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Applications of microbes in industry: Production of primary and secondary metabolites Lesson: Applications of microbes in industry: Production of primary and secondary metabolites Author Name: Dr. Simran Jit & Dr. Nidhi Garg Reviewer: Dr. R.K.Gupta College/ Department: Miranda House , Deshbandhu College, University of Delhi Institute of Life Long Learning Page 0 Applications of microbes in industry: Production of primary and secondary metabolites Table of Contents Introduction Primary metabolites Secondary metabolites Overview of fermentation process Production of: Antibiotics Penicillin Streptomycin Amino acids Lysine Glutamic acid Organic acid Acetic acid Lactic acid Summary Exercise/ Practice Glossary References/ Bibliography/ Further Reading Institute of Life Long Learning Page 1 Applications of microbes in industry: Production of primary and secondary metabolites Introduction Industrial fermentation based on the end-product application can be categorized into four types: 1. Biomass: The end-product is viable cellular material eg, single cell protein, baker’s yeast, probiotic cultures. 2. Extracellular metabolites: Chemical compound intermediates of microbial biochemical pathways are produced and can be divided two groups: a. Primary metabolites (produced during the growth phase of the organism, eg, ethanol, citric acid, glutamic acid, lysine, vitamins and polysaccharides) b. Secondary metabolites (produced during the stationary phase, eg, penicillin, cyclosporin A, gibberellin, and lovastatin). 3. Enzymes and other proteins (intracellular components): A key component of this process is lysis of cells at the end of fermentation. Proteins are typical end products and need to be purified and crystallized. 4. Substrate transformations: Raw material is biologically transformed into a finished product. Generally used for steroid transformations, food fermentations and sewage treatment. This chapter focuses on applications of microbes for industrial production of primary and secondary metabolites. Metabolism in microorganisms involves two pathways: Primary metabolic pathways (PMPs) produce too few end products while secondary metabolic pathways (SMPs) produce a variety of products (Fig 1). There are some similarities between the pathways that produce primary and secondary metabolites, namely that the product of one reaction is the substrate for the next and the first reaction in each case is the rate-limiting step. Also the regulation of secondary metabolic pathways is interrelated in complex ways to primary metabolic regulation. Figure 1: Production of primary and secondary metbolites. Source: Author, ILLL in house Institute of Life Long Learning Page 2 Applications of microbes in industry: Production of primary and secondary metabolites Primary metabolites Involved in growth, development and reproduction. Hence, essential for survival and existence of the organism and reproduction. Formed at the same time as new cells. Production curve follows the growth curve. Formed in trophophase during exponential growth as normal end products of primary metabolism. Also called central metabolites as these maintain normal physiological processes. Cells maintain optimum concentration of all macromolecules (proteins, DNA, RNA etc.). Produced in adequate amount to sustain cell growth for example vitamins, amino acids, nucleosides etc. Overproduction can be genetically manipulated. Auxotrophic (auxo, “increase,” and trophos, ‘‘food’’) mutants having a block in steps of a biosynthetic pathway for the formation of primary metabolite (Fig 2). Growth rate slows down due to limited supply of any other nutrient. Metabolism does not stop but product formation stops. Industrially important for example ethanol, acetone, lactic acid, CO2. Common food supplements, L-glutamate and L-lysine, are produced and purified via the mass production Corynebacterium glutamicum. Citric acid, commonly used in pharmaceutical and cosmetic industries is produced by Aspergillus niger. Figure 2: Over production of primary metabolite. To maximize production manipulation of feedback inhibition pathways is performed. Another approach is to use auxotrophic mutant with defective metabolite production. Source: Author, ILLL in house Institute of Life Long Learning Page 3 Applications of microbes in industry: Production of primary and secondary metabolites Secondary metabolites • Secondary metabolites are not produced until the microbe has largely completed its logarithmic growth phase and entered the stationary phase of the growth cycle. Period of production is called idiophase and metabolites as idiolites. • In the idiophase, cells do not divide but are metabolically active. • Idiolites are organic compounds produced only after considerable number of cells and a primary metabolite have accumulated (end or near the stationary phase of growth). Rather it can be said that these are produced under sub-optimal concentrations of O2, deviations of pH or when primary nutrient source is depleted. • Though idiolites are a characteristic feature of fungal, yeast, actinomycetes and bacterial growth but are not produced by a few strains of E. coli. • In some strains secondary metabolite are produced by further conversion of a primary metabolite. • Alternatively, these may be metabolic products of the original growth medium. • Not necessary for growth, development, and reproduction like primary metabolites. Their production is influenced by environmental factors. • Secondary metabolites are synthesized for a finite period by cells that are no longer undergoing balanced growth. A single microbial type can produce very different metabolites. • Their production is regulated by complex biochemical pathways and some strains can produce a variety of idiolites. For example a strain of Streptomyces can produce a variety of 35 anthracyclines. • Typical examples include antibiotics, toxins and pigments to name a few. • Bacitracin, a peptide antibiotic, derived from Bacillus subtilis, is commonly used as a topical drug. Bacitracin is synthesized in nature via non-ribosomal peptide synthetase (NRPSs). It has a wide spectrum against Gram positive and Gram negative bacteria. Non-essential for growth. • Overproduction of secondary metabolites can be achieved by manipulating larger number of genes (gene cassettes). Institute of Life Long Learning Page 4 Applications of microbes in industry: Production of primary and secondary metabolites Figure 3: Primary and secondary metabolism. During the trophophase, the cell mass increases logarithmically but as the resources become limiting growth rate drops and production stops. Idiolites are special metabolites usually possessing bizarre chemical structures and although not essential for the producing organism's growth in pure culture, they have survival functions in nature. Source: Author, ILLL in house Figure 4: Scheme for industrial production of metabolites. Source: Author, ILLL in house Industrial Production of Microbial Metabolites The process of industrial production of microbial metabolites can summarized as in Fig 4. Basic steps are: 1. Screening, selection, maintenance of source microorganism for the production of target metabolite (primary or secondary). 