Mechanism of zeolite activity in biogas co-digestion

Transcription

1 Department of Physics, Chemistry and Biology Master s thesis Mechanism of zeolite activity in biogas co-digestion Anna Hansson Master s thesis performed at Tekniska Verken in Linköping AB (publ) LITH-IFM-x-EX--11/2487--SE

2 Department of Physics, Chemistry and Biology Mechanism of zeolite activity in biogas co-digestion Anna Hansson Master s thesis performed at Tekniska Verken in Linköping AB (publ) Supervisors Erik Nordell, Tekniska Verken in Linköping AB (publ) Martin Karlsson, Linköping University Examiner Uno Carlsson, Linköping University

3 Avdelning, institution Division, Department Datum Date Chemistry Department of Physics, Chemistry and Biology Linköping University Språk Language Svenska/Swedish Engelska/English Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport ISBN ISRN: LITH-IFM-x-EX--11/2487--SE Serietitel och serienummer ISSN Title of series, numbering URL för elektronisk version Titel Zeolitaktivitetens mekanism vid biogasproduktion Title Mechansim of zeolite activity in biogas co-digestion Författare Author Anna Hansson Sammanfattning Abstract Biogas is a source of renewable energy and is produced at anaerobic conditions. The gas consists mainly of methane (55-70 %) and carbon dioxide (30-45 %). Biogas can be used as vehicle fuel after the gas has been upgraded to a methane content of approximately 97 %. There are several companies in Sweden producing biogas. Svensk biogas AB in Linköping is one of the largest. The company has two biogas production plants; one in Linköping and one in Norrköping. To meet the surge demand for biogas it is not only important to increase the volumetric capacity of the digesters, but also to optimize the process at the existing production plants in different ways. Zeolites have earlier been shown to have a positive effect on anaerobic digestion of certain substrates. The aim of this master s thesis was to investigate if the organic loading rate could be increased and/or if the hydraulic retention time could be reduced by addition of zeolites to a reactor treating slaughterhouse waste as substrate. The aim was further to investigate which substance/substances that zeolites possibly could affect. Addition of the zeolite-clinoptilolite in a continuously stirred lab tank reactor showed significant lower accumulation of volatile fatty acids compared to a control reactor without zeolites added, when the hydraulic retention time was kept low (30 days) and the organic loading rate was high (4.8 kg VS/ (m 3 day)). The same results were observed upon zeolite addition in a batch experiment, which also showed a decreased lag phase. Neither the specific gas production nor the methane concentration was significant affected by addition of zeolites. Furthermore addition of a possible inhibitor (long-chain fatty acids (LCFA)) increased the lag phase further when slaughterhouse waste was used as substrate. The observed results concluded that a metabolite or metabolites produced during the anaerobic degradation is/are the reason to inhibition, an increased lag phase. Nyckelord Keyword Anaerobic digestion, methane, zeolites, clinoptilolite, slaughterhouse waste i

4 Abstract Biogas is a source of renewable energy and is produced at anaerobic conditions. The gas consists mainly of methane (55-70 %) and carbon dioxide (30-45 %). Biogas can be used as vehicle fuel after the gas has been upgraded to a methane content of approximately 97 %. There are several companies in Sweden producing biogas. Svensk biogas AB in Linköping is one of the largest. The company has two biogas production plants; one in Linköping and one in Norrköping. To meet the surge demand for biogas it is not only important to increase the volumetric capacity of the digesters, but also to optimize the process at the existing production plants in different ways. Zeolites, a clay mineral, have earlier been shown to have a positive effect on anaerobic digestion of certain substrates. The aim of this master s thesis was to investigate if the organic loading rate could be increased and/or if the hydraulic retention time could be reduced by addition of zeolites to a reactor treating slaughterhouse waste as a substrate. The aim was further to investigate which substance/substances that zeolites possibly could affect. Addition of the zeolite clinoptilolite in a continuously stirred lab tank reactor showed a significantly lower accumulation of volatile fatty acids compared to that in a control reactor without zeolites added, when the hydraulic retention time was kept low (30 days) and the organic loading rate was high (4.8 kg VS/ (m 3 day)). The same results were observed upon zeolite addition in a batch experiment, which also showed a decreased lag phase. Neither the specific gas production nor the methane concentration was significantly affected by addition of zeolites. Furthermore, addition of a possible inhibitor, long-chain fatty acids (LCFA), increased the lag phase further when slaughterhouse waste was used as a substrate. The conclusion from the observed results is that a metabolite or metabolites produced during the anaerobic degradation is/are the reason to inhibition and an increased lag phase. Keywords: Anaerobic digestion, methane, zeolite, clinoptilolite, slaughterhouse waste ii

