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Biochemical methods of conversion K V Rajeshwari, Fellow and Malini Balakrishnan, Fellow Energy–Environment Technology Division, T E R I, New Delhi Introduction 795 Basics of biochemical processes 795 Aerobic and anaerobic systems for treatment of liquid wastes 815 Aerobic and anaerobic systems for solid waste treatment 839 Nomenclature 852 Glossary 852 References 854 Bibliography 856 Introduction Organic wastes are important resources that can be converted into useful products by adopting suitable bio-processes. Waste materials considered to be bio-resources include animal residues such as cattle dung, poultry litter, municipal solid wastes (including food and vegetable market wastes), industrial organic wastes such as those from food-processing industries, sugar, and plantation industries (for example, tea and coffee processing), leather, distilleries, pulp and paper, and many more (Dhussa and Tiwari 2000). In addition, agricultural wastes and wetland vegetation such as water hyacinth also form an important category of bio-resources. Basics of biochemical processes A biochemical process refers to a number of complex chemical reactions occurring in the living organisms or involving living organisms. The basis of any biochemical reaction is (i) hydrolysis of complex materials in organic matter, such as carbohydrates, proteins, and fats, into simple nutrients or (ii) the synthesis of complex molecules from simple substances. The process is accompanied by the release of energy for growth and maintenance and useful by-products. RB-14.p65 795 30/11/2007, 4:47 PM 14 796 • Renewable energy engineering and technology Food + Micro-organisms → Energy + Products For example, photosynthesis is a biochemical process in which plants utilize solar energy for converting carbon dioxide and water into sugar and oxygen (Figure 14.1). In the process of respiration, organic compounds are converted into complex molecules such as starch, proteins, and fats, which have energy stored in bonds. This energy is released in the form of ATP (adenosine triphosphate) during cellular respiration Glucose + O2 → CO2 + H2O + 36ATP H2O + ATP → ADP (adenosine diphosphate + P + Energy) Fermentation is a biochemical process commonly used in industries for converting organic substrates (for example, carbohydrates) into useful products such as alcohols, acids, and so on. A common example of biochemical conversion is fermentation of milk with lactic acid bacteria. A list of starter cultures in fermentation of milk is given in Table 14.1. Figure 14.1 Photosynthesis process RB-14.p65 796 30/11/2007, 4:47 PM Biochemical methods of conversion • 797 Table 14.1 List of starter cultures Streptococcus Streptococcus Streptococcus Streptococcus cremoris lactis thermophilus diacetylactis Lactobacillus bulgaricus Leuconostoc cremoris Leuconostoc dextranicum Lactobacillus acidophilus Source www.techno-preneur.net/timeis/technology/MaysciTech/DairyProduct.html, last accessed on 25 July 2005 Various food products are obtained from fermentation processes. Most of the industrial food processes are based on fermentation by yeast, fungi, and mould. Alcohol in beer and wine is attributed to fermentation by yeast. Most dairy products are produced by fermentation of milk by microbes, namely lactococcus, lactobacillus, and streptococcus. Beer, a product of alcoholic fermentation of cereal by yeasts, can trace its origin to the Babylonian Empire (Mesopotamia) before 6000 BC. By 2000 BC, many types of beer existed in Babylon. The Egyptians were the first to discover that dough ferment resulted in light delicious bread. Some examples of food fermentation are given in Table 14.2. Classification of micro-organisms Micro-organisms can be broadly classified into plants, animals, and higher and lower protista. Micro-organisms belonging to the plant and animal kingdoms are multicellular, whereas those under protista can either be unicellular or multicellular. Microbes, which are of importance to waste treatment, are protists such as algae, protozoa, fungi, blue-green algae, and bacteria. These can be further classified as eukaryotic cells containing well-defined nucleus