2. Optimization and standardization of growth medium and conditions (w.r.t. choice of fermenter vessel, aeration, temperature, agitation, pH, etc.) for large-scale (fermentation) protocol. Institute of Life Long Learning Page 5 Applications of microbes in industry: Production of primary and secondary metabolites Preparation of the microorganism and the raw materials required for the microorganism to grow and produce the desired product is called upstream processing. 3. Sterilization of the medium, fermenter and ancillary equipment. 4. Active, pure culture in sufficient quantities is used to inoculate growth medium in fermenter. 5. The growth of the organism in the production fermenter under optimum conditions for product formation. Growth of the target microorganism in a large bioreactor (usually >100 litres) with the consequent production (biotransformation) of a desired compound is the phase of fermentation and transformation. 6. The extraction of the product and its purification. 7. Disposal of effluents produced by the process. Purification of the desired compound from either the cell medium or the cell mass is called downstream processing. Production of antibiotics Penicillin This antibiotic was first discovered by Alexander Fleming in 1929 and the producing organism associated with its production was Penicillium notatum. Penicillin (PCN or pen) is the one of most widely used effective antibiotics and is now commercially produced from Penicillium chrysogenum mutant strains NRRL-1951, Wisconsin 54-1255 and AS-P-78. The penicillin produced without addition of any side-chain precursors are referred to as natural penicillin (Penicillin G (intravenous use) and V (oral use)) (Fig 5). The conventional penicillin has been replaced by newer versions eg. Ampicillin, methicillin, carbenicillin (See Fig 9). The basic structure of the penicillin is 6-aminopenicillinic acid (6-APA) which consists of a thiazolidine ring with a condensed β-lactam ring. Depending upon the acyl moiety at position 6 there are various penicillins. The acyl group to be attached to the ring is either added in the growth medium producing biosynthetic penicillins or appended chemically to 6-APA obtained by deacetylation of penicillin through acylases forming semi-synthetic penicillins. Institute of Life Long Learning Page 6 Applications of microbes in industry: Production of primary and secondary metabolites Figure 5: Structure and Function relationship of Penicillin: Source: Yusof M, Mei Yi DC, Enhui JL, Mohd Noor N, Wan Ab Razak W, Saad Abdul Rahim A. An Illustrated Review on Penicillin and Cephalosporin: An Instant Study Guide for Pharmacy Students. WebmedCentral PHARMACEUTICAL SCIENCES 2011;2(12):WMC002776 doi: 10.9754/journal.wmc.2011.002776. Institute of Life Long Learning Page 7 Applications of microbes in industry: Production of primary and secondary metabolites Value addition: Did you Know? Heading text: Action of penicillin Institute of Life Long Learning Page 8 Applications of microbes in industry: Production of primary and secondary metabolites Diagram depicting formation of cross-links in the bacterial cell wall by a penicillin binding protein (PBP, an enzyme) and subsequent suicide inhibition by penicillin. 1. The bacterial cell wall consists of strands of repeating N-acetylglucosamine (NAG) and Nacetylmuramic acid (NAM) subunits. The NAM subunits have short peptide chains attached to them. (The exact composition of these can vary. The proximal alanine is usually L-ala and the distal two are usually D-ala.) 2. The PBP binds the peptide side chains and forms the cross-link with the expulsion of one D-Alanine from one peptide side chain. 3. The PBP dissociates from the wall once the cross-link has been formed. 4. When penicillin is administered to the cell in the culture medium, it enters the active site of the PBP and reacts with the serine group which is important in its enzymatic activity. 5. The beta-lactam ring of penicillin (represented here as the top of the "P") is irreversibly opened during the reaction with the PBP. Penicillin remains covalently linked to the PBP and permanently blocks the active site. As a result PBP is unable to bind the NAG and NAM sites due to penicillin occupied sites hence no cross linking takes place and cell wall remains porous. Source: https://commons.wikimedia.org/wiki/File:Penicillin_inhibition.svg Institute of Life Long Learning Page 9 Applications of microbes in industry: Production of primary and secondary metabolites Value addition: Did you Know? Heading text: Discovery of ‘Mould Broth Filtrate’-First antibiotic Body text: Alexander Fleming discovered mould growing on a bacterial culture in the 1920s and Dr. E.B. Chain and Sir Howard Florey worked as a team that brought curative power to millions. For this discovery all three were conferred Nobel Prize in 1942 in Medicine and Physiology. Source: https://www.youtube.com/watch?v=VGC5JOLQoGo Penicillin inhibits the cell wall synthesis in growing bacterial cells by binding with penicillinbinding protein (PBP) of the bacterial cell wall involved in the trans-peptidation reaction (cross linking) of the peptide side chains of the adjacent peptidoglycan in the bacterial cell wall. Due to the defective cell wall, the bacteria are unable to withstand the osmotic shocks and are ultimately lysed. Production of Penicillin In general, commercial penicillin is produced by fed-batch fermentation process using bioreactors of 30K-250K capacity. The fermentation temperature is maintained around 2527C, pH 6.5-7.0 with a constant supply of oxygen. The fermentation medium is composed of a carbon source like lactose and a nitrogen source like corn steep liquor. The media used for the production of penicillin contains corn steep liquor solids 3.5%, lactose 3.5%, glucose 1%, calcium carbonate 1%, potassium di-hydrogen phosphate 0.4%, edible oil 0.25% and penicillin precursors (Fig 6). Additional supplements like yeast extract, soy meal, ammonium salts are also added. In addition, side chain precursors like phenyl acetic acid