Petra Brunec Razvoj metod za določanje FOS/TAC vrednosti za spremljanje stabilnosti procesa presnove v bioplinskih napravah Diplomsko delo Maribor, ju

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1 Petra Brunec Razvoj metod za določanje vrednosti za spremljanje stabilnosti procesa presnove v bioplinskih napravah Diplomsko delo Maribor, junij 214

2 Razvoj metod za določanje vrednosti za spremljanje stabilnosti procesa presnove v bioplinskih napravah Diplomsko delo univerzitetnega študijskega programa Študent: Študijski program: Smer: Predvideni strokovni naslov: Mentor: Komentor: Mentorja v tujini: Petra Brunec univerzitetni, Kemijska tehnologija biokemijska tehnika un. dipl. inž. kem. tehnol. red. prof. dr. Darinka Brodnjak Vončina doc. dr. Irena Petrinić mag. Zia-Ur Rehman prof. dr. ing. Roland Span Maribor, junij 214

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4 i IZJAVA Izjavljam, da sem diplomsko delo izdelala sama, prispevki drugih so posebej označeni. Pregledala sem literaturo s področja diplomskega dela po naslednjih geslih: Vir: SienceDirect ( Gesla: Število referenc determination of parameter in anaerobic digesters 14 determination of volatile fatty acids in anaerobic digesters 86 determination of total inorganic carbon in anaerobic digesters 52 Vir: Digitalna knjižnica Univerze Ruhr v Bochumu ( Gesla: Število referenc biogas 8 anaerobic digestion 1 Skupno število pregledanih člankov: 6 Skupno število pregledanih knjig: 9 Maribor, junij 214 Petra Brunec

5 ii Acknowledgments This diploma thesis is my final product of my studies at Faculty of chemistry and chemical engineering. I would like sincerely thank to my supervisor M.Sc. Zia-Ur Rehman to take over the mentorship for my diploma thesis and for all his advice and support. I would like show my gratitude to the Prof. Dr. - Ing. Roland Span, for giving me an opportunity to work at Chair of Thermodynamics at Ruhr University Bochum and Dr. - Ing. Mandy Gerber for her help and for giving me an opportunity to do my experimental work at Biogas laboratory. I would also like to thank my coordinators Dr. Sabine Kareth and Prof. Dr. - Ing. Zorka Novak Pintarič who are responsible for my Erasmus Exchange. I would like to thank my mentor in Slovenia Prof. Dr. Darinka Brodnjak Vončina, who despite of a lot of work took me under her mentorship and helps me with expert advice. Finally my sincere thanks go to my parents and my sister for their constant help and support during my studies. I am thankful for all the encouragement, critic when needed and for being always there.

6 iii Razvoj metod za določanje vrednosti za spremljanje stabilnosti procesa presnove v bioplinskih napravah Povzetek Obnovljivi viri energije imajo dandanes vse pomembnejšo vlogo pri oskrbi z energijo ter zmanjševanju obremenjevanja okolja. Bioplin postaja v zadnjem obdobju vse pomembnejši na področju izkoriščanja alternativne energije v svetu in pri nas. Proizvodnja električne in toplotne energije iz bioplina postaja ena najpomembnejših oblik uporabe bioplina, v prihodnosti pa se bodo možnosti uporabe še razširile. Proces nastajanja bioplina je posledica povezanih procesnih korakov, pri katerih se prvotna snov stalno deli na manjše enote. V vsak posamezni korak so vključene specifične skupine mikroorganizmov. Bioplin kot zmes plinov nastaja z anaerobno presnovo organskih snovi oziroma odpadkov v enostavnejše komponente. Pridobivamo ga lahko skoraj iz vseh organskih materialov, ki vsebujejo zadosten delež ogljika. Vsebuje največ 5-7 % metana, 3-4 % oglikovega dioksida, vodikovega sulfida, amonijak ter dušik. Učinkovitost in stabilnost anaerobne presnove v bioplinski napravah je odvisna od nekaterih ključnih parametrov, zato je bistvenega pomena, da se ti parametri redno spremljajo, saj le s tem ustvarjamo primerne pogoje za anaerobne mikroorganizme. Delež proizvedenega bioplina je odvisen od vstopnih organskih snovi, vrednosti ph, vrednosti, temperature, zadrževalnega časa v reaktorju, od prisotnosti in količine inhibitorjev, vrste procesa in izvedbe bioplinske naprave. V tem diplomskem delu bomo poskušali razviti in izboljšati različne metode za določitev vrednosti, s pomočjo pripravljenih standardnih raztopin. vrednost opisuje vsebnost hlapnih maščobnih kislin glede na skupni anorganski ogljik oziroma pufersko kapaciteto karbonata. Povečano razmerje označuje zakisanost fermentorja. Med potekom fermentacije se bakterije preobremenijo in dodatek vhodnega substrata mora biti omejen. S pomočjo meritev vrednosti lahko enostavno spremljamo potek fermentacije ter nadzorujemo dodajanje vhodnih substratov. Ključne besede:, hlapne maščobne kisline, skupni anorganski ogljik, puferska kapaciteta karbonata, bioplin, anaerobna presnova, metan UDK: : (43.2)

7 iv Development of a method for the determination of of a biogas digestate Abstract Nowadays renewable energy sources have increasingly important role in terms of energy supplies and in reducing environmental pollution. In recent years biogas is becoming more and more important in development of alternative energy in the world. Production of electric and heat energy from biogas is becoming one of the most important forms of biogas usage. In the future the possibility of biogas usage will be even more increased. The formation process of a biogas is associated with several process steps in which the original substance is continuously divided into smaller units. In each step the different specific microorganisms are involved. Biogas is produced by anaerobic digestion with anaerobic bacteria or fermentation of biodegradable materials. It is derived from any organic material containing a sufficient amount of carbon. Biogas contains 5-7 % methane, 3-4 % carbon dioxide, hydrogen sulphide, ammonia, and nitrogen. Efficiency and stability of anaerobic digestion in biogas plants are dependent on certain key parameters. It is important that those parameters are monitored regularly, because only with that we can provide appropriate conditions for anaerobic microorganisms and anaerobic digestion to stay stable. The proportion of the biogas production is dependent on the input of organic material, the ph value, value, the temperature, the retention time in the reactor, the presence and quantity of the inhibitor, the type of process and the design of biogas plants. The aim of this diploma thesis is development and improvement of different methods for determination of values based on prepared standard solutions. The value describes the ratio of volatile fatty acids in the digester to the total inorganic carbon or buffer capacity and indicates the acidification of the fermenter. During acidification, the bacteria are overloaded and the substrate input must be restricted. With the help of value measurements the fermentation process and additions of input substrates can be monitored very easily. Key words:, volatile fatty acids, total inorganic carbon, buffer capacity, biogas, anaerobic digestion, methane UDK: : (43.2)

