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In this article we will discuss about:- 1. Biochemistry of Anaerobic Fermentation 2. Microbiology of Anaerobic Fermentation 3. Kinetics of Anaerobic Fermentation 4. Hydrogen Production by Hydrogen Producing Bacteria of Digester Sludge.
Biochemistry of Anaerobic Fermentation:
Anaerobic fermentation basically involves three phases, namely, hydrolysis, an acid phase and the methane phase. Hydrolysis phase covers break-down of large molecules into smaller ones by enzymes that are decomposable by bacteria. During the acid phase, complicated molecules such as proteins, fats and carbohydrates are decomposed by acid-forming bacteria into organic acids, carbon dioxide, hydrogen, ammonia and some impurities.
Organic acids are mainly short chain fatty acids. It should be noted that bacteria can utilise only hydrogen, carbon dioxide and formic acid (H.COOH). Volatile fatty acids formed during fermentation include formic acid (H.COOH), acetic acid (CH3.COOH), and propionic acid (CH3.CH2.COOH), butyric acid (CH3.H2.CH2.COOH), valeric acid (CH3.CH2.CH2 COOH), isovaleric acid (CH3.CH2.CH2.COOH) and cacproic acid (CH3.CH2.CH2.CH2.CH2.COOH).
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Proportion of different volatile acids formed is shown in Fig. 2.2. During methanogenic phase, methane-forming bacteria convert fatty acids into methane. In simple terms the anaerobic fermentation process can be described by a set of following chemical equations.
Methane is also formed from some alcohols and other organic compounds, anaerobic oxidation of hydrogen and carbon dioxide and reduction of carbon dioxide to methane. Normally in a biogas plant all three phases occur in balanced fashion and any imbalance tends to lower gas yield.
Complete details of biochemistry of anaerobic fermentation process are shown in Fig. 2.4. If acids produced during acid phase are excessive and not fully utilised in other chemical reactions, it retards methane formation. For optimum results, pH of digester contents needs to be maintained between 6.8 and 7.2.
Microbiology of Anaerobic Fermentation:
Methane bacteria can be divided into four broad categories covering rod, coccus, sarcina and spiral form. Linked with these four categories there are four broad morphological group of methane bacteria which includes rod-shaped sporulating cells such as methanobacillus; rod-shaped non-sporulating cells such as methanobacterium, spherical cells in sarcina arrangement such as methanosarcina; and spherical cells not in sarcina arrangement such as methanocoocus. Types of methane bacteria helpful for anaerobic fermentation are given in Table 2.1.
These bacterias help to oxidise hydrogen by using available carbon dioxide. Reproductive cycle time of methane bacteria is found to vary from 4 to 6 days. There are some species whose reproductive cycle time is shorter than this period.
These include methanobacterium, thermoautotrophicum, M. arbophicum and Hungattii. Among them reproductive cycle time of M. thermoautotrophicum is even shorter than three hours and hence this is particularly appropriate for breeding methane bacteria. Methane bacteria utilise hydrogen electron donor to support their growth and breeding.
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Most of the above species of methane bacteria are sensitive to temperature and are most effective at 35°C (308°K) and they become ineffective at temperatures below 10°C (283 °K). It should be noted that whereas acid-forming bacteria are not very sensitive to oxygen, reproduce and multiply fast, the methane-forming bacteria are quite sensitive to oxygen and are slow to reproduce and multiply in its presence.
As methane-forming bacteria cannot use nitrogen or carbon directly available from sources such as carbohydrates and proteins, intermediate acid-forming phase is desirable in between in order to maintain correct balance between the two bacterial populations. This in turn depends on temperature, level of acidity, and feedstock composition.
As bacteria use carbon atoms nearly thirty times faster than the nitrogen, desirable carbon nitrogen ratio in feedstock is having more carbon than optimum, anaerobic fermentation decreases as all nitrogen is used; similarly if too much nitrogen is present digestion stops when the carbon is exhausted and excess nitrogen is lost as ammonia.
During the acid phase, pH of digester contents normally drops to a value marginally below 6 in two weeks. After two weeks as digestion continues further and ammonia is formed, pH again rises to around 7 with methane formation and then further upto 8.2. At this stage mixture is regarded to be fairly well buffered and fresh feedstock can be added without any harmful effect. The bacteria are most efficient when they have intimate contact with feedstock particles.