or phenoxyacetic acid are also added to the medium. Institute of Life Long Learning Page 10 Applications of microbes in industry: Production of primary and secondary metabolites Corn steep liquor is an important ingredient in the broth; it supplies the fungus with nitrogen and growth factors. Since high levels of glucose repress penicillin production, lactose (from whey) is added to the corn steep liquor in large amounts as a carbon source. Figure 6: Summary of penicillin production and recovery. Source: Author, ILLL in house The media is placed at 25-27ºC with pH maintained at around 5.5-6.0. Lyophilized spores of Penicillium are used as inoculum. For better production of penicillin, development, of loose mycelial pellets is however preferred. The penicillin fermentation process can be divided into two phases: a vegetative growth phase (around 40 h) followed by penicillin production phase (150-180 h). (Fig 7) Institute of Life Long Learning Page 11 Applications of microbes in industry: Production of primary and secondary metabolites 1. Growth phase. The growth phase lasts for about 40h during which the cell mass increases very rapidly and oxygen demand is very high. 2. Penicillin production phase. The biomass production is greatly reduced and rate of penicillin production increases. As the lactose approaches limiting levels and cell densities in the fermenter become very high, addition of low levels of glucose maximize penicillin yield. Various other media components are fed during this phase to extend the penicillin production for 120-180h. Figure 7: Production of Penicillin. During the growth phase, very little penicillin is produced, but once the carbon source has been nearly exhausted, the penicillin production phase begins. By supplying additional carbon and nitrogen at just the right times, the production phase can be extended for several days. Source: Author, ILLL in house Recovery of penicillin This process is referred to as downstream processing. For this, the fermentation broth is first chilled and passed through rotary filter to remove biomass (Fig 6). The filtrate is acidified to pH 2.0-2.5 with H2SO4 and the penicillin is extracted with butyl acetate. Penicillin is extracted from solvent (butyl acetate) into aqueous buffer with pH 7.0. Aqueous fraction is acidized again and re-extracted into butyl acetate. To recover penicillin, the extract is crystallised by addition of sodium or potassium hydroxide or acetate. The penicillin GK+ salt crystals are washed with acetone by centrifugation and dried under vacuum. The penicillin crystals obtained are more than 90% pure. Semisynthetic penicillins The unstable nature of β-lactam ring makes penicillin molecule susceptible to metabolism. Thus, one of the major drawbacks is that penicillin is not orally active and must be injected. The present drug development focuses on i) to increase chemical stability for oral administration; ii) Institute of Life Long Learning Page 12 Applications of microbes in industry: Production of primary and secondary metabolites to increase resistance to β -lactamases and iii) to increase the range of activity. Analogs of penicillin are produced by varying the carboxylic acid during fermentation or by the semisynthetic approach (Fig 9). These analogs have increased stability and are effective against wide range of pathogens. Semi-synthetic penicillins are broad-spectrum antibiotics and most of them can be taken orally and thus do not require injection. Natural penicillin is treated to yield 6-APA which is further modified chemically by the addition of a side chain (Fig 8). Figure 8: Derivation of natural penicillin to produce semisynthetic and biosynthetic variants. Source: Author, ILLL in house Institute of Life Long Learning Page 13 Applications of microbes in industry: Production of primary and secondary metabolites Figure 9: Analogs of Penicillin. Streptomycin Source: Author, ILLL in house Streptomycin is a broad spectrum antibiotic (antimycobacterial) belonging to oligosaccharide antibiotic/aminoglycoside family. Streptomycin was discovered by Schatz, Bugie, and Waksman in 1944 from Streptomyces griseus isolated from soil. It was the first effective treatment against Mycobacterium tuberculosis. It is also effective against some Gram positive bacteria primarily Staphylococcus aureus and is mainly used against the pathogenic bacteria resistant to penicillins. This antibiotic is also active against other diseases, e.g., plague (Pasteurella pestis), brucellosis (Brucella abortus), tularemia Francicella tularieusis). Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to codon misreading followed by complete or partial inhibition of protein synthesis and eventually death of microbial cells. Humans have ribosomes which are structurally different from those in bacteria, so the drug does not have this effect in human cells. Institute of Life Long Learning Page 14 Applications of microbes in industry: Production of primary and secondary metabolites Figure 10: Isolation and screening of antibiotic producers. Source: Author, ILLL in house The isolation and screening of novel antibiotic producer is outlined in Fig 10. The first phase is the isolation for antibiotic producing strains for example of Streptomyces. This is done on selective media hence most colonies obtained are of Streptomyces. When the indicator strain is overlaid, some strains which show sensitivity as measured by Zone of Inhibition are picked up. These colonies represent antibiotic producing strains. Common indicator strains include Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumoniae. Then, the producer strain colony is picked and streaked to test for its antibiotic spectrum. It is streaked over one-third of plate and incubated. After good growth is obtained, the bacterial species (5 in this case) are streaked perpendicular to the producing organism, and the plate was further incubated. The failure of several species to grow near the producing organism indicates that it produced an antibiotic active against these bacteria. Figure 11: Structure of Streptomycin. Source: Abdul Rahim AS, Muhamad Sayuti M, Hau KC, Ee DC, Wan Zaki W, Raskitar N, et al. An Illustrated Review About Aminoglycosides. WebmedCentral PHARMACEUTICAL SCIENCES 2011;2(12):WMC002744 doi: 10.9754/journal.wmc.2011.002744 Production of Streptomycin Institute of Life Long Learning Page 15 Applications of microbes in industry: Production of primary and secondary metabolites Streptomycin is derived from the bacterium Streptomyces griseus.Streptomycin has three constituents namely; N-methyl L-glucosamine, Streptose and streptidine (Fig 11). The present day streptomycin