with a membrane or prokaryotic cells without a nuclear membrane. Bacteria and blue green algae are prokaryotic, whereas the algae, protozoa, and fungi are eukaryotic. Table 14.2 Examples of food fermentation RB-14.p65 Product Substrate Micro-organism Chocolate Cacao bean Bread Coffee Sauerkraut Soy sauce Flour Coffee bean Cabbage Soyabean Saccharomyces cerevisiae Candida rugosa Kluyveromyces marxianus S. cerevisiae Erwinia dissolvens Leuconostoc plantarum Aspergillus oryzae 797 30/11/2007, 4:47 PM 798 • Renewable energy engineering and technology Bacteria can be classified on the basis of their shape and can be cylindrical, rod-shaped, spherical, and helical. In waste utilization, various groups of micro-organisms act on the substrate to be treated by one of the four processes, namely, aerobic, anoxic, anaerobic, and a combination of the aerobic/anoxic or anaerobic processes. Each process can be further subdivided into attached or suspended growth or a combination of both. The biochemical action of the microbes by any of the above routes results in the degradation of carbonaceous and nitrogenous matter. Classification of micro-organisms can also be based on the use of carbon for growth. Autotrophic organisms are those that utilize carbon dioxide as a source of carbon, while organisms utilizing organic carbon are known as heterotrophic. Autotrophic organisms are further classified into photosynthetic or chemosynthetic, depending on their source of energy, that is, the sun or inorganic oxidation–reduction reactions. In addition to classification based on source of energy and carbon, the oxygen requirement for growth leads to another category of classification of micro-organisms, namely aerobic (those that require oxygen), anaerobic (those that can survive in the absence of oxygen), and facultative (organisms that can exist under both aerobic and anaerobic conditions). Organisms that require the presence of oxygen for their metabolism are also known as obligate aerobes. Almost all animals, fungi, and several bacteria are obligate aerobes. Organisms for which the presence of molecular oxygen is toxic are known as obligate anaerobes. These are involved in acid fermentations, acetogenesis, and methanogenesis. Facultative anaerobes can use available oxygen and also other electron acceptors such as nitrate in absence of oxygen. Yeast and human cells are examples of facultative micro-organisms. Table 14.3 lists the organisms that are obligate aerobes, obligate anaerobes, and facultative anaerobes. Table 14.3 Examples of different kinds of aerobic and anaerobic micro-organisms Obligate aerobes Facultative anaerobes Obligate anaerobes Bacillus subtilis Bdellovibrio spp. Bordetella pertussis Legionella spp. Mycobacterium leprae Mycobacterium tuberculosis Pseudomonas spp. Bacillus anthracis Corynebacterium diphtheriae Escherichia coli Lactobacillus spp. Klebsiella spp. Salmonella sp. Staphylococcus aureus Streptococcus spp. Clostridium botulinum Clostridium tetani Source Abedon (1998) RB-14.p65 798 30/11/2007, 4:47 PM Biochemical methods of conversion • 799 Enzymes in biochemical processes Cell growth and multiplication of micro-organisms are facilitated by enzymes that are extracellular or intracellular. These enzymes act as catalysts during the absorption of substrate or nutrient by the cell and also during the conversion of substrate into product. The reaction is represented by the following equation. [E] + [S] → [E][S] → [P] + [E] where [E] is the enzyme, [S] the substrate, [E][S] the enzyme–substrate complex, and [P] is the product. The activity of enzymes depends on the substrate, its concentration, pH, and temperature. The optimum pH for enzymatic activity is 7 and the optimum temperature is 35–40 ºC. At higher temperatures, enzymes get denatured. The action of enzymes can be explained in a simplified manner by the lock-and-key mechanism (Figure 14.2). Each enzyme is specific to a specific substrate and has an active site where the substrate binds and reaction occurs. The conversion of a substrate into a product can be a singlestep or a multi-step reaction, catalysed by a series of enzymes. For example, rennet, an enzyme preparation containing chymosin, is