8 v LIST OF CONTENTS 1. Introduction Theoretical background Biogas Anaerobic digestion Hydrolysis Acidogenic phase Acetogenic phase Methanogenic phase Type of substrate Organic loading rate Foaming ph value Alkalinity Volatile fatty acids Methods for determination of values Method according to Nordmann Method according to Kapp Method according to Yang and Anderson Materials Hydrochloric acid Sulphuric acid Acetic acid Carbonate Calcium carbonate Sodium carbonate Equipment and methods Titrator Titroline Titrations Titration method according to Nordmann Titration method according to Yang and Anderson Titration method according to Kapp Results... 28

9 vi 5.1. Titrations with H 2SO CaCO 3 and CH 3COOH as a standard solution Na 2CO 3 and CH 3COOH as a standard solution Titrations with HCl CaCO3 and CH3COOH as a standard solution Na 2CO 3 and CH 3COOH as a standard solution Dilution of samples (CaCO 3) Dilution of sample (Na 2CO 3) Discussion Conclusions Literature Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Curriculum Vitae... 64

10 vii LIST OF FIGURES Figure 2-1: 4 litres biogas pilot plant... 2 Figure 2-2: Biochemistry of methane production... 4 Figure 2-3: Formation of monomers... 5 Figure 2-4: Optimum volume load in a bioreactor... 8 Figure 2-5: Carbon dioxide-hydrogencarbonate-carbonate buffer system... 1 Figure 2-6: Development of ph and values through days Figure 2-7: Inhibition through acetic acid Figure 2-8: Relation of acid consumption and FOS value Figure 3-1: Hydrochloric acid Figure 3-2: Sulphuric acid Figure 3-3: Acetic acid... 2 Figure 3-4: Calcium carbonate Figure 3-5: Sodium carbonate Figure 4-1: Titrator Titroline Figure 4-2: Electrode Figure 4-3: Titration curve Figure 5-1: value Figure 5-2: Comparison of method with fixed methods Figure 5-3: value Figure 5-4: Comparison of methods with fixed methods... 3 Figure 5-5: value Figure 5-6: Comparison of methods with fixed methods Figure 5-7: Figure 5-8: Comparison of methods with fixed methods Figure 5-9: value Figure 5-1: Comparison of methods with fixed methods Figure 5-11: value Figure 5-12: Comparison of methods with fixed methods Figure 5-13: value Figure 5-14: Comparison of methods with fixed methods Figure 5-15: value Figure 5-16: Comparison of methods with fixed methods Figure 5-17: value Figure 5-18: Comparison of methods with fixed methods... 4 Figure 5-19: value Figure 5-2: Comparison of methods with fixed methods Figure 5-21: value Figure 5-22: Comparison of methods with fixed methods Figure 5-23: value Figure 5-24: Comparison of methods with fixed methods... 45

11 viii LIST OF TABLES Table 2-1: Components in biogas... 3 Table 2-2: Types of bacteria present during degradation phases... 4 Table 2-3: Rules for the assessment for ratios... 7 Table 2-4: Volatile fatty acids properties Table 3-1: Physical properties of 38 % hydrochloric acid Table 3-2: Physical properties of sulphuric acid Table 3-3: Physical properties of acetic acid... 2 Table 3-4: Physical properties of calcium carbonate and sodium carbonate... 21

12 ix Glossary VFA volatile fatty acids FOS concentration of volatile fatty acids TAC (TIC) totale anorganisches carbonat (total inorganic carbon) Nordmann I, II titration methods with two ph end points Nordmann I, II fix titration methods with one ph end point (fixed method) Kapp I, II titration methods with three ph end points Kapp I, II fix titration methods with one ph end point (fixed method) Yang and Anderson titration method with two ph end points Yang and Anderson fix titration method with one end point (fixed method)

13 Introduction 1 1. Introduction The major concern for most societies these days is the use and availability of energy. Biogas energy offers a lot of advantages. Biogas plants can be built quickly and simply. Unlike the other energy sources biogas is a renewable resource. Biogas is produced during the anaerobic bacterial decomposition of organic materials. The anaerobic digestion is the natural microbiological process and it is defined as a process where organic materials are available and degraded in absence of oxygen. Anaerobic digestion consists of a complex series of reactions with a wide range of microorganisms involved. The organic materials are being degraded into compounds like NH 4+, S 2-3-, PO 4 and water. Products of anaerobic digestion are gases carbon dioxide and methane which are the main compounds of a biogas. The methane fermentation can be divided up into four phases. These four phases are hydrolysis, acidogenesis, acetogenesis and methanogenesis. All the phases are related closely with each other s. In the first phase anaerobic bacteria use enzymes to decompose large molecular organic substances into smaller molecular compounds. During the second phase acid forming bacteria continue the decomposition of compounds into the acids, CO 2, hydrogen sulphide and ammonia. In the next phase the acid bacteria form the acetate, CO 2 and hydrogen. The last phase of the fermentation procedure is methanogenesis. In the methanogenetic genesis the methane forming bacteria produce CO 2 and methane, known as a biogas and this is the last step of a degradation process. (1) Anaerobic digestion comprises several groups of microorganisms in a complex process. Some microbial groups are slowly growing and sensitive to change in operating conditions. Therefore, special knowledge and process parameters are required for an optimal fermentation process. One of these parameters has taken in more detailed studies in this diploma thesis. (2) The aim of this diploma thesis was application of different methods for determination of values. The value describes the contents of volatile fatty acids in relation to the buffer capacity of carbonate. The parameter is one of the parameters which can be determined quite easily on inexpensive and time saving way. Because of its simple determination the parameter has by now become a very popular parameter for the control of biogas plant stability.