producers are the mutants of Streptomyces derived for higher yields with yield as high as 25,000 units per ml. The industrial production of streptomycin is carried out using submerged fermentation processes. As the Streptomyces mutants are genetically unstable, the spores are maintained as soil stocks or lyophilized and are used for inoculating sporulation medium, which is then transferred to germinator where biomass is increased for inoculating fermenters. The fermentation media consists of glucose, starch, dextrin, soy meal, corn steep liquor, sodium sulphate. The streptomycin fermentation requires high aeration and agitation. The fermentation is carried out at 28-30ºC with pH maintained at 7.6-8 for good productivity. The fermentation lasts for 5-7 days with of yield of 1-3 g/L of the fermentation broth (Fig 12). Figure 12: Streptomycin production by S. griseus. Glucose and other carbohydrates have been reported to interfere with antibiotic synthesis and this effect depends on the rapid Source: Author, ILLL in house The streptomycin fermentation proceeds through three phases (Fig 12 and 13): 1. In first phase, the organism produces proteases which digest the soybean meal and release ammonia and carbohydrates. These are utilized for increasing the biomass. Glucose is slowly utilized and net production of streptomycin is low during this phase. The pH of the medium increases from 6.7 or 6.8 to 7.5 or higher. This phase lasts for 24h. 2. The next phase is the idiophase or the stationary phase during which maximum streptomycin (secondary metabolite) is produced. It ranges from 24h to 6-7 days. Rapid utilization of ammonia and glucose occurs with no mycelial growth and pH during this phase remains fairly constant at 7.6 to 9.0. 3. In the last phase (death phase) the sugars have been completely depleted in the medium and streptomycin production ceases completely. The ammonia released due to the cell Institute of Life Long Learning Page 16 Applications of microbes in industry: Production of primary and secondary metabolites lysis raises medium pH. Fermentation broth is generally harvested before the last phase begins. Figure 13: Phases of streptomycin production. Source: Author, ILLL in house Recovery of Streptomycin On completion of fermentation, the mycelium is separated from the broth by filtration. The remaining liquid is then percolated through cation exchange resin columns where streptomycin gets adsorbed and is finally eluted by washing with buffer as streptomycin sulphate. Further impurities are removed by treating it with sodium hypochlorite, EDTA and activated carbon. The purified streptomycin sulphate solution is concentrated under vacuum and dried aseptically. Institute of Life Long Learning Page 17 Applications of microbes in industry: Production of primary and secondary metabolites Production of amino acids Lysine L-Lysine (Fig 14) is an essential amino acid as it is not synthesized by humans and hence has to be obtained from the diet. Cereals and vegetables also have low lysine content and are often fortified with lysine supplements. It is also produced for use as animal feed. Figure 14: Structure of Lysine. Abbreviated as Lys or K, is an α-amino acid. Lysine's codons are AAA and AAG. Source:https://commons.wikimedia.org/wiki/File:Lysine-zwitterion-2D.png Production of Lysine Lysine is produced in a two-step process by combination of two bacterial strains: A. Lysine-histidine double auxotrophic mutant Escherichia coli and B. Aerobacter aerogenes. Firstly, E. coli is grown in molasses based medium containing glycerol, corn-steep liquor, and (NH4)2HPO4 to produce diaminopimelic acid (DAP). Apart from glycerol, ethanol or alkanes supplemented with soybean hydrolysates can also be used as carbon sources. The temperature is maintained at 28ºC. After three days of fermentation under optimized conditions of temperature and pH in aerated stirred tank rectors, the entire fermentation broth is then incubated with Aerobacter aerogenes at 35ºC, which decarboxylates the DAP to L-lysine. Institute of Life Long Learning Page 18 Applications of microbes in industry: Production of primary and secondary metabolites Figure 15: Biotransformation of Diaminopimelic acid (DAP) to lysine by DAP decarboxylase. Source: Author, ILLL in house Another industrial method utilizes one step single strain approach. For this, Corynebacterium glutamicum (homoserine auxotrpohs) and Brevibacterium (methionine-threonine double auxotrophs) are used and production of L-lysine is controlled at the level of the enzyme aspartokinase. In this method Aspartate is used as the precursor and through series of biochemical pathways as shown in Fig 16 excess lysine feedback inhibits the activity of this enzyme. Figure 16: Single step production of lysine using mutant Corynebacterium glutamicum. In the biochemical pathway leading from aspartate to lysine; lysine can feedback-inhibit activity of the enzyme aspartokinase causing cessation of lysine production. S-aminoethylcysteine (AEC) an analog of lysine is identical to lysine in structure except that a sulfur atom replaces a methyl (-CH2) group. AEC normally inhibits growth, but AEC-resistant mutants of C. glutamicum have an altered allosteric site on their aspartokinase and grow and overproduce lysine because feedback inhibition no longer occurs. Source: Author, ILLL in house To achieve higher production values mutants of C. glutamicum are used in which aspartokinase is no longer subject to feedback inhibition. Such mutants can be generated by repeated culturing in a medium containing lysine analog S-aminoethylcysteine (AEC). AEC binds to the allosteric site of aspartokinase and inhibits activity of the enzyme. However, AEC-resistant mutants can be obtained easily and synthesize a modified form of aspartokinase whose allosteric site no longer recognizes AEC or lysine. Institute of Life Long Learning Page 19 Applications of microbes in industry: Production of primary and secondary metabolites In such mutants, feedback inhibition of this enzyme by lysine is nearly eliminated. A typical AEC-resistant mutant of C. glutamicum can produce over 60 g of lysine per litre in industrial fermenters. Industrial production is carried out as batch process in aerated stirred tank reactors. A variety of carbon sources such as sugarcane molasses, acetate, ethanol or alkanes supplemented with soybean hydrolysates can be used. Gaseous ammonia or ammonium salts are used as nitrogen source and urea along with other growth factors such as L-homoserine or L-threonine and L-methionine. The addition of ammonia and urea also helps in maintaining a neutral pH around 7. It is critical to