Hydrolysis Phase:
During hydrolysis phase, large molecules are broken down into smaller sizes by enzymes which in turn are decomposable by bacteria. During enzymatic hydrolysis, polymers are converted into soluble monomers that act as substrates for microorganisms during acid phase in which soluble organic compounds are converted into organic acids. Sewage sludge on dry weight basis broadly comprises hemicellulose 6 per cent, cellulose 34.5 per cent, lipids 14 per cent etc.
Thus cellulose forms the largest fraction of carbohydrates in organic matter. Not only sewage but other wastes like animal manure and crop residues mainly contain carbohydrates along with lipid, proteins, and inorganic material. In organic wastes carbohydrates are mostly in the form of cellulose and other components of plant fiber such as hemicellulose and lignin. Thus, for breaking these components during anaerobic fermentation, bacteria need to possess what is known as cellulolytic, lipolytic and proteolytic activity.
Cellulolytic activity is helpful in reducing complex raw materials into simple and soluble organic components. Cellulose comprises polymerised glucose units as chains of indefinite length and complex branching patterns. Cellulolytic bacteria help to reduce chains and branches to dimeric and then to monomeric sugar molecules which are finally converted to organic acids.
Cellulolytic bacteria is of two types mesophilic and thermophilic. Mesophilic bacteria are most active when digestion ranges from 30°C to 40°C whereas thermophilic bacteria are most active between 50°C and 60°C. For efficient working of both these bacteria, pH value of digester content should be between 6 and 7. As acid formation begins, pH tends to drop which can also be controlled by adding lime.
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When activities of acid forming and methane forming bacteria are in balance, pH of digester contents stabilises around 7. Rate of hydrolysis is somewhat slower than subsequent rate of acid and methane formation. This indicates that conversion of cellulose and other complex raw materials to simple monomers is a kind of rate limiting operation. Rate of hydrolysis depends on nature of substrate, bacterial concentration, pH and temperature of digester contents.
Carbohydrates form an important part of raw material for anaerobic fermentation. Semi-cellulose is mostly in the form of polycondense pentose. It can be hydrolysed into pentose and small traces of hexose, xylose, arabinose, mannitol and glactose. Pectin which is present in traces is another kind of polycondense pentose. Pectin can be hydrolysed into galactose, gum sugar, xylose, galacturonic acid, methyl alcohol and acetic acid.
Starch can be hydrolysed to form glucose, lignin is difficult to biodegrade and various celluloses have different biodegradability. With the formation of butyric and propionic acids as intermediate products, cellulose finally gets transformed into methane and carbon dioxide which are the main constituents of biogas.
Acid Phase:
The monomeric components released during hydrolysis phase act as substrate for acid-forming phase. Acid phase can be viewed as periodic during which complex organic materials are converted into simpler acids such as volatile fatty acids. Acetic acid is one common by-product of digestion of fats, starch and protein.
Methane bacteria are strictly anaerobic and can produce methane either by fermenting acetic acid to form methane and carbon dioxide, or by reducing carbon dioxide to methane using hydrogen gas or formate produced by other bacteria. Acetic acid is by far the single most acid prone to react with methanogenic bacteria resulting in methane formation. Acetic acid accounts for 70 per cent of methane produced.
Methane-Forming Phase:
Methanogenic bacteria are selective in their reaction with substrate components. Methanogenic bacteria react with acetic acid, methanol, carbon dioxide and hydrogen to produce methane. Presence of oxygen retards the activity of methanogenic bacteria. Not only oxygen but even products like nitrites and nitrates which contain oxygen inhibit the activity of methanogenic bacteria. Methanogenic bacteria are sensitive to the pH of digester contents.
The optimum pH lies between 7 and 7.2 though gas production is fairly stable for pH between 6.6 and 7.6. When pH drops below 6.6 there occurs a significant inhibition of methanogenic bacteria, and when it drops to a value of 6.2, digester contents become toxic to these bacteria. Acid-forming bacteria continue to be active even when pH level drops below 6.2 and in fact acidogenic bacteria produce acids until the pH drops to 4.5-5.0.