used to catalyse changes in milk proteins for gel formation during preparation of cheese. Metabolic pathways of various substrate conversions in a cell are very complex due to multiple reactions. For example, formation of ethanol from pyruvate in yeast and other micro-organisms is catalysed by two types of enzymes. In the first step, pyruvate is converted into acetaldehyde, which is catalysed by pyruvate decarboxylase. The second step is the reduction of acetaldehyde to ethanol, catalysed by alcohol dehydrogenase. Figure 14.2 Lock-and-key mechanism of enzyme and substrate RB-14.p65 799 30/11/2007, 4:47 PM 800 • Renewable energy engineering and technology Pyruvate + H+→Acetaldehyde + CO2 + + Acetaldehyde + NADH + H → ←Ethanol + NAD The glycolytic pathway for conversion of glucose into pyruvate along with energy production in the form of ATP is catalysed by different enzymes for each step (Stryer 1986; Figure 14.3). For details on glycolysis, Krebs’ cycle and other biochemical path ways, any standard book on biochemistry (e.g. Stryer 1986) can be referred. Enzymatic activity is maximum at optimum temperature and pH. An acidic or alkaline pH affects the shape of enzyme molecules and inactivates the site at which reaction occurs (Figure 14.4). Figure 14.3 Glycolytic pathway Biochemical methods of conversion • 801 Figure 14.4 Effect of pH on enzymatic reaction Figure 14.5 Optimum temperature in enzyme-catalysed reaction At low temperatures, molecules of the substrate do not have enough energy for the reaction to take place. At high temperatures, molecules are highly energetic and cause degeneration of enzyme shape and its activity. Most enzymes involved in the metabolic reactions of the human body work at body temperature. Enzymes involved in the thermophilic reactions work at a higher temperature (Figure 14.5). In addition to the optimum temperature and pH, enzyme activity is also affected by the presence of various elements. The enzyme systems of acetogenic and methanogenic bacteria, for example, formate dehydrogenase and hydrogenase, involved in waste treatment processes require the presence of selenium, tungsten, and nickel (Table 14.4). RB-14.p65 801 30/11/2007, 4:47 PM 802 • Renewable energy engineering and technology Table 14.4 Examples of enzymes dependent on trace elements Trace element Enzyme Bacteria Selenium Hydrogenase Formate dehydrogenase Glycine reductase Nicotinic acid hydroxylase Xanthine dehydrogenase Formate dehydrogenase Carbon monoxide dehydrogenase Hydrogenase Methanococcus spp. Clostridium thermoaceticum Clostridium pasteurianum Clostridium thermoaceticum Methanobacterium thermoautotrophicum Acetobacterium woodii Tungsten Nickel Source Malina and Pohland (1992) Energy requirement for growth and maintenance The conversion of various nutrients in a cell results in the generation of energy, which is stored in the form of ATP. The release of energy from ATP for various purposes results in the generation of ADP, which can again absorb energy to form ATP. In addition to the carbon source, micro-organisms, particularly bacteria, require other nutrients like nitrogen and phosphorus (Figure 14.3). In the process of glycolysis, glucose is converted into glucose-6phosphate, fructose-6-phosphate, and fructose 1,6-diphosphate by the addition of phosphorus from ATP. Fructose 1,6-diphosphate produces dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by the action of aldolase. ATP is again released when pyruvate is formed from 1,3diphosphoglycerate. Pyruvate formed from glycolysis is further processed through the Kreb’s cycle in mammalian cells for additional ATP production through oxidative phosphorylation. This is an aerobic respiration process, also known as the citric acid cycle, in which pyruvate is converted into simpler compounds and citrate is formed as the first intermediate. Figure 14.6 shows the Kreb’s cycle depicting the conversion of pyruvate through TCA (tricarboxylic acid) pathways. The oxidative decarboxylation of pyruvate to form acetyl CoA (coenzyme A) is the link between glycolysis and the citric acid cycle. Pyruvate + CoA + NAD+ Acetyl CoA + CO2 + NADH In Kreb’s cycle, decarboxylation of pyruvate occurs in the presence of