5 Sammanfattning Biogas är en förnyelsebar energikälla som bildas under anaeroba förhållanden och består till största del av metan (55-70%) och koldioxid (30-45 %). För att biogas ska kunna användas som fordonsbränsle måste gasen uppgraderas till en metankoncentration på cirka 97 %. I Sverige finns flera företag som producerar biogas, varav Svensk biogas AB i Linköping är en av de största producenterna. Företaget har två biogasanläggningar, en i Linköping och en i Norrköping. För att möta dagens och framtidens efterfråga av biogas måste produktionskapaciteten öka men lika viktigt är det att produktionen i befintliga anläggningar blir mer effektiva. Tillsats av zeoliter har i tidigare experiment visat sig ha positiva effekter på rötningsprocessen. Detta examensarbetes mål var att undersöka om belastningen kunde ökas samtidigt som uppehållstiden förkortades i en kontinuerlig reaktor vid tillsats av zeoliter när slakteriavfall användes som substrat. Vidare var målet att undersöka vilka substanser som zeoliterna möjligen kunde interagera med eller vad som eventuellt frisattes. Tillsats av zeoliten clinoptilolite i en kontinuerlig reaktor visade signifikant lägre koncentrationer av flyktiga fettsyror jämfört med en kontrollreaktor utan zeolittillsats. Detta observerades när uppehållstiden var kort (30 dagar) och samtidigt med en hög belastning (4,8 kg VS/ (m 3 dag)). I ett batchförsök där zeoliter tillsattes observerades även där lägre koncentrationer av flyktiga fettsyror, i detta försök förkortades även lagfasen jämfört med en serie utan zeoliter. Varken den specifika gasproduktionen eller metankoncentrationen påverkades signifikant av zeolittillsats. Lagfasen förlängdes ytterligare vid tillsats av långa fettsyror i batchförsöket när slakteriavfall användes som substrat. Tillsammans med resultat från ett annat batchförsök kunde det konstateras att den inhiberande faktorn och den förlängda lagfasen, var en metabolit/metaboliter som bildades under den anaeroba nedbrytningen. Nyckelord: Anaerob nedbrytning, metan, zeoliter, clinoptilolite, slakteriavfall iii

6 Acknowledgements I would like to thank the research and development group at Tekniska Verken in Linköping AB (publ) who gave me the opportunity to do my master s thesis in your group. You welcomed me with open arms and included me in your research, you always answered my questions (more and less important ones?!) which is admirable. First of all I would like to thank my supervisors Erik Nordell and Martin Karlsson. Erik for his patience with all my questions especially the questions regarding Excel. For the discussions we had about results, theory and experiments and the help with the report. Martin for his help with experiment setups and the discussions about the results, for his help with the report and for his help with chemicals. Thanks to Jan Moestedt for the help at the laboratory and for valuable discussions regarding results and experiments. To Yasna Calderon for the help at the laboratory and the nice days we had in the basement. Thanks to my examiner Uno Carlsson for his help with the report and the administrative parts before submission. I would like to thank my opponent Gabriela Baeza for reading my report and critical examination of it. Thanks for the years we have studied together (days and late nights), it would not have been as fun without you. To Helen Wolrath and Karin Edén for the help with analysis of longchain fatty acids. Thanks to Gösta Hydén for the help with the strainer. Thanks to all employees at Tekniska Verken in Linköping AB s (publ) laboratory, for the help with my samples and for letting me use the equipment. Thanks to Lovisa Fricot Norén who helped me with the administrative parts at Tekniska Verken in Linköping AB (publ). I would also like to thank my family and friends who always asked about the project, how the worked proceeded and how the reactors felt. Thanks to Tobias who always believes in me and is always supporting me. iv

7 Table of content 1 Introduction Aim Hypotheses Delimitations and method Theoretical background Biogas The microbiology in the biogas process Anaerobic digestion Hydrolysis Acidogenesis Acetogenesis The methanogenesis Syntrophic acetate oxidizer Homoacetogenesis Biogas substrates Slaughterhouse waste Thin stillage Process parameters Total solids and volatile solids Degree of degradation Organic loading rate and hydraulic retention time Specific gas production Gas composition Alkalinity and ph Volatile fatty acids and long-chain fatty acids Total Kjeldahl nitrogen and ammonium-nitrogen Metals Inhibition of the anaerobic process Long chain fatty acids Ammonia Other inhibition parameters Zeolites Clinoptilolite Zeolites in anaerobic digesters Material and methods Semi-continuously stirred tank reactor experiment The used zeolite v

8 3.1.2 Gas production and methane content Batch reactor experiments Inoculum and nutritional media Material and equipment Batch reactor experiment Batch reactor experiment Measured parameters Volatile fatty acids and long-chain fatty acids Total solids and volatile solids Alkalinity and ph Methane content Total Kjeldahl nitrogen and ammonium-nitrogen Hydrogen sulphide Metals Result and discussion Semi-continuously stirred tank reactor experiment Gas production and methane content Volatile fatty acids Alkalinity and ph Total Kjeldahl nitrogen and ammonium nitrogen Total solids and volatile solids Hydrogen sulphide and metals Discussion about the semi continuously stirred tank experiment Batch reactor experiment Batch reactor experiment Discussion about the experiments Further experiments Conclusions References Appendix I- Setup of batch experiment 1... I Appendix II- Setup of batch experiment 2... II Appendix III- Total gas production in the CSTR experiment... III vi

9 Abbreviations and definitions Anaerobic digestion: BA: Biogas: Clinoptilolite: CSTR: GC-FID: HRT: Inoculum: LB: LCFA: NB: Nm 3 OLR: Reactor: SAO: TA: TS: VFA: VS: Zeolite: Microbial degradation of organic matter during the absence of oxygen Bicarbonate alkalinity A gas composed mainly of methane and carbon dioxide A natural zeolite Continuously stirred tank reactor Gas chromatography- flame ionization detector Hydraulic retention time, duration of a particle is in the reactor Sample from a running reactor, consists of microorganisms, used when starting new plants and experiments Linköping biogas Long-chain fatty acids, fatty acids with 12 or more carbon atoms Norrköping biogas Normal cubic meter Organic loading rate, the organic amount supported to the reactor A sealed container Syntrophic acetate oxidizer Total alkalinity Total solids Volatile fatty acids, fatty acids with less than 12 carbon atoms Volatile solids A clay mineral vii