maintain biotin content in the medium >30 μg/L for optimal lysine production and lower concentrations result in the accumulation of L-glutamate instead of L-lysine. To shorten the lag phase during fermentation larger ~ 10% inoculum is used. Lysine production starts in the early log phase of growth and through the stationary phase and a maximum of production 40-45 g/L lysine is achieved in 60h. Recovery of Lysine Lysine is recovered by acidification of the cell free fermentation broth to pH 2.0 with hydrochloric acid. This acidified mixture is then passed through cation exchange column where L-lysine gets adsorbed in the ammonium form. Bound L-lysine is then eluted from column with dilute ammonia solution. The eluate is re-acidified and crystallized as L-lysine hydrochloride. Glutamic acid Kyowa Hakko, Japan was the first company to start the production of L-glutamic acid (monosodium glutamate, MSG) by fermentation using Corynebacterium glutamicum. Breviacterium, Microbacterium and Arthrobacter are some other glutamic acid producing bacteria. These bacteria are Gram positive, nonsporulating, non-motile, require biotin. Importantly, these lack or have little α-ketoglutarate dehydrogenase activity and show high glutamate dehydrogenase activity (Fig 17). Isocitrate dehyderogenase catalyses the decarboxylation and dehydrogenation of isocitrate to α-ketoglutarate in tricarboxylic acid (TCA) cycle. This α-ketoglutarate is the key precursor, which is converted into L-glutamic acid via reductive amination with free NH4+ ions, catalysed by glutamate dehydrogenase. The strains used for commercial production of glutamic acid lack or have very low α-ketoglutarate dehydrogenase activity. Institute of Life Long Learning Page 20 Applications of microbes in industry: Production of primary and secondary metabolites Figure 17: Enzymes involved in the biosysnthesis of L-glutamate Source: Author, ILLL in house Production of Glutamic acid The glutamic acid bacteria in normal growth conditions synthesize glutamic acid intracellularly and do not secrete it out of the cell because of the rigid cell wall. However, the permeability of the bacterial cells can be enhanced by: i. restricting the formation of normal phospholipid biosynthesis using biotin deficient media, ii. limiting oleic acid in oleic acid auxotrophs, iii. limiting glycerol in glycerol auxotrophs, iv. addition of surfactants (e.g. Tween 60) or by adding C16 - C18 saturated fatty acids, v. addition of penicillin in case of biotin rich media to weaken the cell wall thus rendering high permeability for secreting glutamic acid in the culture broth and vi. limiting biotin, an essential co-factor. Deficiency of biotin affects fatty acid biosynthesis, membrane formation falters, permeability is affected and intracellular export of glutamic acid is altered. Stirred tank reactor up to 450 m3 is used for industrial production of glutamic acid. The fermentation is carried out aerobically at 30-37°C, depending on the microorganisms used. Glucose, fructose, sugar cane and sugar beet molasses, and starch hydrolysates are some carbon sources used in production of L-glutamic acid. Institute of Life Long Learning Page 21 Applications of microbes in industry: Production of primary and secondary metabolites Penicillin or fatty acid derivatives (e.g. Tween 60) are added in the sugar cane or sugar beet molasses based medium upsetting the cell wall synthesis of these bacteria as these carbon sources contain high biotin (0.02-0.12 mg/Kg) content favoring the formation of cell membrane with high lipid content. Acetate, methanol, ethanol, acetaldehyde, or n-alkane have also been employed as carbon source in the production of L-glutamic acid by bacteria, but still cane sugar molasses or starch hydrolysates are the main carbon sources. Ammonium salts or ammonia are generally used as nitrogen source. In case of glutamic acid bacteria having high urease activity, urea can also be used as nitrogen source in the medium. Oxygen supply is necessary for glutamic acid production and under oxygen deficiency, excretion of lactate and succinate occurs, whereas excess oxygen results in ammonium ion deficiency, ceasing the growth and production of α-ketoglutarate, thus lowering the Lglutamic acid yield in both cases (Fig 18). Medium pH during fermentation is maintained at 7-8 by the addition of alkali/ammonia. L-glutamic acid starts accumulating from the mid-way of the fermentation process which normally lasts for 30-35 h and finally L-glutamic acid level reaches to 100g/L in the fermentation broth in case of Brevibacteriumdivaricatum. In acidic pH with excess ammonia, glutamine is produced instead of L-glutamic acid. Glutamic acid bacteria convert 50-60% of the added carbon source to L-glutamic acid under the optimal culture conditions. Recovery of Glutamic acid At the end of the fermentation the broth contains l-glutamate in the form of its ammonium salt. L-Glutamic acid is recovered from the fermentation broth by separating the cells from the culture medium and passing the broth through anion exchange resin. L-Glutamate ions bind to the resin and ammonia is released. Elution is performed by NaOH to form monosodium glutamate (MSG). MSG crystals are then prepared and further purified to food-grade quality (Fig 19). Institute of Life Long Learning Page 22 Applications of microbes in industry: Production of primary and secondary metabolites Figure 18: Biosynthetic production of Glutamic acid production by Corynebacterium glutamicum. Bold arrows represent major carbon flows while the dashed lines indicate reactions that are being used to a lesser extent. On left growth occurs by the glyoxylate bypass to provide critical intermediates in the TCA cycle. On the right pathway after growth is depicted. As can be seen most of the substrate carbon is processed to glutamate. Source: Author, ILLL in house Institute of Life Long Learning Page 23 Applications of microbes in industry: Production of primary and secondary metabolites Figure 19: An overview of L-glutamate (Mono Sodium Glutamate) production. Source:http://glutamicacid.wikispaces.com/?responseToken=0a62912f0da7aaf080bf8a89fb7aef 5d9 Production of Organic acid Acetic acid (Vinegar) Acetic acid (CH3COOH) is the principal constituent of vinegar. It is also known as ethanoic acid, ethylic acid, vinegar acid, and methane carboxylic acid (Fig 20). Glacial acetic acid is the pure compound (99.8%), as distinguished from the usual water solutions known as acetic acid. Vinegar is used as a flavouring agent in salads and other foods, and because of its acidity, it is also used in pickling. Foods pickled with high concentrations of vinegar