When anaerobic fermentation proceeds in balanced state, pH automatically comes within desired range due to various biochemical reactions. It should be noted that volatile organic acids produced during acid-forming phase tend to lower the pH, a phenomenon which is countered by destruction of volatile acids and reformation of bicarbonate buffer during the methane-forming phase.
In the event of imbalance, acid formers become more active than methane formers resulting in volatile fatty acid accumulation. This problem can be alleviated by adding lime which tries to arrest drop in pH. Chemicals such as ammonium hydroxide can also be used as buffering agents but it should be added in requisite quantity only otherwise it may release excessive ammonia and ammonium ions which are toxic to methanogenic bacteria.
Common toxins include ammonia (> 1500-3000 mg/l of total ammonia nitrogen at pH > 7.4); ammonium ion (> 3000 mg/l of total ammonia nitrogen at any pH); soluble sulphides (> 50 – 100 mg/l, possibly > 200 mg/l); and soluble salts of metal such as copper, zinc and nickel.
Alkali an alkaline-earth metal salts of sodium, potassium, calcium or magnesium may be either stimulatory or inhibitory for methane bacteria depending upon their concentration. Toxic effect of harmful material can often be appreciably reduced by permitting only gradual build up in their concentration which helps bacteria to acclimatize. It is possible to minimise the adverse impact of soluble sulphides and ammonia on bacterial activity by allowing their concentration to grow gradually.
Concentrations of soluble sulphides varying from 50 to 100 mg/l can be tolerated with little or no acclimatisation, but concentrations above 200 mg/l are considerably toxic. Heavy metal ions such as copper, zinc and nickel are toxic at concentrations as low as few ppm. However, when these concentrations are upto 100 mg/l (nearly 100 ppm) or more, no toxic effect is reported because at such concentrations they react with soluble sulphides and thus contain toxicity.
There are some organic materials as well which act toxic to anaerobic fermentation. Their harmful effect depends on their concentration and rate of generation. For example, alcohols act toxic at high concentrations but when introduced gradually at low concentrations, they are easily degraded, and if added in controlled manner their addition is non-toxic.
Biodegradation of protein or urea in digester leads to the production of ammonia. Ammonia provides nitrogen to bacteria and its presence upto a certain level is desirable as it acts stimulatory to the bacterial activity. If ammonia concentration exceeds desired limit, it becomes toxic or inhibitory to bacterial activity. It is to be underscored that ammonium ion exists in equilibrium with dissolved ammonia gas NH+4 <=> NH3 + H+ and the latter is inhibitory at a much lower concentration than ammonium ion.
Degree of impact of ammonia on bacterial activity depends on ammonia concentration and pH of the digester contents. When pH is high, there occurs increased concentration of dissolved ammonia gas as compared to ammonium ions which again causes inhibition. It has been found that if ammonia concentration exceeds 3000 mg/l or ranges between 1500 and 3000 mg/l it acts inhibitory to bacterial activity.
Process temperatures have considerable bearing on microbial activity. During mesophilic fermentations, as temperature falls below the optimum range of 33-38°C, microbial activity declines following increase in minimum solids-retention time for process stability. Same phenomenon takes place when temperature of digester contents during thermophilic fermentation falls below the desired range. Effect of sudden temperature changes is more-deeper during thermophilic than mesophilic fermentation.
During conversion of carbohydrates to carbon dioxide and methane, almost equal volumes of each gas are produced. As carbon dioxide is soluble in water, not all of the carbon dioxide produced during fermentation is released as part of it gets dissolved in water. Carbon dioxide is also known to react with hydroxyl ion to form bicarbonates whose formation depends on alkalinity, temperature and composition of digester contents.
Hydroxyl ion is mainly produced following reaction of ammonia with water to form ammonium hydroxide during deamination of biodegradable protein. Protein content of substrate thus affects amount of carbon dioxide formed. Amount of carbon dioxide associated with liquid stream as bicarbonate declines with rise in temperature and decrease in pH.
Retention time of slurry in digester also affects proportion of carbon dioxide produced. Based on experimental studies for a given substrate, anaerobic fermentation at shorter retention times produces more carbon dioxide during acid phase and a gas with higher methane content during methane forming phase.
