Microbial Metabolic Pathways!
Biological degradation of recent biomass and organic chemicals during solid waste or wastewater treatment proceeds either aerobic (respiration), anaerobic (methanogenesis) and sometime anoxic (denitrification).
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In aerobic process, organic compounds like carbohydrates, proteins, fats, or lipids are digested using aerobic bioreactors like activated sludge system, that leads to the formation of carbon dioxide, water, and a significant amount of surplus sludge. Oxygen in pure form or air must either be supplied by aeration or by injection in to the reactor.
Capacity for oxygen transfer and the stripping efficiency for carbon dioxide from respiration are two important factors in oxygen supply. It is stripping of CO2 which is common process required to prevent a decrease value of pH and to remove heat energy. During respiration, denitrification process occurs in which chemically bound oxygen supplied in the form of nitrate or nitrite and yields dinitrogen anaerobic organisms, such as methanogenes or sulphate reducers use nitrate as bulk mass to reduce redox potential.
If anaerobic zones are formed in sludge floes of an activated sludge system, e.g., by limitation of the oxygen supply, methanogens and sulphate reducers may develop in the centre of sludge floes and produce small amount of methane and hydrogen sulphide. Both aerobic and anaerobic micro-organisms are consuming the waste material as feed (carbon and energy source) and metabolise waste components in to valuable products by using different metabolic pathways.
1. Hydrolysis of Cellulose by Aerobic and Anaerobic Micro-Organisms:
Cellulose, hemicellulose and lignin are the major structural compounds of plants. Cellulose is the most abundant biopolymer on earth. Cellulose fibres are formed of linear chains of 120-1300 glucose units linked together by glycosidic bonds.
These fibres are arranged in a matrix of hemicelluloses, pectin, or lignin. The hemicelluloses consist mainly of xylans or glucomannans, which have side chains of acetyl, gluconuryl, or arabinofuranosyl units. Hemicellulose and pectin or lignin make a cover and protect cellulose.
To make cellulose fibres available to micro-organisms, first of all hemicellulose, pectin, or lignin matrix must be degraded microbiologically or solubilised chemically. Cellulose degradation is naturally occurring in the presence of oxygen in soil and also in the absence of oxygen in the rumen of ruminants.
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There are some microbial genera containing cellulolytic micro-organisms, e.g., genera of fungi; Trichoderma, Phanaerochaete, Neocallimastix, Piromyces, and genera bacteria, Cellulomonas, Pseudomonas, Thermomonospora (aerobic cellulose degraders) and Clostridium, Fibrobacter, Bacteroides, Ruminococcus (anaerobic cellulose degraders). The anaerobic digestion of cellulose can be done by following steps, such as hydrolytic, fermentative, acetogenic, and methanogenic steps.
i. Hydrolysis and Fermentation:
Hydrolysis of cellulosic material is catalysed by the group of micro-organisms, used for fermentation also. The distinction of the two phases is of more theoretical than practical relevance. Hydrolysis is the rate limiting step for other steps followed by fermentation, as hydrolysed monomers will be substrate for fermentation process. Hemicellulose and pectin are hydrolysed ten times faster than lignin rich cellulose.
ii. Acetogenesis and Acidogenesis:
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In the acidification reaction vessel, hydrolysis of polymers to monomers is normally slower than fermentation of monomers to fatty acids and other fermentation products. Due to this, no sugar monomers can be detected as residue during steady state operation.
Oxidation of fatty acids like propionate or n-butyrate, is the rate-limiting step for methane forming step in reaction vessel. Fatty acid degradation is the slowest reaction overall in a two stage methane reactor fed with carbohydrate containing wastewater from sugar production and require larger reaction vessel size to hold fatty acid that contains part for long time.
Thus, the methane reactor has to be larger than the acidification reactor to permit longer hydraulic retention times.
The rate of cellulose degradation depends on the available form of the cellulose in the wastewater. If cellulose is strongly bonded with lignin, lignin prevents access of cellulases enzyme produced by micro-organism, to the cellulose fibres. If cellulose is in a crystalline form, cellulases enzyme can easily attach to it, and then hydrolysis can be done relatively fast.
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iii. Methanogenesis:
In this step, produced acids are consumed by methanogenic micro-organisms to convert acids in to methane and CO2. For methanogenesis process, acetogenesis became the rate-limiting process, leading to propionate and butyrate formation. Naturally plant material contains cellulose in form of lignin encrusted. Due to the highly restricted access to these complexes by cellulases, hydrolysis of cellulose is the rate-limiting step in its degradation to methane and CO2.
2. Anaerobic Degradation of Starch:
Carbohydrates are made up of homopolymers or heteropolymers of hexoses, pentoses, or sugar derivatives. They occur in either soluble form or as particles, forming grains or fibres of various sizes. Starch metabolism is done by hydrolysis, which is done by amylase enzyme produced by hydrolytic bacteria to form soluble monomers or dimmers after hydrolytic cleavage.