can be stored unrefrigerated for years. Institute of Life Long Learning Page 24 Applications of microbes in industry: Production of primary and secondary metabolites Figure 20: Structure of acetic acid. Source: https://commons.wikimedia.org/wiki/File:Acetic-acid-2D-flat.png Production of Acetic acid Acetic acid producing bacteria are Gram-negative bacteria which belong to the family Acetobacteriaceae, and to the alpha-subclass of Proteobacteria. The recognized genera are: Acetobacter, Acidomonas, Gluconobacter, Gluconacetobacter, and Kozakia. These are obligate aerobes, motile rods that carry out the incomplete oxidation of alcohols and sugars, leading to the accumulation of organic acids as end products. The acetic acid bacteria are tolerant of acidic conditions and most strains thrive well at pH values lower than 5. Two important biochemical changes take place in the production of vinegar (Fig 21): A. Alcoholic fermentation of a carbohydrate. B. Oxidation of the alcohol to acetic acid. Figure 21: Acetic acid (vinegar) production. In the first step ethanol is oxidised to acetaldehyde by alcohol dehydrogenase (ADH). Second oxidation by the same enzyme produces acetic acid. ADH donates electrons directly to ubiquinone (UQ) in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria. Source: Author, ILLL in house Different starting substrates eg., dilute ethanol solutions, fermented rice, fruit juices, hydrolysed starchy material, sugar syrups, alcoholic apple juice (hard cider) are used depending on the type of vinegar to be produced. The production of ethanol from a carbohydrate source is carried out at 30-32C using anaerobic yeast Saccharomyces cerevisiae. The alcohol produced by this fermentation act as a substrate for the second step carried out by strictly aerobic bacteria. Institute of Life Long Learning Page 25 Applications of microbes in industry: Production of primary and secondary metabolites The flagellated Acetobacter and Gluconobacter are principal acetic acid producing microorganism. While Acetobacter is able to further oxidize acetic acid forms to CO2, for Gluconobacter acetic acid is a “deadend”product (Fig 22). A B Figure 22: Acetic acid producing bacteria: (A) Acetobater is a Gram negative bacterium capable of converting alcohol into acetic acid. The aerobic rod shaped bacterium grows in temperatures around 30C and found in any alcoholic niches such as flowers, soil and water. (B) Gluconobacter partially oxidize carbohydrates and alcohol through oxidative fermentation. Used to synthesis vitamin C, D-gluconic acid and ketogluconic acids. Source:http://microbiologyglossary.wikispaces.com/Acetobacter_acetihttp://www.micronaut.ch/ shop/gluconobacter/ In the reaction depicted in Figure alcohol acts as the electron donor and is oxidised to acetic acid by Gluconobacter. Thermophilic bacteria can utilize cellulose instead of alcohol as substrate while Acetobacterium woodii and Clostridium aceticum can synthesize acetic acid from hydrogen and carbon dioxide. Since aerobic microbes employed for the acetic acid production, oxygen demand is very high during fermentation. Three processes are used for commercial vinegar production. 1. Open-vat method: In this process, wine is placed in shallow vats to facilitate exposure to the air. At the surface of the liquid, the acetic acid bacteria develop as a slimy layer. The process is continued over a period of several months at room temperature, the fruit juices ferment into alcohol and then oxidized into acetic acid. Although this original process is still used but it is not very efficient as the bacteria contact both the air and substrate only at the surface (Fig 23). Alcohol is poured into wooden barrels in which holes have been drilled on the top as well as ends. To initiate the process a starter vinegar stock is added and the barrel is filled to a level just below the holes on the ends. The barrel is then covered with nets and allowed to sit for several months. The room temperature is kept at approximately 29°C. Institute of Life Long Learning Page 26 Applications of microbes in industry: Production of primary and secondary metabolites Figure 23: Open-vat method of vinegar production. This method was used until nineteenth century. In this method fermented fruit juices are exposed to the airborne acetic acid producing bacteria. Thereafter the contents are removed by filtration. Since wild type strains are used, the final extract also contains a mixture of other acids such as lactic and propionic acid besides vinegar. Source: Author, ILLL in house 2. Trickle method or generator method: This method is used for the production of distilled and industrial vinegar and operated in a continuous fashion. Loosely packed twigs or wood shavings are arranged as tall oak vats or column and filled with charcoal or grape pulp. These provide a surface for bacteria to grow to form a biofilm using trickling fluid as a substrate. These wood shaving are not consumed and can last from 5 to 30 years, depending on the kind of alcoholic liquid used in the process. Alcoholic liquid is trickled over the top of the vat and slowly drips down the fillings. The process is aerated with dual oxygen supply through punched holes at the top and perforations at bottom. A stream of air is supplied from bottom which then passes upward to provide oxygen to aerobic bacteria. The process is continued for several weeks after which vinegar is poured off from the bottom of vat into storage tanks. This may be percolated again to complete oxidation of alcohol and hence maximise production. The vat is maintained within the temperature range of 15-34C (Fig 24). Institute of Life Long Learning Page 27 Applications of microbes in industry: Production of primary and secondary metabolites The major advantage of this method is that it is low on cost. In addition, ease of maintenance, small space requirement and high acetic acid concentrations of the product are other benefits of this method. However, disadvantages of the process are: long start-up time, loss of ethanol by volatilization, and production of slime-like material by Acetobacter. Figure 24: Production of vinegar by continuous fermentation. Alcoholic juice is trickled through wood shavings and air is passed from the bottom and moves upward. Acetic acid bacteria colonize the wood shavings form a biofilm that oxidizes alcohol to acetic acid. The dilute acetic acid is pooled in the collecting chamber. It is recycled through the generator. After the acetic acid content reaches 4% (the minimum concentration to be labelled as vinegar), the pooled product is drained off. This submerged, batch process using Frings acetator