14 Theoretical background 2 2. Theoretical background 2.1. Biogas Biogas is one of the most favorable energetic products with the most positive impact on the environment because it does not contribute to the increase the greenhouse gases in the atmosphere. (2) During the anaerobic digestion, biogas is produced by microbial degradation of organic materials such as sludge, sewage sludge, bio waste, food waste in the absence of oxygen. (1) Figure 2-1: 4 litres biogas pilot plant (45) Biogas is mainly composed of methane and carbon dioxide, but it also contains small amounts of other components. It consists of around 55 % to 7 % methane, 3 % to 45 % carbon dioxide and 1 % nitrogen. Typical biogas also contains hydrogen sulphide and other sulphur compounds such as sulphate, sulphite, undissociated and dissociated forms of hydrogen sulphide, ammonium, compounds such as siloxanes, aromatic and halogenated compounds, heterocyclic compounds, ketones, terpenes and halogenated aliphatics. The compounds present in biogas are shown in Table 1. Even though the amount of these compounds are very low compared to methane and carbon dioxide they have strong influence on production of biogas and on further use of biogas, they also have huge impact on environment such as greenhouse effect and stratospheric ozone depletion. (1) (3) In terms of energy utilization, the concentration of methane gives us the quality of biogas, which can be improved with the reduction of the incombustible components in the biogas such as ammonia, oxygen, and nitrogen and water steam. (1) (3)

15 Theoretical background 3 Table 2-1: Components in biogas (1) Combustible components Incombustible components Component Symbol Percentage content of gas (% by vol.) Methane CH Hydrogen H 2.5 Hydrogen sulphide H 2S -.5 Carbon dioxide CO Water steam H 2O 1-5 Oxygen O Ammonia NH Nitrogen N 2-5 The ratio of carbon dioxide to methane can be only partly controlled. It depends on a lot of factors: (1) The materials, which are rich in fat, can improve the quality of gas. With longer time of exposure of biomass the anaerobic degradation will improve. Near the end of residence time the amount of methane increases disproportionally. The fermentation is more evenly and faster if the material in the bioreactor is well and homogeneously activated. A higher amount of liquid and the higher pressure in the bioreactor reduces the level of CO 2 in gas phase because the higher content of CO 2 will be dissolved in water. A higher temperature during the fermentation process decreases the concentration of CO 2 dissolved in water. The material for the fermentation process has to be completely decomposed. The substrate has to be prepared that it expedites and intensifies the decomposition. Biogas has similar properties as a natural gas, therefore it can be used for the same purpose. Resulting energy from the gas offers a lot of advantages. It is renewable energy source, which can solve the problems of rising energy prices, biogas plants can be built quickly, simply and for much less money than nuclear, coal, oil or other power plants. It also provides an excellent source of energy that is friendly to environment, with less carbon dioxide and methane emissions and it reduces the amount of organic waste. (4) In biochemical aspect, the production of biogas involves a complex reaction that take place under anaerobic conditions in the presence of highly ph sensitive microorganisms.

16 Theoretical background 4 Table 2-2: Types of bacteria presents during degradation phases (5) Hydrolysis Acidogenesis Acetogenesis Methanogenesis Bacillus sp. Bacillus sp. Acetobacterium woodii Methanobacterium spp. Bacteroides sp. Bacteroides sp. Peptostreptococcus sp. Methanococcus sp. Clostridium sp. Clostridium sp. Methanosarcina barkeri Pseudomonas sp. Pseudomonas sp. Methanosaeta sp. Clostridium aceticum Methanospirillum Micrococcus sp. Micrococcus sp. Methanumbacterium sp Anaerobic digestion Anaerobic digestion of organic matter is a natural biological process of a biogas production. It is a complex four step process and depends on the balanced activities of microorganisms. (6) The individual phases are operated by different groups of bacteria, which partially degrade together specific components of substrates, which they are not able to degrade on their own. The interdependence of microorganisms is a key factor for the biogas process. The main four steps involving in anaerobic degradation of organic mass are hydrolysis, acidogenetic genesis, acetogenetic genesis and methanogenetic genesis. (7) (8) Figure 2-2: Biochemistry of methane production (17) Hydrolysis Hydrolysis is the first step of anaerobic degradation, during which the complex polymers like carbohydrates, lipids, nucleic acids and proteins are converted into smaller units as mono- and oligomers. (1)

17 Theoretical background 5 Figure 2-3: Formation of monomers (1) Hydrolytic microorganisms excrete hydrolytic enzymes, converting biopolymers into simpler and soluble compounds such as short-chain sugars, fatty acids and glycerine, purines and pyridines as it is shown below. (2) Lipids lipase fatty acids, glycerine Eq. 2-1 Polysaccharide cellulase cellobiase xylanase amylase monosaccharide Eq. 2-2 Proteins proteasse amino acids Eq. 2-3 The hydrolysis of carbohydrates takes place within few hours and the hydrolysis of proteins and lipids within few days. (1) The products resulted from hydrolysis are further decomposed by the involved microorganisms and used for their own metabolic processes. (1) Acidogenic phase During the acidogenesis, the hydrolyzed compounds are fermented by anaerobic acidogenic bacteria into volatile fatty acids (butyric acid, propionic acid, acetic acid, valeric acid), alcohols, hydrogen and carbon dioxide. The concentration of intermediate hydrogen ions (H + ) impacts on fermentation products of acidogenesis. The higher is the partial pressure of hydrogen, the fewer compounds as acetate will be produced. (1) (9) amino acids, sugars, fatty acids acidogens Intermediate components(propionate, ) Eq Acetogenic phase Products from acidogenic phase, which cannot be directly converted to methane by methanogenic bacteria, serve as methanogenic substrates. In this phase acetogenic bacteria oxidize volatile fatty acids and alcohol into methanogenic substrates like acetate, hydrogen and carbon dioxide. The product hydrogen increases the hydrogen partial pressure. That can have a huge effect on metabolism of acetogenic bacteria and can inhibit their process. When partial pressure is low, acetogenic bacteria convert mainly H 2, CO 2 and acetate or butyric, capronic, propionic and valeric acids when hydrogen partial pressure is high. However in the next phase methanogenic bacteria can only process acetate, CO 2 and H 2. During the degradation it can be seen the