CoA, resulting in the formation of acetyl CoA. This is catalysed by pyruvate dehydrogenase complex. Acetyl CoA condenses with oxaloacetate to form citrate that isomerizes to isocitrate, which is further oxidized and RB-14.p65 802 30/11/2007, 4:47 PM Biochemical methods of conversion Figure 14.6 Kreb’s cycle RB-14.p65 803 30/11/2007, 4:47 PM • 803 804 • Renewable energy engineering and technology decarboxylated to α-ketoglutarate. Further, oxidative decarboxylation reaction results in the production of succinyl CoA that is converted to succinate, which is further oxidized to fumarate. Fumarate on hydrolysis forms malate and on further oxidation, converts to oxaloacetate. All these reactions are catalysed by various enzymes (see Stryer 1986 for details). Bacterial growth and kinetics The growth of bacteria in a batch system follows a sigmoidal curve and includes four distinct stages, namely lag phase, log phase (or growth phase), stationary phase, and death phase. The different phases, along with corresponding changes in substrate concentration, are shown in Figure 14.7 (a). The lag phase is the time required for adaptation of bacteria to the growth medium. The log or growth phase is the stage in which bacteria multiply by consuming nutrients available in media. The growth phase can be further divided into three stages: log growth phase, declining growth phase, and endogenous phase. In the log growth phase, abundant nutrients are available for the bacteria. This is followed by the declining growth phase in which the nutrient supply becomes limited. The endogenous phase results in the metabolism of cell protoplasm due to the non-availability of food. During the stationary phase, there is a balance between the growth and the decay of cells. Finally, the death phase indicates increased lysis of cells with the cell decay rate predominating the growth rate. Figure 14.7(a) Schematic diagram of bacterial growth curve RB-14.p65 804 30/11/2007, 4:47 PM Biochemical methods of conversion • 805 As mentioned in earlier sections, in addition to the formation of various products through biochemical conversion of resources, a major application of biological processes is in the utilization of waste water and solid wastes. The following section focuses on the kinetics and types of biochemical reactions for waste degradation. Mixed cultures Mixed cultures are microbial cultures, involving two or more micro-organisms. The bacterial consortium used in the biological waste treatment is a typical example of mixed cultures. This is a complex system as each microorganism has a distinct growth pattern depending on the external parameters like pH, temperature, substrate, availability of oxygen, and presence of other micro-organisms. Growth rates of different micro-organisms in a mixture culture are shown in Figure 14.7(b). Growth of the mixed cultures in batch systems simultaneously follows the three steps: oxidation of organic matter, auto-oxidation of cells for energy, and growth of cells. These three processes are depicted using the terms COHNS for organic matter and C5H7NO2 to represnet cell tissue as follows (Metcalf and Eddy 1993). Figure 14.7(b) Growth curves of different microorganisms in mixed cultures RB-14.p65 805 30/11/2007, 4:47 PM 806 • Renewable energy engineering and technology Oxidation COHNS + O2 + bacteria → CO2 + NH3 + other end products + energy Synthesis COHNS + O2 + bacteria + energy → C5H7NO2 (New cell tissue) Endogenous rrespir espir ation (aut o-o xidation) espiration (auto-o o-oxidation) C5H7NO2 + 5O2 → 5CO2 + NH3 + 2H2O + energy As an example, anaerobic digestion of waste involves a consortium of bacteria, which can be broadly divided into three main groups. Fermenting bacteria (also termed acidifying or acidogenic bacteria) These cause hydrolysis and acidogenesis of the substrate. The exo-enzymes released from these micro-organisms hydrolyse polymeric matter like proteins, fats, and carbohydrates into smaller units. These, in turn, undergo an oxidation–reduction process resulting in the formation of the VFA (volatile fatty acids) as well as some carbon dioxide and hydrogen. Acetogenic bacteria These break down the products of the acidification step to form