organism, and consequently higher costs and lower profits. 3. 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 the reasons given above. 4. Its end products should not include toxic and other undesirable materials, especially if these end products are for internal consumption. 5. The organism should have reasonable genetic and physiological stability. An organism that mutates easily is an expensive risk. It could produce undesired INDUSTRIAL MICROBIOLOGY (MICR404) 12  products if a mutation occurred unobserved. The result could be reduced yield of the expected material, production of an entirely different product, or even a toxic material. 6. The organism should lend itself to a suitable method of product harvest (downstream) 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 due to the bacterial diameter of approximately 1 µm, while yeasts are approximately 5 µm. 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. 7. Wherever possible, organisms that have physiological requirements that 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. 8. 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, yet high-yielding, strains of the organism. 9. Where practicable, the organism should not demand large amounts of oxygen as aeration (through greater power demand for agitation of the fermenter impellers, forced air injection, etc.) contributes to about 20% of the cost of the finished product. 10. Lastly, the organism should be easily amenable to genetic manipulation to enable the establishment of strains with more acceptable properties. INDUSTRIAL MICROBIOLOGY (MICR404) 13