18 Theoretical background 6 connection between acetogenic bacteria which are H 2 producing organisms and methanogenic H 2 consumed bacteria that means that acetogenesis and methanogenesis usually run parallel, as symbiosis of two groups of organisms. (1) (9) Intermediate compounds acetogens acetic acid, carbon dioxide, hydrogen Eq Methanogenic phase The final step in conversion of organic substances into methane and carbon dioxide is methanogenesis. Methanogenesis must strictly operate under anaerobic conditions. Methogenic bacteria utilize hydrogen, acetic acid and carbon dioxide to form methane and carbon dioxide. They are very sensitive to environmental changes, if any occurs. 7 % of methane arises from acetate, while remaining 27-3 % arises from conversion of hydrogen and carbon dioxide. (2) (6) The methanogens are classified into two major groups: (2) 1. The acetate converting methanogens acetic acid methanogenic bacteria methane + carbon dioxide Eq The hydrogen utilizing hydrogenotrophic methanogens methanogenic bacteria hydrogen + carbon dioxide methane + water Eq. 2-7 Methanogenesis is the slowest reaction in the anaerobic digestion, but it is also the most significant biochemical reaction of the process. (2) 2.3. An optimum fermentation process depends on numerous parameters, which must be carefully monitored to ensure the effective degradation of organic matter. The growth and activity of anaerobic microorganisms is significantly influenced by conditions such as exclusion of oxygen, type of substrate, constant temperature, ph-value, nutrient supply, stirring intensity, presence and amount of inhibitors, retention time and a many others. Some of them are described in subsections below. (1) value indicates amount of volatile acids in proportion to the buffer capacity of carbonate. It is one of the parameters, apart from temperature and ph value, which can be determined on location, in an inexpensive and time-saving way. (1) (11) The titration method for determining FOS and TAC in biogas plant was first introduced by Scott McGhee in To determine fatty acid concentration McGhee tried to develop a method in which the consumption of acids varies with the ph measured between different titration end points. According to Nordmann analyses, which were based on acid and alkaline capacity and other analytical parameters the

19 Theoretical background 7 titration method for determination of volatile fatty acids and buffer capacity contents were developed. After Nordmann many new different methods, which were based on his experiments, were developed in order to determine the quotient of acids and buffer capacity in fermentation substrate. (11) The relation of both parameters, FOS which stands for Flüchtige Organische Saüren, i.e. volatile organic acids and is measured in mg HAc/l and TAC stands for Totale Anorganisches Carbonat, i.e. total inorganic carbon which is measured in mg CaCO 3/l has by now become a very popular parameter for the control of biogas plant stability. In general the value of ratio for stable process should be somewhere around.15 to.45. The lower values will lead to alkalosis, which will raise the ph values and decrease the contents of organic acids and the higher values are indicators for acidosis, which is reflected by an excessive accumulation of organic acids. (11) Table 2-3: Rules for the assessment for ratios (12) Background Measure >.6 Highly excessive biomass input Stop adding biomass.5-.6 Excessive biomass input Add less biomass.4-.5 Plant is heavily loaded Monitor the plant more closely.3-.4 Biogas production at a maximum Keep biomass input constant.2-.3 Biomass input is too low Slowly increase the biomass input <.2 Biomass input is far too low Rapidly increase the biomass input The titration procedures developed so far are quite different. Among others there are methods by: DiLallo and Albertson (1961), McGhee (1967), Nordmann (1977), Colin (1984), Kapp (1984), Ripley et al. (1986), Powel and Archer ( 1989), Moosbrugger et al. (1992,1993). Methods are different in the way of titration end point determination and the way of calculation the ratio. (11) Type of substrate The type of substrates determines the anaerobic decomposition. They must be taken into consideration in the anaerobic process. Bio-wastes most often consist of several components, such as biodegradable organic fraction, a combustible and inert fraction. If a substrate components of vital importance runs out, the microorganisms stop their metabolism. Biodegradable organic fractions are for example kitchen scraps, food residue, grass and they are the most appropriate substrates for anaerobic degradation process. The combustible fraction includes slowly degrading lignocellulosic organic substances containing wood or paper. Stones, glass, sand and metal represent non-biodegradable or inert fraction of bio-waste and they should be removed or recycled. The removal of inert fraction is important for anaerobic digestion, in the other hand digester volume and energy consumption for process will increase. According to the composition of the substrates, intermediate products of the decomposition can also inhibit the degradation process. For example, the degradation of fats can increase content of volatile fatty acids, which can limit the further anaerobic degradation. With the decomposition of proteins, methane fermentation can also be decreased by the formation of ammonia and hydrogen sulphide. (1) (9)

20 Theoretical background Organic loading rate In construction of biogas plant it is important to take into consideration the technical and economical aspect. The methane output depends on the digester load. Complete digestion of organic matter input and the resulting maximum output of biogas, would require a long retention time of organic matter and therefore also correspondingly large volume of a biogas reactor. In practice digester type and size are based on the compromise between maximum yield of gas and justifiable economy. (1) Figure 2-4: Optimum volume load in a bioreactor (1) Organic load is an important factor of the biogas plant, which shows how much dry matter can be loaded into the biogas reactor per volume and time unit. (2) Where; B R = m c V R Eq B R = organic load (kg/d m 3 ) - c = concentration of organic matter (%) - m = mass of organic matter fed per time unit (kg/d) - V R = digester volume (m 3 ) Every biogas plant has its own optimal ratio, in general the normal values should be around.3 to.4. This must be determined by long-term observation and regular checks, because not every substrate gives the same values. For example, plants that make use of renewable raw materials usually require a ratio of.4 to.6 for their stable operation process. (1) Foaming It was studied that several parameters could potentially contribute to foaming in anaerobic digesters. Some of them are growth of filamentous organism, organic overloading accumulation of volatile fatty acids, and inadequate mixing of the digesters. (1)