10 1 Introduction Fossil fuel is a finite and a non-renewable energy source and is the most used fuel globally. It is important to develop the existing methods, using renewable energy sources. One reason is the way fossil fuels affect the global warming. Non-renewable energy sources have been formed during thousands of years and, when using them for example as vehicle fuel, the carbon dioxide emissions is extra addition to the atmosphere. Using biogas as vehicle fuel also releases carbon dioxide, however the emissions are already included in the natural circulation of carbon (Svensk biogas, 2011; Deublein, et al., 2008). Biogas is produced at anaerobic conditions by different microorganisms. The microorganisms degrade organic matter such as proteins, carbohydrates and fat to finally methane, carbon dioxide and water. Compared to aerobic respiration less biomass is produced during anaerobic digestion. Biogas consists mainly of methane (55-70 %) and carbon dioxide (30-45 %). To use biogas as vehicle fuel the gas has to be upgraded to a methane concentration of approximately 97 %. Furthermore the byproduct (digestate) can be used as a fertilizer since it contains high amounts of nutrients such as phosphate and nitrogen (Figure 1) (Svensk biogas, 2011; Energigas Sverige, 2011; Bryant, 1979; Gerardi, 2003; Zinder, 1984). There are several companies producing biogas in Sweden, one of them is Svensk Biogas AB, owned by the municipal company Tekniska Verken in Linköping AB (publ). The company has two biogas production sites; one in Norrköping (NB) and one in Linköping (LB). In 2010, these two plants produced approximately Nm 3 upgraded biogas (Nordell, 2011). The biggest of them is the LB plant where slaughterhouse waste is the mainly substrate used, in the NB plant residuals products from an ethanol production plant (thickened thin stillage) is the main substrate. In early 2010 the site in Linköping expanded from two digesters of m 3 capacity to three digesters with a total capacity of m 3. One reason for the expansion is due to the cooperation with Östgötatrafiken, which has decided to run all their buses on renewable fuels. Another reason of the expansion is the increased request of biogas for private traffic (Svensk biogas, 2011; Östgötatrafiken, 2011). To meet the increasing demand for biogas it is important to increase both capacity and efficiency of existing plants. Even though the process today has a high yield, it is important to increase the yield further in both plants in order to get the most of the substrates. This master s thesis aim is to investigate if the organic loading rate can be increased and if the retention time can be shortened by addition of zeolites in the reactors. Previous studies have shown a shortened lag phase with a zeolite addition when digesting slaughterhouse waste in batch conditions (Nordell, 2009; Nordell, et al., 2010). 1

11 Figure 1. The figure shows the carbon cycle of different substrates and products during the anaerobic digestion. 1.1 Aim Earlier studies indicate that zeolites have the capacity to either inactivate some inhibiting substances, alternatively to release ions that affect the anaerobic digestion in a positive way. The aim of the present thesis is to investigate, in a continuously stirred tank lab reactor with slaughterhouse waste used as substrate, if the positive effects of zeolites can be used to increase the organic loading rate and at the same time reduce the retention time by addition of clinoptiolite. That is, to increase the effiency of biogas production from slaughterhouse waste. The aim is further to determine which inhibiting substance/substances that is possibly adsorbed to clinoptilolite (zeolite) upon addition to a batch process. 1.2 Hypotheses If the zeolites can adsorb and inactivate inhibiting substances the positive effects of zeolite can be used to increase the organic loading rate while, concomitantly, the hydraulic retention time can be reduced without any negative effects on the process. The 2

12 hypothesis is further that the inhibiting substance is either a metabolite produced during the anaerobic digestion or a substance already presented in the slaughterhouse waste substrate. The hypotheses will be rejected or confirmed by the answers to the following questions: How are the methane production and/or gas production affected with addition of zeolites? Does zeolite addition prevent volatile fatty acids and long-chain fatty acids accumulation? Can the organic loading rate be increased without any decrease in the yield of degradation? Is the lag phase shortened if slaughterhouse waste is treated with zeolites prior to the start-up of a batch experiment? Is the lag phase affected when zeolites are presented during various times during a batch experiment? 1.3 Delimitations and method The work of this master s thesis comprises a semester of 30 ECST credits, 20 weeks full time. Information has been found in books, scientific articles, conference documents, patents, websites and from earlier experiments performed at Tekniska Verken i Linköping AB (publ). The experiments presented in this project have been performed at Tekniska Verken in Linköping AB s laboratory, if nothing else is mentioned. Since the experiment has been performed at a laboratory scale, problems with up-scaling have not been investigated. Investigations such as the economical feasibility have not been evaluated. Since the master s thesis comprises 20 full weeks it was not possible to investigate the effects for longer than a few retention times. However, it would have been favourable if possible to investigate the experiment for a longer time. The experiments were performed at 38 ᵒC, mesophilic conditions, meaning that the result has not been evaluated according to thermophilic conditions. 3