acetate. In addition, hydrogen and carbon dioxide (in the case of odd-numbered carbon compounds) are also produced. Methanogenic bacteria These convert acetate or carbon dioxide and hydrogen into methane. Other possible methanogenic substrates like formate, methanol, carbon monoxide, and so on are of minor importance in most anaerobic digestion processes (Figure 14.8). In addition to these three main groups, hydrogen-consuming acetogenic bacteria are always present in small numbers in an anaerobic digester. They produce acetate from carbon dioxide and hydrogen and, therefore, compete with the methanogenic bacteria for hydrogen. Also, the synthesis of propionate from acetate, coupled with the production of a longer chain VFA, occurs, to a limited extent, in anaerobic digestion. Competition for hydrogen can also be expected from sulphate-reducing bacteria in case of sulphate-containing wastes. The different types of reactions during anaerobic digestion are given below (Malina and Pohland 1992). Hydrogenotrophic reactions (oxidation of hydrogen) 4H2 + CO2 → CH4 (methane) + 2H2O 4HCOOH (formic acid) → CH4 + 2H2O + 3CO2 4(CH3CHOHCH3) (2-propanol) + CO2 → CH4 + 4CH3COCH3 + 2H2O RB-14.p65 806 30/11/2007, 4:47 PM Biochemical methods of conversion • 807 Figure 14.8 Steps in anaerobic digestion of waste Source Malina and Pohland (1992) Aceticlastic reaction (breakdown of acetate) CH3COOH (acetic acid) → CH4 + CO2 Disproportionation reactions (oxidation of substrate) 4CH3OH (methanol) → 3CH4 + CO2 + 2H2O 4CH3NH2 (methylamine) + 3H2O → 3CH4 + CO2 + 4NH4+ (ammonium ion) 2(CH3)2S (dimethyl sulphide) + 2H2O → 3CH4 + CO2 + H2S (hydrogen sulphide) CH4 + 2O2 → CO2 + 2H2O Organic wastes typically consist of proteins (40%–60%), carbohydrates (25%–50%), and oils and fats (8%–12%). At this elemental level, the organic matter consists of carbon, hydrogen, oxygen, and nitrogen. The aggregate organic matter in wastes and waste water can be quantified by measuring BOD (biochemical oxygen demand), COD (chemical oxygen demand), and TOC RB-14.p65 807 30/11/2007, 4:47 PM 808 • Renewable energy engineering and technology (total organic carbon). BOD determination involves measurement of the oxygen used by microorganisms in the biochemical oxidation of organic matter. The test procedure for BOD is given in standard books such as Metcalf and Eddy, 1993. COD measurement involves the measurement of oxygen-equivalent of the organic matter that can be oxidized chemically using dichromate in an acid solution, as outlined in Chapter 12. The theoretical COD can be determined if the chemical formula of the organic matter is known. This is illustrated in the examples provided below. Example 1 Estimate the COD (chemical oxygen demand) of (i) glucose, (ii) cell biomass, (iii) methane, and (iv) sewage sludge with the following composition. Carbon (31.1%), hydrogen (4.2%), oxygen (24.3%), nitrogen (3.3%), and sulphur (1.1%). Solution (i) Glucose A single molecule of glucose requires 6 moles of oxygen to break down into carbon dioxide and water. The complete oxidation of glucose is represented by the following equation. C6H12O6 + 6O2 → 6CO2 + 6H2O Hence, COD of glucose is calculated as 6 × 32/180 g of O2/g of glucose = 1.07 g O2/g glucose (ii) Cell biomass For oxidation of the cell biomass, 5 moles of oxygen are required as represented by the following equation C5H7NO2 (cell biomass) + 5O2 → 5CO2 + NH3 + 2H2O Thus, COD of the cell biomass = 5 × 32/113 g of O2/g of cell biomass = 1.42 g O2/g cells. (iii) Methane The complete oxidation of methane is represented as CH4 + 2O2 → CO2 + 2H2O The COD of methane, which is the amount of oxygen required for oxidation of each mole of methane = (2 × 32)/16 = 4 g O2/g of methane RB-14.p65 808 30/11/2007, 4:47 PM Biochemical methods of conversion • 809 (iv) Sewage sludge The molecular formula is estimated by dividing the weight per cent of each element by the atomic weight and dividing the resulting number by the moles of carbon. From the elemental composition given for sewage sludge, the number of moles can be calculated as Carbon 31.1/12 Hydrogen 4.2/1 Oxygen 24.3 /16 Nitrogen 3.3/14 Sulphur 1.1 /32 = = = = = 2.59 4.2 1.51 0.235 0.15 For molecular formulae