21 Theoretical background 9 Temperatures and ph can inhibit the growth of the bacteria in the anaerobic digesters. Accumulation of volatile fatty acids can be observed when methane production decreases. The increase of VFA accumulation could be the cause of higher foaming potential in anaerobic digesters. Using the ph as the control parameter is not so advisable. By the time the ph drastically changes it may be too late to avoid the changes in anaerobic digestion. When this happens, the methane production will drop dramatically and the foaming will occur. The better parameter for monitoring the stability of an anaerobic digestion is volatile fatty acid concentration. The VFA determination gives the result immediately. The VFA accumulation in the anaerobic digester reflects an imbalance between the acid producing and acid consuming bacteria. If the digester is overloaded and contains a high VFA concentration, more than the methane producing bacteria can consume, biogas production will quickly rise and this will increase the foaming potential of anaerobic digestion. (1) (13) ph value ph value is a measure of acidity or alkalinity of a solution. It is normally relatively easy to measure and it is one of liquid-phase parameter, which can be measured online. The change in ph can be an indicator or cause of process imbalance, since the microorganisms can only function in a specific range of ph. The optimum ph range for methane forming microorganisms is between The ph value of the anaerobic digester substrate influences the growth of methanogenic microorganisms and effects on disintegration of some compounds (ammonia, sulphide, organic acids). During the decomposition, for optimal process control, different ph levels are required for acidogenesis and for methanogenesis. For methanogenesis is it important to adjust the higher ph values, around 7 and for acidogenesis the ph values can be lower. (1) (2) (9) (1) The most appropriate ph interval for mesophilic digestion is between 6.5 and 8. and the process is severely inhibited if the ph-value decreases below 6. or rises above 8.3. The solubility of carbon dioxide in water decreases at increasing temperature. The ph-value in thermophilic digesters is therefore higher than in mesophilic ones, as dissolved carbon dioxide forms carbonic acid by reaction with water. (2) Accumulation of volatile fatty acids can lower the ph value below 6.5. Increased activity of hydrolytic bacteria occurs further lowering of ph and possibly end of fermentation. A too strong acidification is solvable with carbon dioxide-hydrogencarbonate-carbonate buffer system which is shown in Figure 2-5. (1) During fermentation process a lot of carbon dioxide is produced and escapes into the air. With lower ph value more CO 2 dissolved in the substrates and all CO 2 is present as free molecules. At higher ph value the CO 2 dissolved in the substrates has form of carbonate. Neither of these two values is appropriate. Increasing ph values have impact on more CO 2 dissociating out of the gas and decreasing ph value means that more CO 2 are released into the environment. (1)

22 Theoretical background 1 CO 2 H 2 CO 3 H + +HCO 3-2H + + 2CO 3 2- Eq. 2-9 Figure 2-5: Carbon dioxide-hydrogen carbonate-carbonate buffer system (1) A too weak acidification can be also avoided by ammonia-ammonium buffer system. With decreasing ph value, ammonium ions are formed with release of hydroxyl ions and with increasing the ph value, more free ammonia molecules are formed. (1) NH 3 + H 2 O NH OH - Eq. 2-1 NH 3 +H + + NH 4 Eq The value of ph in anaerobic reactors is mainly controlled by the bicarbonate buffer system. Both bicarbonate and ammonia buffering systems can be overloaded by feed of particularly rapidly acidifying wastewaters or organic materials, by toxic substances, by decrease in temperature or by too high volume load in the bioreactor. (1) The ph value inside digesters depends on the partial pressure of CO 2 and on the concentration of alkaline and acid components in the liquid phase. If accumulation of base or acid occurs, the buffer capacity tries to prevent these changes in ph, up to a certain level. When the buffer capacity of the system is exceeded, drastic changes in ph values occur and can completely inhibit the anaerobic digestion process. First sign of acidification is the rise of propionic acid concentration. The measures for the prevention of fermentation process disturbance must be run immediately but when the drastic change of ph finally occur the fermentation process can be completely disrupted and will be too late to avoid the upset. (2) Only determination of the ph value is not enough to operate a biogas plant safely. This is particularly true for plants utilising a fermentation substrate with a high buffering capacity, since even a strong accumulation of organic acids will not necessarily lead to a decrease of the ph value. Analysis of the single organic acids is a common practice to assess the operational stability of biogas plants but these analyses are quite complex and expensive. For this reason, the determination of value has been recognised as enough accurate value for evaluation of fermentation processes. With determination of value the process disturbances can be recognised at an early stage and can be immediately

23 Theoretical background 11 solved. In the Figure 2-7 the development of ph and values over days in a maize silage plant is illustrated. (14) Figure 2-6: Development of ph and values by days (14) Alkalinity Alkalinity is the most important parameter for indicating the state of anaerobic digestion system. The fundamental problem in the anaerobic digestion process is that equilibrium has to be maintained between acid and methanogenic fermentation. While this equilibrium exists, the concentration of intermediate products from the conversion of organic material into biogas will be low and at any time the conversion of hydrolysed products to the final products is substantially complete. If the equilibrium is disrupted, there will be an accumulation of acid and consequently the ph of the digester will decrease. The methanogenesis requires a ph near the neutral value. The decrease of ph might lead to a reduction of the methane production and to further accumulation of acids. As the consequence, the anaerobic digester process may fail down. The digester will only return to its activity when the ph of the reactor is restored to a value near neutral ph, which can be effected by addition of alkalinity to buffer the effect of acids. Performing alkalinity and volatile fatty acids tests on the anaerobic digester is an important part of effective digester operations. The acid and alkalinity ratio will change before the ph begins to drop. (1) (15) From all of previous explanation follows that alkalinity is a better alternative than ph for indicating VFA accumulation, since the increased VFA will directly consume alkalinity before large ph changes. With the titration to ph 4.3 total alkalinity (TA) is measured. Titration to this ph point not only measure bicarbonate buffering capacity of the sample, but also the volatile fatty acids (VFA) buffering. With pk 4.7 of acetic acid and pk 4.9 of propionic acid, which is below the normal digester operating ph, their buffering is not useful part of the alkalinity. To prevent VFA buffering from being included in the alkalinity measurement, Jenkins suggested that digester supernatant is titrated to end point 5.75, which is known as partial alkalinity (PA). Measuring the partial alkalinity can be one way of indirectly measuring the VFA accumulation. (15) (16) (17) (18) However, this relationship is not observed during VFA accumulation in response to ammonia overload, as the ammonia adds alkalinity to the system. According to all this the determination of value