13 2 Theoretical background 2.1 Biogas The organic matter is degraded by several different microorganisms. A typical content of the produced gas in a co-digestion plant is methane (55-70 %) and carbon dioxide (30-45 %). The methane content varies depending on several different factors such as the temperature and which substrate that has been used (Benjaminsson, 2011; Gerardi, 2003; Energigas Sverige, 2011; Deublein, et al., 2008). When the gas is used as vehicle fuel the gas has to be upgraded to a methane concentration of 97 ±2 %. Upgrading the biogas to vehicle fuel also requires removal of impurities such as hydrogen sulphide (Table 1). After the biogas has been upgraded, it is compressed and stored at 200 bars which is 200 times higher compared to normal pressure (Svensk biogas, 2011; Deublein, et al., 2008). Table 1. Impurities in the rawgas (Deublein, et al., 2008). Component Content (of volume) H 2 S % H 2 O 1-5 % N % The by-product from the anaerobic process can be used as bio-fertilizer since it contains high amounts of both nitrogen and phosphate, which is necessary for plants. Studies have shown that the microflora is positively affected by the bio-fertilizer. Certified biofertilizer according to SPCR 120 certify that the fertilizer is free from both Salmonella and EHEC. In the LB plant the incoming substrate has been heated to C to eliminate those pathogens (Avfall Sverige, 2011; Svensk biogas, 2011). 2.2 The microbiology in the biogas process There are several types of microorganisms active during the anaerobic process, which all require organic matter (substrate) for their survival. However the substrate does not only need to contain organic matter, the microorganisms also requires nutrients. Part of the substrate is used as building blocks; anabolism, for example to produce new cells and the remaining part of the substrate is finally degraded to methane and carbon dioxide, catabolism (Figure 2). Methane-forming bacteria have several enzyme systems which require trace elements, especially cobalt, iron, nickel and sulphur (Gerardi, 2003; Schnürer, et al., 2009). Nutrients needed during anaerobic digestion can be hydrogen, carbon dioxide, and organic components such as polysaccharides, fatty acids and protein. The cells consist mainly of carbon (Table 2) (Gerardi, 2003; Schnürer, et al., 2009). 4

14 Nutrients: carbon sources, energy source, trace elements etc. Catabolism/ Anabolism Products: carbon dioxide, hydrogen, methane, alcohols Figure 2. The metabolism of the cell modified according to Schnürer et al Table 2. Composition of a bacterial cell (Schnürer, et al., 2009). Component C O N H P S K Na Ca Mg Fe Others % of dry weight In contrast to aerobic respiration, that uses oxygen as an electron acceptor, anaerobic digestion with both fermentation and anaerobic respiration, requires electron acceptors other than oxygen in the anoxic process. The fermentation usually uses organic compounds as electron acceptors whereas the anaerobic respiration uses carbon dioxide, or inorganic electron acceptors such as manganese (Mn 4+ ), iron (Fe 3+ ), nitrite (NO 3- ) and sulphate (SO 4 2- ). The electron acceptors can be sorted due to how much energy that is produced, where oxygen releases most energy and carbon dioxide releases least energy; O 2 >Fe 3+ >Mn 4+ >NO 3- >SO 2-4>CO 2 (Schnürer, et al., 2009). 2.3 Anaerobic digestion In the anaerobic digestion process, an energy-rich end product will be obtained while less biomass is formed compared to aerobic digestion. Aerobic digestion oxidizes most of the substrate to water and carbon dioxide during production of biomass. The aerobic oxidation of glucose to carbon dioxide has a ΔG 0 of kj which can be compared to the ΔG 0 of the anaerobic oxidation of glucose to methane and carbon dioxide which is kj, about one-seventh of that of aerobic oxidation (Bryant, 1979; Zinder, 1984). The anaerobic digestion is a complex process and can be grouped into four different main stages; hydrolysis, acidogenesis, acetogenesis and methanogenesis (Figure 3). Also seen in Figure 3 is the conversion of carbon dioxide and hydrogen to acetate and vice versa. In each of these stages different microorganisms are active. The different 5

15 microorganisms work in sequence, one microorganism s products is another one s substrate, except of course for the final product methane. Therefore the microorganisms have to be synchronized, if not, the process can be imbalanced and different inhibitory compounds can accumulate. The microorganisms can be divided in to two different groups depending on their optimal temperatures; mesophilic and thermophilic organisms prefer temperatures between - C and - C, respectively (Gerardi, 2003; Deublein, et al., 2008). Complex organic matter (Proteins, fat, polysaccharides etc.) Hydrolysis Monosacharides (Amino acids, sugars, peptides etc.) Acidogenesis Intermediate products (Alcohols, fatty acids etc.) Acetogenesis Homoacetogenesis H 2 + CO 2 Acetate Hydrogenotrophic methanogenesis SAO CH 4 + CO 2 Acetoclastic methanogenesis Figure 3. A schematic picture of the anaerobic digestion. 6