Carbon 2.59/2.59 = 1 Hydrogen 4.2/2.59 = 1.62 Oxygen 1.51/2.59 = 0.58 Nitrogen 0.235/2.59= 0.09 Sulphur 0.15/2.59 = 0.06 Hence, the molecular formula is CH1.62 N0.09 O0.58 S0.06 Molecular weight = 12 × 1 + 1 × 1.62 + 14 × 0.09 + 16 × 0.581 + 32 × 0.06 = 26.096 For estimating the COD of sewage sludge, the balancing equation for the oxidation of the sewage sludge is CH1.62 N0.1 O0.6 S0.06 + 0.995O2 → CO2 + 0.1NH3 + 0.59H2O + 0.06H2S Hence, COD = (0.995 × 32)/26.096 = 1.22 g of O2/g of sewage sludge. Example 2 Estimate the theoretical methane yield for 1 g COD of organic matter. Solution Density of methane is 0.7167 g/litre. Hence, 1 g of methane will occupy a volume of 1.4 litres. As the COD of methane is 4 g [Example 1 (iii)], 1.4 litres of methane will be equivalent to 4 g of COD. 810 • Renewable energy engineering and technology Thus, 1 g of COD of methane is equal to 1.4/4 litre or 0.35 litre of methane. Theoretical methane yield is thus 0.35 litre/g COD. Growth kinetics of micro-organisms It is important to understand the kinetics of bacterial growth for arriving at an optimal design of the reactor size and hence the cost. It is possible to have an optimum growth of micro-organisms through proper control of the environmental conditions such as pH, temperature, aerobic or anaerobic conditions, substrate level, additional micronutrients, and so on. The digestion period and hence the retention time is dependent on the growth rate of micro-organisms and the rate at which the substrate can be metabolized to give stable products. Simple kinetics in batch culture (growth phase) The microbial growth rate can be described by the equation ...(14.1) where rm is the rate of growth of micro-organisms (g/m3/day), µ is the specific growth rate (day–1), and X is the microbial concentration. Microbial concentration, also called biomass concentration, is usually measured by VSS (volatile suspended solids). See Chapter 12 for measurement of VSS. In case of substrate limitation, where the growth rate is affected by the limited concentration of nutrients or substrate, the cell growth is represented by the Monod equation as ...(14.2) where µmax is the maximum specific growth rate (g new cells/g cells/day), S is the limiting substrate concentration (g/m3), and ks is the half-velocity constant (g/m3). When µ = µmax /2 Equation 14.2 becomes Rearranging the equation, we get ...(14.3) Biochemical methods of conversion • 811 Or ks = S½ The graphical representation of the variation of specific growth rate with substrate concentration is shown in Figure 14.9. Other kinetic models proposed include rm = µmax (constant) rm = µmax XS (first order) (mixed) where S0 is the influent substrate concentration and k is a dimensionless kinetic parameter. (mixed) Substituting for µ from Equation 14.2 in Equation 14.1, the overall rate expression can be represented as Figure 14.9 Variation of growth rate with substrate concentration Reproduced with permission of The McGraw-Hill Companies Source Metcalf and Eddy (1993) ...(14.4) 812 • Renewable energy engineering and technology The growth of microbes is accompanied by depletion of the substrate. The microbial growth rate and substrate depletion rate are thus related by the equation rm = –Y rsub ...(14.5) where Y is the yield coefficient. The substrate concentration is usually measured in COD or BOD. Thus the yield coefficient is expressed in gVSS/gCOD removed. As the concentration of substrate decreases with time, the rate is indicated as negative. Substituting dX/dt from Equation 14.5 in Equation 14.4 gives ...(14.6) The term (µmax/Y) is also denoted k, the maximum specific substrate utilization rate. The decay of cells during the endogenous or death phase is given as ...(14.7) where rd is the rate of biomass decay (g VSS/m /day) and kd is the decay constant (g VSS/g VSS/day). The net rate of increase in biomass concentration will thus be 3 ...(14.8) Substituting for rm from Equation 14.1, we get ...(14.9) Net specific cell biomass growth rate (g VSS/g VSS/day) is ...(14.10) or Some indicative values of kinetic constants are given below. ...