24 Theoretical background 12 is better parameter to for identifying the efficient of anaerobic digestion process. Other authors suggested that the ratio of VFA/TA as an indicator where the healthy digester should have should be between the range of (19) Volatile fatty acids Organic acids are normally present in the substrate and are decomposed during methanation. Stability of anaerobic digestion process depends on their concentration. Volatile fatty acids are mainly acetic acid, propionic acid, butyric acid and valeric acid. They are intermediate compounds, produced during acidogenesis and they are one of the most critical parameters for production of biogas. During the acidogenesis, volatile fatty acids, longer than two carbons, are converted to acetic acid, carbon dioxide and hydrogen, which become methane gas through methanogenesis. The process step must be well balanced to prevent volatile fatty acids accumulation and sudden drop of system ph, which can lead to complete error of the decomposition process. Due to the different types of biomass with different buffer capacities in the digester, accumulation of VFA will not be always expressed as a drop of ph. (2) Figure 2-7: Inhibition through acetic acid (1) Accumulation of VFA must exceed a certain level, that the drop of ph will occur but at that point the concentration of VFA will be so high that the process of anaerobic digestion could be at serious risk. (2) Many authors have studied, investigated and correlated the process stability to the concentration of individual VFA in bioreactor. The most represented were acetic and propionic acids. The influential concentration of acetate affected on imbalance in bioreactor is higher than 13 mm. From some investigators the propionate was suggested as better indicator of process instability. They also try to combine these two acids and Hill proposed that for a stable process it can be used propionate/acetate ratio, which should not be higher than 1.4. Also long-chain (C 4-C 6) acids and their isomers have been suggested as good indicators for stable process. Scientists Hill and Holmberg showed that concentrations of isobutyrate and isovalerate below.6 mm indicate a stable process and the concentrations between.6 mm and.17 mm reflect the process imbalance. (6)

25 Theoretical background 13 However, different anaerobic systems have their own different levels of VFA for a stable process, determined by composition of the substrates or by the operating parameters, so it could not be possible to define an absolute indicated VFA level for stable state of the process. (6) (4) The VFAs can be determined with various methods, such as simple titrations, liquid chromatography procedures (thin layer chromatography, high performance liquid chromatography, ion-exchange chromatography, gas chromatography (GC)) and (ratio between VFA and alkalinity). (1) Table 2-4: Volatile fatty acids properties Structural formula Ion Molar mass pk a Acetic acid CH 3COO Propionic acid C 2H 5COO Butyric acid C 3H 7COO Valeric acid C 4H 9COO Methods for determination of values Method according to Nordmann The stability of an anaerobic process can be assessed with knowledge of the single parameters like volatile fatty acid and buffer capacity or the ratio of those. Nordmann (1977) developed the determination of volatile fatty acids with titration from initial ph value to ph 5 and from ph 5 to ph 4, with.2 M sulphuric acid into filtered sludge sample. The consumption of sulphuric acid up to ph 5 expresses the buffer capacity of the carbon buffer system and between the values ph 5 and ph 4.4 the protons are absorbed by organic acids. The Nordmann equations are based on McGhee linear connection between the acid consumption and FOS value in filtered sludge samples. Relation of acid consumption and concentration of volatile fatty acids in filtered samples and regression are shown in Figure 2-8 below. (21) (22) (11)

26 Theoretical background 14 Figure 2-8: Relation of acid consumption and FOS value Y = X Eq With the linear equation given by McGhee, Nordmann developed the ratio. (11) X = FOS [mg l HAC] Eq Y = B [ml] ph Eq ph = ph 5 ph 4.4 Eq Y = X Eq X = (Y.15 ) Eq X = ( B.15) Eq ph The relation of both parameters gives the value. FOS [ mg HAc l ] = ((B 1.66).15) 5 Eq TAC [ mgcaco 3 l ] = A 25 Eq. 2-2

27 Theoretical background 15 Where; - FOS is concentration of volatile fatty acids in (mg/l HAc), - TAC is buffer capacity in (mgcaco 3/l) - B is consumption of titer acid from ph 5 to ph 4.4 in ml, - A is consumption of titer acid from initial ph of sample to ph 5 in ml Method according to Kapp Kapp founded his procedure on a principle suggested by McGhee (1968). The idea was that the consumption of the acid can be considered as proportional to the content of volatile fatty acids in the solutions if the sample is titrated from ph 5 to ph 4. Between ph 5 and ph 4 acetate subsystem is only present and there are no other weak acid/base subsystems that could have major influence on the titration result. The acetic, propionic, butyric and valeric acids can all be treated together as one parameter, because they all show very similar buffering characteristic and all have pk a around The only - additional buffer system according to Kapp s calculations of VFA is the carbonate subsystem HCO 3 /CO 2; the other buffer subsystems as sulphide, phosphate and ammonium buffer subsystem are assumed to be insignificant. (23) The method according to Kapp (1984) was originally developed for control of mesophilic sludge digesters. The method was tested by Kapp himself using samples of digester sludge, which were analyzed by titration and steam distillation. Kapp s titration approach involves three ph titrations set/end points, ph 5, ph 4.3 and ph 4 in addition to the initial ph of sample. Kapp claimed that accuracy of his method is ± 5 mg/l or ± 1 % for VFA 2 mg/l. (23) The major weakness of Kapp s approach is that it is based on empirical mathematical relationships that were developed under unique conditions (ionic strength, temperature, absence of other weak-acid systems) that are not necessarily generally applicable. The advantages of the method are its simple, single equation calculations of VFA concentration, simple equipment and quick and easy analytical work. (23) (24) (25) VFA = (13134 N VA 5 4 V sample ) 3.8 Alk meas 1.9 Eq Alk meas = VA 4.3 meas N 1 V sample Eq TAC = ( VA 4.3 meas N 1 V sample ) M(CO 3 2 ) Eq. 2-23