16 2.3.1 Hydrolysis In the first step, the hydrolysis, complex biopolymers, such as proteins and polysaccharides, are degraded to monomers such as long-chain fatty acids (LCFA), amino acids and simple sugars such as glucose. The degradation proceeds due to the action of the hydrolytic enzymes; amylases, cellulases, proteases and peptidases which are secreted by different microorganisms (Schnürer, et al., 2009). Common polysaccharides are cellulose, hemicellulose, starch, pectin and glycogen. Cellulose, hemicellulose and starch are important components in different plants. Cellulases are secreted by e.g. the bacterium Cellumonas which is specialised on hydrolysis of cellulose. The covalent bonds are hydrolysed and simple sugars are produced (Figure 4). Pectin is usually found in fruit and vegetables and glycogen is an energy source for animals (Schnürer, et al., 2009). H 2 O R-C-C-R R-C-H Monomers OH-C-R Figure 4. The figure shows the hydrolysis of carbohydrates (Deublein, et al., 2008). During hydrolysis oxygen is produced, which is consumed by the facultative anaerobic microorganisms. This conversion is necessary for the survival of the obligatory anaerobic microorganisms (Deublein, et al., 2008; Zinder, 1984) Acidogenesis In the second step, the acidogenesis or the primary fermentation, the products from the hydrolysis are degraded to smaller compounds by different microorganisms which are both facultative and obligate anaerobic microorganisms. Amino acids can either be degraded separately or in pairs by the Stickland reaction. When amino acids are degraded in pairs by the Stickland reaction, one amino acid works as an electron donor and the other one as an electron acceptor. During this step carbon dioxide, ammonia and acetate are produced (Madigan, et al., 2006; Deublein, et al., 2008). Simple sugars are degraded to volatile fatty acids, carbon dioxide, hydrogen and alcohols. The products formed depend on the microorganisms; propionic acid is for instance produced by the propionic bacterium (Zinder, 1984; Deublein, et al., 2008). LCFAs are degraded through β-oxidation by acetobacter. Two carbon atoms are separated from the chain at a time and acetate is produced. The β-oxidation will continue until only two carbon atoms are left (Deublein, et al., 2008) Acetogenesis The last step before methanogenesis is called acetogenesis, secondary fermentation. In this step fatty acids longer than two carbon atoms and alcohols are degraded to acetate, carbon dioxide and hydrogen. The microorganisms active during acetogenesis can be 7

17 ΔGᵒ' J/reaction called hydrogen producers due to their production of hydrogen (Table 3). The hydrogen producing bacteria require a low hydrogen pressure, below 10-3 atmosphere, therefore they are obligatory coupled to hydrogen consuming bacteria such as hydrogenotrophic methanogens (Zinder, 1984; Deublein, et al., 2008). Table 3. Conversion of propionic acid and ethanol. Low hydrogen pressure makes both reactions favourable (Madigan, et al., 2006). Reaction ΔG 0 a ΔG b C 2 H 5 COOH+ 3H 2 O CH 3 COO - + HCO H + + 3H kj -5.4 kj C 2 H 5 OH + H 2 O C 2 H 3 O H 2 + H kj kj a hydrogen partial pressure 1 atm, normal air pressure b hydrogen partial pressure 10-4 atm Since the methanogenic microorganisms require higher hydrogen partial pressure compared to acetogenic microorganisms they have to live in symbiosis with each other. If not, propionate and butyrate will accumulate and the hydrogen partial pressure will increase and the acetogenesis will be inhibited (Gerardi, 2003; Deublein, et al., 2008) The conversion of propionate to acetate and the conversion of hydrogen and carbon dioxide to methane has a small window (Figure 5) of hydrogen partial pressure. A good indication of the productivity in the reactor is the concentration of propionate since it is often the rate limiting step during the anaerobic process (Deublein, et al., 2008) Favourable for oxidation of propionate and methane formation log(hydrogen partial pressure) atm Figure 5. The figure shows the Gibbs free energy of the oxidation of propionate( ), butyrate ( ) and the methane formation ( ) at different hydrogen partial pressures. The black arrow marks the area where production of methane and oxidation of propionate is favorable. 8

18 2.3.4 The methanogenesis Unlike the microorganisms (bacteria) in the initial fermentation steps, the methane forming microorganisms belong to the domain Archaea. Methanogenic archaea are strictly anaerobic and, it is therefore important to avoid oxygen in the process (Deublein, et al., 2008; Gerardi, 2003). The methane forming bacteria can be divided into three different groups due to their substrate; hydrogenotrophic-, acetoclastic- and methylotrophic methanogens. In a normal process approximately 70 % of the methane is believed to come from acetate and % derives from carbon dioxide and hydrogen whereas 0-3 % orginates from methyl groups (Deublein, et al., 2008; Gerardi, 2003) Hydrogenotrophic methanogens These methanogens produce methane and water from carbon dioxide and hydrogen. Since these microorganisms use hydrogen as a substrate they help to maintain a low hydrogen partial pressure (Demirel, et al., 2008). There are further many hydrogenotrophic methanogens that can utilize formate as a source for electrons for reduction of carbon dioxide to methane (Equation 1) (Khanal, 2008). Equation 1. The conversion of carbon dioxide and hydrogen to methane and water by hydrogenotrophic methanogens. CO 2 + 4H 2 CH 4 + 2H 2 O Acetoclastic methanogens The acetoclastic methanogens convert acetate to methane and carbon dioxide (Equation 2) (Demirel, et al., 2008). While the hydrogenotrophic methanogens prefer higher hydrogen partial pressure, acetoclastic methanogens prefer low hydrogen partial pressures, meaning that not only acetate formation will be favourable at low hydrogen partial pressures but also methane production by acetoclastic methanogens. A high partial pressure will decrease the methane production from acetate (Gerardi, 2003). Equation 2. Acetoclastic methanogens conversion of acetate to methane and carbon dioxide. CH 3 COOH CH 4 + CO Methylotrophic methanogens Methylotrophic methanogens use methyl groups and produce methane from for example methanol, mono-, di-, triethylamine (Equation 3). A methyl carrier helps the methyl group to be reduced to methane (Gerardi, 2003; Khanal, 2008). Equation 3. Methanol and hydrogen conversions to methane and water. CH 3 OH + H 2 CH 4 + H 2 O Syntrophic acetate oxidizer The acetoclastic methanogens can be inhibited by several substances such as ammonia, sodium, volatile fatty acids, heavy metals and sulphide. Schnürer et al 1999 described the anaerobic digestion during high levels of ammonia. Ammonia inhibits acetoclastic methanogens, which is the most common route of methane production. However the 9