can produce 12% acetic acid in about 35 h. Source: Author, ILLL in house 3. Bubble method: It incorporates industrial incubation techniques to introduce and mix air into a fermenter containing the alcoholic substrate and inoculated with acetic acid bacteria. The bubble method is highly efficient; 90–98% of the alcohol is converted to acetic acid. Recovery of Acetic acid The vinegar produced by trickle method has high acetic acid content (14%), and must be diluted with water to bring its acetic acid content to a range of 5-6%. Distilled vinegar is produced by pouring the diluted liquid into a boiler and brought to its boiling point. A vapour rises from the liquid and is collected in a condenser. It then cools and becomes liquid again. This liquid is then bottled as distilled vinegar. Institute of Life Long Learning Page 28 Applications of microbes in industry: Production of primary and secondary metabolites Figure 25: Some applications of acetic acid in cosmetic, food and chemical industry. Source: Author, ILLL in house Lactic acid Lactic acid (2-hydroxypropanoic acid) was discovered and isolated in 1780 by the Swedish chemist Scheele from sour milk and the first organic acid produced microbiologically in 1881 by Charles E. Avery at Littleton, Massachusetts, USA (Fig 26 a). Lactic acid is a highly hygroscopic, syrupy liquid and is technically available in various grades, i.e. technical grade, food grade, pharmacopoeia grade and plastic grade (Fig 26 b). Institute of Life Long Learning Page 29 Applications of microbes in industry: Production of primary and secondary metabolites Figure 26 a: During intense exercise our body cannot provide enough oxygen to allow the complete combustion of glucose to carbon dioxide. Under these conditions, an alternative means of obtaining energy from glucose by converting it to lactic acid. Source:http://2012books.lardbucket.org/books/principles-of-general-chemistry-v1.0/s09-07end-of-chapter-material.html Figure 26 b: Commercial applications of lactic acid. Source: Author, ILLL in house Institute of Life Long Learning Page 30 Applications of microbes in industry: Production of primary and secondary metabolites Figure 27: Lactic acid fermentation. Source:https://eochemistry.wikispaces.com/acid+and+running+-+alek,+adam Lactic acid producing bacteria (LABs) belong to the genera Lactococcus, Streptococcus, Pediococcus, Oenococccus, Leuconostoc and Lactobacillus and have been isolated from natural habitats like plants or fermented foods like dairy and meat products (Fig 27, 28). Materials frequently used include glucose syrups (e.g. derived from starch hydrolysis), maltose-containing materials, sucrose (e.g. from molasses) or lactose (whey). Lactobacillus delbrueckii subsp bulgaricus which utilize lactose as substrate is industrially important organisms used for the production of dairy products like yoghurt, cheese, buttermilk and kefir and is also used to produce lactic acid from whey. Lactobacillus delbrueckii and L. pentosus produce lactic acid when glucose and sulphite waste liquor are used as substarte, respectively. Other biological agents capable of producing lactic acid are also used such as strains of Rhizopus, Escherichia, Bacillus, Kluyveromyces and Saccharomyces. Institute of Life Long Learning Page 31 Applications of microbes in industry: Production of primary and secondary metabolites A B Figure 28: Lactobacillus bulgaricus. (A)It is a gram-positive rod that may appear long and filamentous. It is non-motile and does not form spores. It is regarded as aciduric or acidophilic, since it requires a low pH (around 5.4–4.6) to grow effectively. The bacterium has complex nutritional requirements. (B) Lactic acid production from whey by Lactobacillus bulgaricus. Source: (A)http://mrrohanbio.wikispaces.com/Lactobacillus+Bedlbrueckii (B) Source: Author, ILLL in house Apart from fermenting milk, LABs are also used to ferment vegetables, meat, fish and cereals. These can also improve taste and texture of fermented food. Finally, LABs play an important role in the production of alcoholic beverages. In case of homofermentative bacteria (Lactobacillus sp.), very little substrate is used for producing cell mass and other metabolites and majority of the carbon source is converted to lactic acid, and here the percent conversion of sugars to lactic acid is virtually equivalent to the theoretical yield of two moles of lactic acid per mole of hexose sugar utilized. Production of Lactic acid LABs are categorized into two groups: i) Homolactic fermenters which utilizes the glycolytic pathway and directly reduce almost all their pyruvate to lactate with the enzyme lactate dehydrogenase; and ii) Heterolactic fermenters produce substantial amounts of products other than lactate for example ethanol and CO2 by way of the phosphoketolase pathway. Commercial production of lactate utilizes only homolactic fermenting strains of LABs In both, the starter culture is maintained in skimmed milk. The typical medium employed in the fermentation process consists of 10-15 % dextrose, 10 % calcium carbonate, and small amounts of nitrogenous substances such as malt sprouts and (NH4)2HPO4(0.25 %). LABs have complex nutritional requirement hence medium is supplemented with B vitamins and some amino acids. Incubation temperature is 43-45C for 48 hours. The free lactic acid produced must be neutralized as it is toxic to the bacteria. The pH of the fermenting medium is maintained between 5.5 and 6.5 by addition of calcium carbonate or calcium hydroxide. This precipitates the free lactic acid by forming calcium lactate. Since lactic acid fermentation is an anaerobic process no aeration of the broth is required but agitation is essential for proper mixing the calcium hydroxide to react with lactic acid produced. Institute of Life Long Learning Page 32 Applications of microbes in industry: Production of primary and secondary metabolites Figure 29: Biosynthesis of Lactic acid. Anaerobic fermentation of glucose produces lactic acid in which pyruvate is reduced to lactic acid by lactate dehydrogenase and generating NAD from NADH2 for glycolysis. The LABs produce either D(-)-lactic acid, L(+)-lactic acid or the racemic mixture of both D and L isomers. Source: Author, ILLL in house Recovery of Lactic acid After 48 - 72 hours of fermentation, contents of fermenter are boiled to coagulate protein. The filtrate containing calcium lactate is concentrated by removal of water under vacuum followed by purification. A standard procedure for recovery from rather pure nutrient media is given in Figure 30. Broths from the fermentation of lower quality raw materials require even more extensive