28 Theoretical background 16 Where; - VFA is concentration of volatile fatty acids in (mg/l), - TAC is buffer capacity in (mg/l), - Alk meas is alkalinity in (mol/l), - N is normality of titrant acid in (mmol/l), - VA 5-4 is volume of acid consumed from ph 5 to ph 4 in ml, - VA ph-5 is volume of acid consumed from initial ph of sample to ph 5 in ml, - VA 4.3 meas is volume of acid consumed from initial ph of sample to value 4.3 ph in ml Method according to Yang and Anderson Method according to Anderson and Yang is simple alkalimetric method that can determine bicarbonate and total volatile acid concentrations in anaerobic digesters by a two-stage sequential titration. The method was studied on standard solutions of volatile fatty acids and bicarbonate over a ph 5.5 and 7.65 and it was also used for determination of volatile fatty acid and bicarbonate concentration in effluent samples. The standard solutions were prepared with analytical volatile fatty acids and were neutralized, when was necessary, to their sodium salts with sodium hydroxide and standard bicarbonate solution was prepared with analytical sodium carbonate. N/1 standard sulphuric acid was used as the titrant. The average recovery of volatile fatty acids and bicarbonate by this method from standard solution was found to be 96 % and also the concentration of volatile fatty acid from effluent was coincided well according to samples results obtained using chromatography. (26) Usually, the alkalinity in anaerobic digester is determined by titrating a sample to a ph 4.3. The measured value includes all bicarbonate and about 8 % of volatile fatty acids. The value is known as total alkalinity. Total alkalinity does not always represent the available buffer capacity in digester, because it is also the only parameter, which is usable for neutralization of volatile fatty acids. Beside to this, the measured alkalinities may not be comparable without referring to the ph of the sample. So this total alkalinity disadvantages were leading to explore a new research about alkalinity. The change in the titration end point from ph 4.3 to 6. was proposed. This could measure the alkalinity that includes approximately 7 % of carbonate and nearly 5 % of volatile fatty acid in samples. Jenkins et al. (1983) introduced new alkalinity titration method with an endpoint of ph 5.75 and calculated total bicarbonate alkalinity, with assumption that VFA concentration is insignificant. Ripley et al. (1985) suggested measuring a partial alkalinity (PA) with endpoint of ph 5.75 and intermediate alkalinity (IA) with endpoint of ph 4.3 and the ratio between IA and PA was proposed as indicator to monitoring digester. However, these modifications have improved the sensitivity of alkalinity test in digester monitoring, but neither of them give an accurate measure of bicarbonate concentration. (26) The other useful titration parameter for digester control is also concentration of volatile fatty acids. A number of methods are currently used for their determinations. The Anderson and Yang used a simple two-point titration (i.e. ph 5.1 and ph 3.5) method, which gives a direct measurement of both bicarbonate and volatile fatty acids concentration at the same time. Experiment was done by measuring the initial ph of the sample, than the sample was titrated with standard sulphuric acid to the first ph endpoint 5.1 and after that to the second endpoint 3.5. The

29 Theoretical background 17 concentrations of volatile fatty acids and bicarbonate were calculated using equations below. (26) (27) (28) A 1 = [HCO 3 ]([H] 2 [H] 1 ) [H] 2 +K 1 + [VA]([H] 2 [H] 1 ) [H] 2 +K 2 Eq Where; A 2 = [HCO 3 ]([H] 3 [H] 1 ) [H] 3 +K 1 + [VA]([H] 3 [H] 1 ) [H] 3 +K 2 Eq A 1 and A 2 are molar equivalents of standard acid consumed to the first and second endpoints, - [HCO 3- ] is the bicarbonate concentration, - [H] 123 are hydrogen ion concentration, - [VA] is volatile fatty acid concentration, - K 1 is dissociation constants of carbonic acid, - K 2 is dissociation constant of the volatile fatty acids.

30 Materials Materials 3.1. Hydrochloric acid Titration experiments for determination of values were performed using.1 M hydrochloric acid. Hydrochloric acid is a monoprotic acid with a high acid dissociating constant what means that HCl can dissociate into H + and Cl - ions in water. For every mole of HCl that dissociates in solutions, one mole of H + and Cl - ions are formed which means that normality of used acid was.1. To compare how.2 N solution will have influence on reaction and if this will have impact on acid consumption sulphuric acid as a titer acid was also used. Unlike the HCl, H 2SO 4 is a diprotic acid which dissociate in solutions and every mole of dissociated acid forms 2 H + 2- ions and SO 4. (29) HCl+ H 2 O H 3 O + +Cl - K a = Eq. 3-1 Hydrochloric acid is a versatile chemical used in a variety of chemical processes. The acid is nonflammable, transparent, colourless or slightly yellow liquid. When it has concentration more than 25 % it is fuming strong acid. Hydrochloric acid gas has a strong pungent odor and is strongly corrosive. (3) (29) Figure 3-1: Hydrochloric acid (31) Table 3-1: Physical properties of 38 % hydrochloric acid (32) Molecular mass Boiling point Melting point Density (g/mol) ( C) ( C) (g/l) Sulphuric acid Sulphuric acid was also used as titrant solution for simple titration procedures to determine volatile fatty acids and total inorganic carbonate values, known as parameter. The used concentration of sulphuric acid was.1 M.

31 Materials 19 Sulphuric acid is a colourless, viscous and highly corrosive oily liquid. It is a diprotic acid. The chemistry of sulphuric acid it primarily related on its acidity. The dissociation does not happen at once due to the two dissociation steps with different K a values. (29) (33) H 2 SO 4 H + (aq)+ HSO 4 - (aq) K a =1 1 3 Eq. 3-2 HSO 4 - H + (aq)+ SO 4 2- (aq) K a =1 1-2 Eq. 3-3 The first dissociation is considered to be complete and the second is not. Diprotic acids are especially important in a titration experiment, where a ph versus titrant volume curve will clearly show two equivalence points for acid. The proton activity of sulphuric acid increases with increasing concentration of acid, so pure sulphuric acid is very strong acid media in which most other dissolved substances act as bases. The strong tendency of sulphuric acid to give away a proton is the reason that pure sulphuric acid has an extraordinary affinity to water. The hydration reaction of sulphuric acid is highly exothermic so acid must be always added to the water rather than the water to the acid. (29) (33) Figure 3-2: Sulphuric acid (34) Table 3-2: Physical properties of sulphuric acid (32) Molecular mass Boiling point Melting point Density (g/mol) ( C) ( C) (g/l) Acetic acid For the preparation of standard solutions, which were used in titration procedures to determine values in solutions, acetic acid was used, one of the acid which has a huge impact on production of biogas. Concentrations of acetic acid were known and they were calculated according to amount of carbonates values in standard solutions. Calculations of acetic acid concentrations are shown in Appendix 1.