Kinetics of Anaerobic Fermentation:
Several kinetic models have been developed to describe the anaerobic fermentation process which were developed initially in the context of anaerobic digestion of sewage sludge. J. Monod developed a hyperbolic relationship between the exponential microbial growth rate and substrate concentration. In this model kinetic parameters, namely, microorganism growth rate and half velocity constants are deterministic in nature which help to predict condition when maximum biological activity occurs and when activity ceases.
This model can be used to determine substrate utilisation (rs):
The Monod model suffers from the drawback that one set of kinetic parameters cannot describe biological process at short and long retention times, and that kinetic parameters cannot be obtained for some complex substrates. To alleviate limitations of the Monod model while retaining its advantages.
A.G. Hashimoto, et al., developed an alternative equation which attempts to describe kinetics of methane fermentation in terms of several parameters. According to this equation given below, for a given loading rate S0/θ daily volume of methane per volume of digester depends on the biodegradability of the material (B0) and kinetic parameters µm and K.
Continuous Vs. Batch Process:
Anaerobic fermentation can be carried out as continuous or batch process. Continuous digestion in turn can be carried out either in a plug flow reactor (PFR) or continuous stirred tank reactor (CSTR).
In the plug system each particle has the same residence time in the reactor (digester) while in CSTR there is a broad distribution of residence times. Traditionally CSTR digesters have been used for sewage treatment plants based on continuous anaerobic fermentation. Plug flow reactors though sufficiently developed are still much less in number.
Assuming that digestion process follows first order kinetics, plug flow or continuous stirred tank digestion can be modelled by utilising chemical reaction theory. For first order kinetics-
Where A is the volume of gas produced per mass converted. The rate of gas yield can be determined by their respective derivatives with respect to T. Hence (dG/dT) represents the volume of gas produced per reactor volume per day (VVD).
Thus,
The rate of gas yield (WD) decreases with time and is proportional to e-kT. Experimental results indicate that there occurs first an increase in gas yield with time followed by a decrease. This difference can be attributed to the assumption of first order kinetics which does not hold completely at all times. To elaborate further, this assumes that the microorganism level, volatile acid concentration, pH, nutrients, and other digestion conditions are at appropriate levels for first order kinetics to be applicable which in reality is not true.
Hydrogen Production by Hydrogen Producing Bacteria of Digester Sludge:
Scientists at the CIB (Chengdu Institute of Biology) in China in past also isolated 14 strains which comprised 63.6 per cent of total hydrogen producing bacteria obtained from enriched digester sludge. This included 4 strains of Serratia marcescens (18.2 per cent), 1 strain of Escherichia coli (4.5 per cent), 1 strain of Citrobacter freundii (4.5 per cent) and 2 strains of Clostridium acetobutylicum (9.1 per cent).
While carrying out experiments for analysing the effect of hydrogen producing bacteria on anaerobic fermentation the above strains were mixed with enriched cultures of methane producing bacteria.
The results are summarised in Table 2.2 from which it can be seen that with the mixed cultures, methane contents in biogas increased to appreciably high value of 90.3 per cent with carbon dioxide reduced to insignificant proportions. This is a very welcome improvement in biogas composition and needs to be studied further to make a commercial success.
During hydrolysis and acid-forming phase, a group of bacteria produce hydrogen. Further research needs to be carried out to study kind, number, nutrition, metabolism and behavioural pattern of micro-organisms. Only 20 per cent of hydrogen producing bacteria produce hydrogen. Considerable work has been recently done at the CIB, Academia Sinica in China to study the role of dynamics of hydrogen producing bacteria.
Scientists (China) obtained hydrogen producing algae by isolating them from biogas plant sludge. These bacteria were later on enriched which gave hydrogen yield of more than 50 per cent. A total of 51 bacteria strains were isolated from enriched cultures containing 24 (47 per cent) hydrogen producing bacteria.
As expected hydrogen yield was found to be different from different types of bacteria. Hydrogen-forming bacteria belong to Escherichiaccae and Bacillaceae families. The former category comprises five species, namely Escherichia coli, Enterbacter, Cloacae, Hafnia alvei, Citrobacter freundii and Serratia marcescens whereas the latter comprises only one specie, Glostridium acetobutylicum.