19 study concluded that acetate was converted to hydrogen and carbon dioxide during high amounts of ammonia. The conversion can proceed due to the microorganism syntrophic acetate oxidizer (SAO) (Equation 4). Besides the concentration of ammonia, the concentration of acetate and the activity of the methanogens affect the way methane is produced. The hydraulic retention time (HRT) and the temperature have also been shown to affect the formation of methane from SAO. How common this way of producing carbon dioxide and hydrogen is however not known. Low hydrogen pressure is required for formation of carbon dioxide and hydrogen from acetate (Schnürer, et al., 2009; Schnürer, et al., 1999). Equation 4. SAO conversion of acetate and water to hydrogen and carbon dioxide. CH 3 COOH + 2H 2 O 4H 2 + 2CO Homoacetogenesis Homoacetogenesis can be divided into two groups; heterotrophic acetogens and autotrophic acetogens. Autotrophic acetogens use hydrogen and carbon dioxide to produce acetate. The heterotrophic acetogens can utilize organic substrates such as formate and methanol. Since both methanogens and homoacetogens utilize hydrogen as an electron donor and that Gibb s free energy is similar there are possibilities that there is competition for the hydrogen (Table 4) (Khanal, 2008). Table 4. Conversion of hydrogen carbonate in two different ways; to methane or acetate (Khanal, 2008). Reaction ΔG 0 (kj/reaction) 4H 2 + HCO H + CH 4 + 3H 2 O H 2 + 2HCO H + CH 3 COO - + 4H 2 O Biogas substrates Different organic compounds give different amounts of methane and carbon dioxide; fat is one of the most energy rich compounds in a substrate (Table 5) (Carlsson, et al., 2009; Schnürer, et al., 2009). The ratio between carbon and nitrogen is important. Experiments have shown that a C/N ratio of approximately 30 favours the microorganisms metabolism. A low C/N ratio usually results in ammonium inhibition (section 2.6.2) and an increased ph, which can be toxic to the microorganisms. Higher ratios of C/N have shown a decreased biogas production due to lack of nitrogen. To overcome the problems with high or low C/N ratio, co-digestion of different substrates is favourable to be used (Carlsson, et al., 2009; Schnürer, et al., 2009). Table 5. Different substrates and their biogas and methane yields. Substrate Biogas Methane Methane Nm 3 /kg VS Nm 3 /kg VS % Fat Protein Carbon hydrate

20 2.4.1 Slaughterhouse waste The plant LB mainly uses slaughterhouse waste as a substrate. Slaughterhouse waste is rich of proteins and fat, leading to a high biogas yield and is therefore favourable as a substrate for the biogas process. The substrate contains high amounts of fat which has both advantages and disadvantages. The advantage is a high amount of biogas per kilogram VS and the disadvantage is inhibition of the anaerobic digestion (2.6.1) and a decreased ph. Slaughterhouse waste further contains high amounts of protein. During the degradation of proteins ammonia is released and inhibition can be a fact as, described in section (Carlsson, et al., 2009; Chen, et al., 2008; Heinfelt, et al., 2009; Schnürer, et al., 2009). According to European parliament regulations slaughterhouse waste has to be pretreated in various ways depending on the origin. There are three classes; class 3, low risk material (meat etc.) can be used as a substrate for biogas production if treated at 70 ᵒC for 1 hour in a sealed system which is the case on the plant LB (European Parliament, 2009) Thin stillage Plant NB uses thin stillage as a substrate. The C/N ratio is high in thin stillage and can similar to slaughterhouse waste cause ammonium inhibition in the process. Moreover, during the pre-treatment step in the ethanol production, sulphuric acid is added which causes problems with high levels of sulphide which also can inhibit the anaerobic process (Schnürer, et al., 2009). 2.5 Process parameters In order to maintain process stability it is important to monitor different process parameters. Even a small change for example in temperature or ph can result in process instability and sometimes even in process failure. By controlling certain parameters the anaerobic process can be optimized and process instability be detected early Total solids and volatile solids Total solids (TS) are the dry matter after heating to C for at least 1 hour. It includes both organic and inorganic material. Volatile solids (VS) are the amount of the material that has vaporized at C. VS is usually specified as percentage of TS. It is important to take into account if there are high amounts of volatile organic compounds such as VFA, in the samples because it may give false (too low) results since it can resign during the heating at C and can result in lower TS than the real value (Svensk Standard SS edition 1; Pind, et al., 2003) Degree of degradation The degree of degradation is the amount of the substrate which has been degraded and converted to gas in the reactor. Calculations of the degree of degradation can be done according to Equation 5. 11

21 Equation 5. The equation used to calculate the degree of degradation. Description VSreactor VSsubstrate = the VS of the material coming out from the reactor = the VS of the substrate provided to the reactor Organic loading rate and hydraulic retention time The time the material is in the reactor is called the hydraulic retention time (HRT). HRT is determined by the volume the reactor is feed with. Different substrates require different HRT in the reactor and a general rule is that the HRT can be reduced with increased temperature. A typical co-digestion plant has a HRT of between days although; it is not uncommon with shorter or longer HRT. The LB plant has a HRT between days. The HRT can be calculated according to Equation 6 (Schnürer, et al., 2009). Equation 6. The calculation of the hydraulic retention time (HRT). Description Vreactor Vsubstrate = the volume of the reactor = the volume of the added substrate each day The organic loading rate (OLR) is the organic material supplied to the reactor per unit volume and time. An increased OLR means increased VS supplied to the reactor. The OLR can be calculated according to the Equation 7 (Schnürer, et al., 2009). Equation 7. Calculation of the organic loading rate (OLR). Description TSsubstrate VSsubstrate Vreactor = the TS of the substrate = the VS of the substrate = the volume of the reactor Specific gas production As the gas production is the end product in the anaerobic process the specific gas production is a parameter which gives good information about the process. Thus, to obtain the specific gas production the gas production is normalized according to OLR and reactor volume and, can be used to compare different reactors and substrates with each other. Equation 8 is used to calculate the specific gas production (Schnürer, et al., 2009). 12