3. Anaerobic Degradation of Protein:
Proteins are biological macromolecules, present in soluble and non-soluble form. When protein molecule is present outside the cell at an acidic pH, soluble proteins precipitate in presence of enzymes. For example- precipitation of casein by addition of rennet enzyme. Protein can also be converted in to biogas by methanogenesis.
Hydrolysis of protein is catalysed by several types of protases enzymes that cleave proteins in to amino acids, dipeptides, or oligopeptides. Hydrolysis of proteins requires a neutral or weakly alkaline pH compare to carbohydrate which required acidic pH.
Acidification of protein containing wastewater proceeds optimally at pH values of 7 or higher. Acetogenesis of fatty acids from deamination of amino acids requires a low H2 partial pressure for the same reasons as for carbohydrate degradation. This can be maintained by a syntrophic interaction of fermentative, protein-degrading bacteria and acetogenic and methanogenic or sulphate- reducing bacteria.
4. Anaerobic Degradation of Neutral Fats and Lipids:
Fats and lipids are biopolymers that contribute significantly to the Chemical Oxygen Demand (COD) in sewage sludge, cattle and swine manures, and wastewater from the food processing industry, for example, slaughterhouses or potato chip factories. Hydrolysis is the process to convert fats and lipids in to simple form like fatty acids saturated and unsaturated by use of hydrolytic enzymes like lipases.
To provide a maximum surface for hydrolytic cleavage by lipases or phospholipases, solid fats, lipids, or oils must be emulsified. Glycerol and saturated and unsaturated fatty acids like palmitic acid, linolic acid, linolenic acid and stearic acid are formed from neutral fats.
Lipolysis of phospholipids generates fatty acids, glycerol, alcohols like serine, ethanolamine, choline, inositol and phosphate. Lipolysis of sphingolipids generates fatty acids and amino alcohols like sphingosine, and lipolysis of glycolipids generates fatty acids, amino alcohols, and hexoses such as glucose and galactose.
5. Metabolic pathway for Ethanol Production:
Glucose is first converted in to pyruvic acid by the glycolysis pathway. Now pyruvate is converted in to ethanol molecules after following various steps and intermediate formation. Figure 4.6 shows the pathway of ethanol production. Ethanol is produced from glucose (sugar) in anaerobic condition, commonly used micro-organism is Saccharomyces cerevisiae.
6. Metabolic Pathway for Acetate Production:
Acidic acid producing micro-organisms grow in ethyl alcohol converting ethanol in to acetate as shown in metabolic pathway (Fig. 4.7). This process can be used for commercial production of vinegar from alcohol (wine). Acetobacter aceti produces acetic acid in aerobic condition.
Study on the anaerobic bacteria shows that some micro-organisms like Clostridium thermoacetical, Clostridium thermoautotrophicum etc., have the potential for the large scale production of acetate. One mole of glucose produces three moles of acetate. The flucose molecule is converted in to ethanol first in anaerobic process by Saccharomyces cerevisiae. Now this ethanol molecule is converted anaerobically in to acetate molecule. This conversion can be utilised for commercial production of acetate.
7. Metabolic Pathway for Mixed Acid Production:
In mixed acid fermentation process the six carbon sugar, i.e., glucose is converted in to various organic acids and ethanol. The produced mixture having organic acids like succinic acid, fumaric acid, lactic acid, acitic acid, etc., and ethanol. Different products are produced in this pathway so it is called mixed pathway (Fig. 4.8).
8. Metabolic Pathway for Anaerobic Fermentation:
The pathway representing some valuable products synthesise during anaerobic digestion process. Various products like propionic acid, isopropyl alcohol, ethanol, butyric acid, butanol etc., as presented in the metabolic pathway.
Butyric acid is a four carbon fatty acid which is formed in colon by bacterial anaerobic fermentation. Butyric acid can be found in most cultured dairy products, it can help to improve health of host gut and also help to control diabetes. Metabolic pathways for anaerobic fermentative products are shown in Fig. 4.9.
9. Metabolic Pathway for Biofuel Production:
Biofuels like ethanol, Butanol and isopropanol can be produced from various sources of raw materials like cellulose, starch, proteins and fats. Metabolic pathways for biofuel production are shown in Fig. 4.10.
In first step, hydrolysis of these raw materials is done by use of various micro-organisms for different sources for example fungal species like Trichderma viride, Trichoderma reesei converts cellulosic raw materials in to glucose, Bacillus amyloliquifaciens produces amylase enzyme that is necessary for the hydrolysis of starchy material, Bacillus subtilis produced protease which hydrolyses the protein molecules, Bacillus cereus, Acinetobacter radioresistens, etc., produces lipase enzyme that catalyses the fat molecules.
After hydrolysis of cellulose, starch, protein and fat, sugar is produced. This sugar or glucose is now finally converted in to ethanol by the use of Saccharomyces cerevisiae in anaerobic fermentation process. The produced ethanol can be utilised as fuel.