(14.11) Biochemical methods of conversion • 813 Acid producing bacteria Methane producing bacteria µmax Y kd > 1.33 day –1 0.54 gVSS/gCOD utilized 0.87 day –1 > 1.33 day –1 0.14 gVSS/gCOD utilized 0.02 day–1 The VSS in any bioreactor includes the cell biomass, lysed cells, or cell debris due to the decay of the cells, and non-biodegradable VSS present in the feed or substrate. Effect of temperature Temperature affects the biological reactions through its influence on microbial metabolism, gas transfer rates, and settleability of solids. The microbial metabolic reactions are catalysed by enzymes that are influenced by temperature as explained earlier. The gas transfer rates are influenced by temperature due to its effect on diffusivity and viscosity of the media. Temperature also affects the settleability of solids by causing deflocculation of sludge. The effect of temperature on reaction rate can be derived from the Vant Hoff–Arrhenius relation ...(14.12) where k is the reaction rate constant, T is the temperature (ºC), E is the activation energy constant in J/mole, and R is the ideal gas constant (8.314 J/ mol ºC). Integration of Equation 14.12 gives ...(14.13) where k2 is the rate constant at temperature T2 and k1 is the rate constant at T1. As the waste treatment reaction occurs at ambient temperature, the term E/RT1T2 can be considered constant C. This transforms Equation 14.13 into ...(14.14) ...(14.15) 814 • Renewable energy engineering and technology where η = eC Choosing a reference temperature of 20 ºC we get ...(14.16) ...(14.17) where kT is the reaction rate constant at a temperature T ºC, k20 is the reaction rate constant at a temperature 20 ºC, and η is the temperature activity coefficient. Effect of nutrients Certain inorganic elements are required for growth, including nitrogen, sulphur, phosphorus, magnesium, calcium, iron, sodium, and chlorine. Micronutrients such as zinc, manganese, molybdenum, selenium, cobalt, and nickel, and growth promoters, namely amino acids and vitamins, may also be required in certain cases. A note on terminology As mentioned earlier, substrate concentration is quantified as BOD and COD in waste waters. But as waste waters contain varieties of substrates some more terms are used to quantify, such as biodegradable COD (bCOD) or biodegradable soluble COD (bs COD). The waste waters contain colloidal and particulate matter, part of which may be biodegradable. Bacteria cannot consume the particulate substrates directly. These are first hydrolysed enzymatically to convert into soluble substrates. The bCOD can be a measure of particulate substrates also. The bacterial concentration, also termed often as active biomass, is commonly measured as TSS (total suspended solids) and VSS (volatile suspended solids). In waste water treatment plants in which part of the sludge produced is recycled, the term MLVSS (mixed liquor volatile suspended solids) is used to define biomass concentration in the reactor input. The solids consist of bacteria, nbVSS (nonbiodegradable volatile suspended solids) and iTSS (inert total suspended solids). The biomass yield Y can be estimated from stoichiometry considerations. For example, if we assume that the soluble substrate can be represented as glucose (C6H12O6), and bacterial cells can be represented as C5H7NO2, a stoichiometric equation can be written as 3C6H12O6 + 8O2 + 2NH3 → 2C5H7NO2 + 8CO2 + 14H2O Biochemical methods of conversion • 815 The yield then can be obtained as = 0.42 g.cells/g.glucose consumed In the above, 113 and 180 are the molecular weights of bacterial cells and glucose respectively. The COD of glucose can be determined by the oxidation reaction C6H12O6 + 6O2 → 6CO2 + 6H2O = 1.07 gO2/g. of glucose The yield in terms of COD will then be The actual yield in any given biological treatment process will be less than the figures given above. Biomass yield can also be estimated from considerations of bioenergetics (see Metcalf and Eddy 2003). Aerobic and anaerobic systems for treatment of liquid wastes Different types of bioreactors for waste water treatment Batch reactor It is a simple vessel, with continuous mixing of contents of the reactor with no inflow or outflow of materials. The H/D (height/diameter) ratio is generally kept at 1:3 (Figure 14.10). Figure 14.10 Batch reactor