22 Equation 8. Calculation of the specific gas production. Description Vgas OLR Vreactor = the volume of the produced gas = the organic loading rate = the volume of the reactor ) Gas composition The gas composition is a good tool for monitoring the anaerobic process. A decreased amount of methane and an increased amount of carbon dioxide is an indication of process instability, indicating that the methanogenic microorganisms are probably inhibited. It is also of interest to investigate the amount of hydrogen sulphide (H 2 S) in the gas. A high proportion of hydrogen sulphide in the gas can be indications of a substrate with high concentrations of certain amino acids, sulphate or sulphide (Schnürer, et al., 2009) Alkalinity and ph Alkalinity is a way of estimating the buffer capacity in a liquid. A high alkalinity gives a good buffer capacity and thereby a stable ph. Alkalinity can be measured as total alkalinity (TA) and bicarbonate alkalinity (BA). TA includes bicarbonate, ammonia and VFA. BA includes bicarbonate and ammonia. Bicarbonate has a pka 1 at 6.35 and a pka 2 at Ammonia has a pka at 9.25 and VFA has pka lower than ph 5. BA is the most common way of monitoring the alkalinity since it excludes VFA. Exclusion of VFA is made by titrating only to a higher ph (5.4) than their pka. A stable process usually has a BA between mg HCO - 3/ l. At the LB plant the BA is around mg HCO 3 - / l, there are high amounts of ammonium in the digesters which has to be taken into account. High amounts of VFA result in a high TA, and accumulation of VFA is an indication of an instable process and not a good sign. A strong acidification is avoided with the carbon dioxide/ hydrogen bicarbonate/ carbonate buffer system (Equation 9) which is most effective around ph 6.5. A weak acidification is avoided by the ammonia/ ammonium buffer system (Equation 10) which is most effective around ph 10 (Pind, et al., 2003). Equation 9. The carbon dioxide/ hydrogen bicarbonate/ carbonate buffer system. CO 2 H 2 CO 3 H + + HCO 3 - H + + 2CO 3 - Equation 10. The ammonia/ ammonium buffer system. NH 3 + H 2 O NH OH - NH 3 + H + NH + 4 The ph is a measure of the proton activity, which reflects all cations and anions in the solution. ph can thereby indicate changes in the chemical balance, such as acids, bases, anions and cations produced or consumed during metabolic activity. Since ammonia, VFA and carbonate can stabilize the ph or at least affect the ph it can be difficult to interpret the ph results. Due to this ph is only used together with other parameter (Pind, et al., 2003). 13

23 The methanogenic microorganisms are sensitive to changes in the ph value. Their optimum ph is between 6.5 and 7.2. The acidogeneic microorganisms are more tolerable compared to methanogens, as they can function in the ph range between 4.0 and 8.5. It is important to optimize the ph for methanogenic microorganism since they often constitute the bottleneck in the anaerobic process. The VFA produced during the anaerobic digestion reduces the ph. This is counteracted by the methanogens which consume acetate and produce carbon dioxide, a part of the alkalinity (Deublein, et al., 2008; Khanal, 2008; Pind, et al., 2003; Schnürer, et al., 2009) Volatile fatty acids and long-chain fatty acids During hydrolysis, acidogenesis and acetogenesis different types of fatty acids are produced. Finally these acids are converted to carbon dioxide and methane. Accumulation of volatile fatty acids (VFA) will be an effect for example if the methanogens are inhibited. Overloading can also be a reason of accumulation of fatty acids and is due to that the hydrolytic and acidogenic microorganisms grow faster than the methanogens. The conversion of acetate to methane and carbon dioxide will then become the rate limiting step. Accumulation of fatty acids leads to a decreased alkalinity and thereby a decreased ph. The fatty acid concentration is therefore a good indication of the stability of the process. Fatty acids can be divided into volatile fatty acids (VFA) and long-chain fatty acids (LCFA). Thus VFA is a product from LCFA and analysis of LCFA can thus indicate process instability in earlier stages such as the acidogenesis. Both VFA and LCFA can be analysed by gas chromatography (GC) and high-performance liquid chromatography (HPLC) (Jonsson, et al., 2002; Schnürer, et al., 2009) Total Kjeldahl nitrogen and ammonium-nitrogen Ammonia and ammonium are produced during the degradation of protein. It is therefore of interest to monitor the ammonia and ammonium in the anaerobic process. The monitoring can be done by total Kjeldahl nitrogen (TKN) and ammonium-nitrogen. TKN gives information of the organic nitrogen, ammonia and ammonium and ammonium nitrogen gives information about the amount of ammonium and ammonia in the sample. During the ammonium nitrogen analysis the sample is treated with base meaning that all ammonia will be converted into ammonium. Ammonia concentration can then be calculated from the ammonium nitrogen value, according to Equation 12 (Tecator, AN 300 SV, version 2) Metals Many metals are trace elements, meaning that they are required for the survival of the microorganisms. Therefore it can be relevant to monitor if there is enough metals for the microorganisms. 2.6 Inhibition of the anaerobic process The methane producing archaea and the acid producing bacteria differ widely, for example: methane producing microorganisms require a high hydrogen partial pressure whereas acid producing microorganisms require a low hydrogen partial pressure. Failure in the anaerobic process is often caused by instability between these two groups. 14

