Industrial Biotechnology

1. Industrial Biotechnology Lecturer Dr. Kamal E. M. Elkahlout Assistant Prof. of Biotechnology

2. CHAPTER 2 Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology

3. LIVING THINGS: THREE DOMAINS • 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) 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.

4. According to the currently accepted classification living things are placed into three groups: • Archae, Bacteria, and Eukarya. • Archae and Bacteria are procaryotic while Eucarya are eucaryotic.

6. TAXONOMIC GROUPING OF MICRO-ORGANISMS IMPORTANT IN INDUSTRIAL MICROBIOLOGY ANDBIOTECHNOLOGY • Biotechnologically important Mos are found mainly among the bacteria and eukarya. • But processes used in industrial microbiology and biotechnology are dynamic. • At present organisms from Archae are not used for industrial processes, but that may change in future. • 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 physiological properties may put a member of the Archae at an advantage over contaminants.

7. 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: • i. Microorganisms grow rapidly (15 minutes) in comparison with plants and animals (6 months to 12 years). • ii. The space requirement for growth microorganisms is small. • iii. Microorganisms are not subject to the problems of weather. • iv. Microorganisms are not affected by diseases of plants and animals.

9. Bacteria • Bacteria are described in two compendia, • Bergey’s Manual of Determinative Bacteriology and • Bergey’s Manual of Systematic Bacteriology. • The first manual is designed to facilitate the identification of a bacterium whose identity is unknown. • The companion volume (on Systematic Bacteriology) records the accepted published descriptions of bacteria, and classifies them into taxonomic groups.

10. The bacterial classification is based on 16S RNA sequences, following the work of Carl Woese, and organizes the Domain Bacteria into 18 groups (or phyla). • The bacterial phyla used in industrial microbiology and biotechnology are found in the Proteobacteria, the Firmicutes and the Actinobacteria.

12. The Proteobacteria • The Proteobacteria are a major group of bacteria. • It is named after Proteus, the Greek god, who could change his shape. • They 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.

13. All are Gram-negative, with an outer membrane mainly composed of lipopolysaccharides. • Many move by 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 facultative or obligatory anaerobic and heterotrophic, but there are numerous exceptions. • Proteobacteria are divided into five groups: (alpha), (beta), (gamma), (delta), (epsilon). • The only organisms of current industrial importance in the Proteobacteria are Acetobacter and Gluconobacter, which are acetic acid bacteria and belong to the Alpha-proteobacteria. • Zymomonas belongs to the Alpha-proteobacteria, and has the potential to become important industrially Mos. • It produces abundant amounts of alcohol, but its use industrially is not yet widespread.

14. The acetic acid bacteria • 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. • 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.

15. iv. Their property of ‘under-oxidizing’ sugars is exploited in the following: • a. The production of glucoronic acid from glucose, galactonicaicd from galactose and arabonic acid from arabinose; • b. The production of sorbose from sorbitol by acetic acid bacteria (Fig. 2.5), 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.

17. The Firmicutes • The Firmicutes are a division of bacteria, all of which are Gram-positive. • Originally the Firmicutes were taken to include all Gram-positive bacteria. • 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. • 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, • Those with 50% or more are in Actinobacteria.

18. Fermicutes contain many bacteria of industrial importance and are divided into three major groups: • i. spore-forming, • ii. nonspore • forming, and • iii) wall-less (this group contains pathogens and no industrial organisms, e.g., mycoplasmas.)

19. 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 have the 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. • The genes for the toxin produced by B. thuringiensis are also being engineered into plants to make them resistant to insect pests. • Clostridia on the other hand are mainly pathogens of humans and animals.

20. Non-spore forming firmicutes • The Lactic Acid Bacteria: The non-spore forming low G+C members of the firmicutes • Theyare 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 ofthe most widely studied bacteria because of their importance in the production of some foods, and industrial and pharmaceutical products.

21. They lack porphyrins and cytochromes, do not carry out electron transport phosphorylation. • Gaining 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.

22. 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 5-C xylulose-5 -phosphate from the Pentose pathway. • The 5-C xylulose is split into glyceraldehyde 3-phosphate (3-C), which leads to lactic acid, and the 2-C acetyl phosphate which leads to ethanol (Fig. 2.6).

24. 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.

25. 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 bulgaricusis able to use lactose while Lactobacillus helveticusis able to use both lactose and galactose. Lactobacillus amylophylusand Lactobacillus amylovirusare able to ferment starch. • Lactobacillus lactiscan ferment glucose, sucrose and galactose and Lactobacillus pentosushas been used to ferment sulfite waste liquor.

31. The Actinobacteria • The Actinobacteriaare 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 of the group are the Actinomycetes and Corynebacterium. • Corynebacteriumsppare important industrially as secretors of amino acids (Chapter 21). • The rest of this section will be devoted to Actinomycetes.

32. 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 1.0 μin diameter (never more than 1.5 μ), whereas fungi are at least twice that size in diameter. • They 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.

34. Eucarya: Fungi • Fungi are Eucaryawhich 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) • Rhizopusand Mucorare used for producing various enzymes • Ascomycetes • Yeasts are used for the production of ethanol and alcoholic beverages • Clavicepspurperea is used for the production of the ergot alkaloids

35. Fungi Imperfecti • Aspergillusis important because it produces the food toxin, aflatoxin, while Penicilliumis well-known for the antibiotic penicillin which it produces. • Basidiomycetes • Agaricusproduces the edible fruiting body or mushroom • Numerous useful products are made through the activity of fungi, but the above are only a selection.

38. 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. • 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.

39. 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.

40. 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.

41. 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 bacterial diameter is approximately 1 μ, yeasts are approximately 5 μ. • 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.

42. 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 Bdellovibriosppor 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.

43. 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.