32 Materials 2 Acetic acid is the most important and simplest carboxylic acid. It is colourless, combustible, hygroscopic, corrosive liquid with very pungent odor. Acetic acid is a weak monoprotic acid with a low dissociating constant. (35) CH 3 COOH CH 3 COO - +H + K a = Eq. 3-4 Acetic acid is a very stable substance. It resists the action of reducing and oxidizing agents and does not readily decompose when heated. For these reasons it is a useful solvent for organic substances. Acetic acid has the usual properties of acids, such as reacting with bases to form salts. (36) Figure 3-3: Acetic acid (36) Table 3-3: Physical properties of acetic acid (32) Molecular mass Boiling point Melting point Density (g/mol) ( C) ( C) (g/l) Carbonate All experimental work was made on standard solutions with known concentration of acetic acid and calcium or sodium carbonate. According to calculated value, different amounts of calcium carbonate or anhydrous sodium carbonate were added for determination of TAC value in standard solutions. The calculations are shown in Appendix 1. Both carbonates were used because of their difference in solubility in water and their different initial ph values. The sodium carbonate aqueous solutions are strongly alkaline, with the ph more than 1. ph of calcium carbonate was between 7 and 8. According to their different initial ph it was assumed that the different amounts of CO 2 will escape from the samples. According to higher ph value of sodium carbonate samples it was assumed that less CO 2 will escape. Escaping of CO 2 will change the equilibrium and more hydrogen carbonate will convert into CO 2. HCO 3 + H + CO 2 + H 2 O pk a =6.3 Eq. 3-5

33 Materials 21 Table 3-4: Physical properties of calcium carbonate and sodium carbonate (32) Formula Molecular mass Boiling point Melting point Density (g/mol) ( C) ( C) (g/l) CaCO decomposes Na 2CO decomposes Calcium carbonate Calcium carbonate is the most common calcium compound with low solubility in water. It appears in three crystal modifications as calcite, aragonite and valerite. The most common and stable is calcite. Other industrially important source minerals which are predominantly calcium carbonate are lime, chalk, marble and travertine. (37) (38) Like all carbonates, calcium carbonate is sensitive to acids. Calcium carbonate causes a unique reaction with acids. Upon contact with an acid, no matter the strength, it produces carbon dioxide. (38) Reaction with hydrochloric acid; CaCO HCl CaCl 2 + CO 2 + H 2 O Eq. 3-6 Reaction with sulphuric acid; CaCO 3 + H 2 SO 4 CaSO 4 + CO 2 + H 2 O Eq. 3-7 Reaction with acetic acid; CaCO 3 +2 CH 3 COOH Ca(CH 3 COO) 2 + CO 2 + H 2 O Eq. 3-8 Calcuim carbonate is very difficult to dissolve in water. Solubility in pure water is 13 milligrams per liter. (38) CaCO 3 + CO 2 + H 2 O Ca(HCO 3 ) 2 Eq. 3-9 But the presence of carbon dioxide can increase solubility of calcium carbonate by more than 1 times. (38) Figure 3-4: Calcium carbonate (39)

34 Materials Sodium carbonate Sodium carbonate is hygroscopic, odorless white powder. Sodium carbonate crystallizes from water to form three different hydrates, decahydrate, monohydrate and anhydrous sodium carbonate. When the crystals of decahydrate (Na 2CO 3 1 H 2O) lose water, they are formed into monohydrate (Na 2CO 3 H 2O). When the monohydrate is heated to 1 C it changes to anhydrous sodium carbonate (Na 2CO 3), also known as soda ash. (37) (3) Also like all of carbonates, sodium carbonate liberates carbon dioxide with reactions with acids. (4) Reaction with hydrochloric acid; Na 2 CO 3 +2 HCl 2NaCl+ CO 2 + H 2 O Eq. 3-1 Reaction with sulphuric acid; Na 2 CO 3 + H 2 SO 4 Na 2 SO 4 + CO 2 + H 2 O Eq Reaction with acetic acid; Na 2 CO 3 +2 CH 3 COOH 2 NaCH 3 COO+ CO 2 + H 2 O Eq Sodium carbonate is more soluble in water than calcium carbonate. Solubility of sodium carbonate is 3.7 g/1 g water at 25 C. (32) Na 2 CO H 2 O 2NaOH + H 2 CO 3 Eq Figure 3-5: Sodium carbonate (41)

35 Equipment and methods Equipment and methods 4.1. Titrator Titroline 6 For standard titration procedure for determination of values in the samples titrator Titroline 6 was used. The titrator can be used for analyses in food, water, wastewater and environmental applications. According to high resolution and precise ph and mv measuring electrode, it is possible to determine a wide range of parameters quickly, reliably and precisely. Features of titrator include temperature inputs for ph, redox or photometric titrations and polarisable electrode input for set end point applications. It also includes default standard methods for determinations of alkalinity, total alkalinity and parameters. Titration measurement of ph, mv and µa can be done with self-selecting end point or with present end points. The dosing and filling speed can be optimaly adjusted to the dosing solution. The piston burette can be filled with 5, 1, 2 or 5 ml of titer solutions. Calculation of results is performed after titrations and they are displayed on display screen. If there are two results, such as in case of two ph end points titration, it can be entered two results texts. (42) The titrator Titroline 6 is easy to use and control and its determinations are sufficiently precise for laboratory analyses. (43) Figure 4-1: Titrator Titroline 6 (43) Figure 4-2: Electrode (43)