purification steps, including pre-purification by filtering the hot calcium lactate solution, and its repeated re-crystallisation. Alternatives used are solvent extraction (e.g. using isopropyl ether, 2-butanol or tri alkyl tertiary amines in organic solvents), or esterification with methanol and subsequent distillation. Ammonia can be used as an alternative to calcium bicarbonate and lactic acid can be recovered by esterification. But this increases the cost of fermentation considerably. Institute of Life Long Learning Page 33 Applications of microbes in industry: Production of primary and secondary metabolites Figure 30: With the addition of lime or chalk, the raw materials are fermented in a fermenter and crude calcium lactate is formed. Calcium is removed as calcium sulphate (gypsum) by adding sulphuric acid to the crude concentrated calcium lactate resulting in crude lactic acid. The crude lactic acid is further purified and concentrated to get L (+) lactic acid. Source: Author, ILLL in house Institute of Life Long Learning Page 34 Applications of microbes in industry: Production of primary and secondary metabolites Summary Industrial fermentation can be applied for the production of i) Biomass; ii) Extracellular metabolites iii) Enzymes and other proteins; and iv) Substrate transformations. Several of the metabolites produced by yeast, fungi and bacteria are useful to us food supplements & alternative; cosmetic ingredients; antibiotics and as input to industry for producing a variety of commercially important products. Industrially important strains have been derived from prototrophic strains by classic technique of medium replacement or through genetic manipulations to maximise production yields. Primary metabolites are produced during active period of growth and are essential for growth and reproduction. Secondary metabolites are produced in response to specific environmental conditions and during stationery phase of growth. Primary metabolites Amino acids Vitamins Nucleic acid Polysachchrides Ethanol Secondary metabolites Antibiotics Pigments Toxins Alkaloids Steroids Lactic acid Polymeric substances eg gums, rubber Large scale fermentation purposes are specifically adjusted to microbial growth conditions. Downstream processing i.e., recover, purification, packaging and shipment are of equal significance. For production of penicillin, a submerged fermentation process is followed with high aeration and agitation with 25-27ºC with pH maintained at around 5.5-6.0. Lyophilized spores of Penicillium are used as inoculum. The industrial production of streptomycin is carried out using submerged fermentation processes. Streptomyces griseus is the principal strain for industrial production. The fermentation is carried out at 28-30ºC with pH maintained at 7.6-8. The fermentation lasts for 5-7 days with of yield of 1-3 g/L of the fermentation broth. L-Lysine can be produced in a two-step process by combination of Lysine-histidine double auxotrophic mutant Escherichia coli and Aerobacter aerogenes. Industrial method utilizes one step single strain approach majorly relying on Corynebacterium glutamicum. An important feature of industrial production of L-glutamic acid (monosodium glutamate, MSG) using Corynebacterium glutamicum is permeabilization of rigid cell wall which otherwise is an intracellular aggregate. Institute of Life Long Learning Page 35 Applications of microbes in industry: Production of primary and secondary metabolites Lactic acid producing bacteria or LABs is used to ferment milk, vegetables, alcoholic beverages meat, fish and cereals. These can also improve taste and texture of fermented food. Exercise/ Practice 1. Define the following: a. Auxotrophs b. Idiolites c. Homofermentative LAB d. Biosynthetic penicillin. 2. How are industrial microorganisms different from microorganisms in nature? In what ways are they similar? 3. List three examples of primary metabolites and give their source organism. 4. Compare and contrast the production of natural, biosynthetic, and semi-synthetic penicillins. 5. What are the factors that influence production of secondary metabolites? 6. Explain how can Corynebacterium glutamicum be used for production of L-Glutamic acid and L-Lysine. 7. Explain different methods for the production of vinegar. 8. Write a short on LABs. 9. How are antibiotic producing strains isolated and screened. Institute of Life Long Learning Page 36 Applications of microbes in industry: Production of primary and secondary metabolites Glossary Fermentation: Anaerobic catabolism of an organic compound in which the compound serves as both an electron donor and an electron acceptor and in which ATP is usually produced by substrate-level phosphorylation. Metabolome: The total complement of small molecules and metabolic intermediates of a cell or organism. β-Lactam: Antibiotic a member of a group of antibiotics including penicillin that contain the four-membered heterocyclic β-lactam ring. Broad-spectrum antibiotic: An antimicrobial drug useful in treating a wide variety of bacterial diseases caused by both gram-negative and gram-positive bacteria. Industrial microbiology: Large-scale use of microorganisms to make products of commercial value. LAB: Lactic acid producing bacteria. Institute of Life Long Learning Page 37 Applications of microbes in industry: Production of primary and secondary metabolites References/ Bibliography/ Further Reading 1. Brock Biology of Microorganisms (13th Edition) by: Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark. Benjamin Cummings. 2. Microbiology: Principles and Explorations (8th Edition) by: Jacquelyn G. Black. John Wiley & sons, Inc. 3. Crueger and A. Crueger; Biotechnology: A Textbook of Industrial Microbiology (Eng. Ed. T. D. Brook). Sinaeur Associates, 1990. 4. L. E. Casida, Jr.; Industrial Microbiology. Wiley Eastern Ltd. Suggested Readings 1. Derkx PM, Janzen T, Sørensen KI, Christensen JE, Stuer-Lauridsen B and Johansen E. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology. Microbial Cell Factories 2014, 13(Suppl. 1):S5 doi:10.1186/1475-2859-13-S1S5 Further Readings 1. Garg N, Jit S. 2015. Fermentation Process. https://drive.google.com/file/d/0B0Izh6GcIA_DV2ViRmJnNW5seXM/view?pli=1. 2. Garg N, Jit S. 2015. Fermentation Products and DownstreamProcessinghttps://drive.google.com/file/d/0B0Izh6GcIA_DMVdfWVBBMzNUVDA/v iew?pli=1. Useful Web Links https://www.mdpi.com/1422-0067/13/5/5482/xml http://www.sciencedirect.com/science/article/pii/S1687850714000314 Institute of Life Long Learning Page 38 Applications of microbes in industry: Production of primary and secondary metabolites Institute of Life Long Learning Page 39