24 The most common cause is drastic changes in OLR, but is sometimes caused by an inhibitory compound, which can be the reason to process instability or even process failure. These compounds can be found in the substrate such as slaughterhouse waste which contains high amounts of ammonia and long-chain fatty acids. Process instability is usually indicated when there is a decrease in specific gas production and degree of degradation, volatile fatty acid accumulation and loss of methane formation (Chen, et al., 2008) Long chain fatty acids Long-chain fatty acids, LCFA, are composed of a hydrophilic head (a carboxyl group) and a hydrophobic aliphatic tail. The tail can vary in length and degree of saturation; 12 or more carbons are defined as LCFA. Long-chain fatty acids are produced during the hydrolysis of oils and fats. Stearic acid, palmitic acid and oleic acid are examples of LCFA (Table 6). Those and other LCFA are favourable for methane production if they are added in favourable proportions. Over 90 % of the methane potential from lipids comes from LCFA. According to Sousa et al known syntrophic microorganisms can degrade fatty-acids with 12 carbon atoms or more. They belong to the families of Syntrophomonadaceae and Syntrophaceae. Of these, three can degrade unsaturated long-chain fatty acids (Battimelli, et al., 2009; Cavaleiro, et al., 2010; Koster, et al., 1987; Sousa, et al., 2009). Table 6. Various long-chain fatty acids (LCFA). LCFA Stearic acid Palmitic acid Oleic acid Molecular formula C 17 H 35 COOH C 15 H 31 COOH C 17 H 33 COOH The hydrolysis of fat-rich substrates can proceed well during anaerobic digestion. Ester bonds are broken and glycerol and long-chain fatty acids are produced during the hydrolysis (Figure 6). LCFA adsorbs to cell surfaces and are transported into the cell where the degradation occurs through beta-oxidation (Equation 11) (Battimelli, et al., 2009; Koster, et al., 1987; Cavaleiro, et al., 2010; Sousa, et al., 2009). CH 2 OCOR 1 R 1 COOH CH 2 OH CHOCOR 2 + 3H 2 O R 2 COOH + CHOH CH 2 OCOR 3 R 3 COOH CH 2 OH Neutral fat Long-chain fatty acids Glycerol Figure 6. Hydrolysis of neutral fat to long-chain fatty acids and glycerol (Hanaki, et al., 1981). Equation 11. The beta-oxidation of LCFA (Lalman, et al., 2001). C n H 2n O 2 + 2H 2 O C n-2 H 2n-4 O 2 + C 2 H 4 O 2 + 4H + 15

25 Only a few millimolar of LCFA can be enough to inhibit the anaerobic process. Gram positive microorganisms have been shown to be sensitive to LCFA, whereas gram negative microorganisms do not seem to be affected to the same degree. Since the methanogens have a similar cell wall as gram positive microorganisms it is not unusual that they are affected as well. The degradation of LCFA can become rate limiting because of the slow growth of the LCFA-consuming microorganisms and because they require low hydrogen partial pressure. To overcome the problems it is important to investigate the substrate composition regarding to LCFA and high levels can be reduced by co-digestion with other substrates. The mass transfer from solid to liquid phase has also been shown to be rate limiting. LCFA has also a lower density compared to water and can thereby accumulate at the surface of the sludge (Battimelli, et al., 2010; Roy, et al., 1985; Heinfelt, et al., 2009). Foam can occur during degradation of LCFA and causes problems with the process. This can affect the bioavailability and the toxicity and can cause problems such as polluted gas and foam overflow (Salminen, et al., 2002). LCFA adsorbs easily to surfaces especially cell membranes, which affects the cells transport system and the protective function (Chen, et al., 2008; Masse, et al., 2002; Ahmed, et al., 2001). The LCFA toxicity has been shown to correlate with the physical characteristics of the sludge, such as specific surface area and size distribution. The toxicity effect is also affected by temperature and the presence of calcium and magnesium salt. Roy et al 1985 showed decreased levels of inhibition caused by long-chain fatty acids with addition of calcium ions. According to Angelidaki et al 1990 they found it favourable with bentonite (clay mineral) addition during an experiment with high amounts of LCFA. The authors have two suggestions: bentonite binds to LCFA or ions are released and have a positive effect on the degradation (Chen, et al., 2008; Rinzema, et al., 1994; Roy, et al., 1985) Ammonia Ammonia is released when amino acids are degraded in the anaerobic process. During hydrolysis proteins are converted to amino acids or peptides. In the next step, fermentation, the amine group is released and ammonia and ammonium are produced. Ammonia (NH 3 ) and ammonium (NH 4 + ) is in equilibrium, and the ratio between them depends on the temperature and ph (Equation 12). Higher temperature results in higher concentrations of ammonia. Thermophilic processes are therefore more sensitive compared to mesophilic (Figure 7). Both thin stillage and slaughterhouse waste contains a great amount of proteins which is the main reason to the high ammonium nitrogen levels in plant LB and NB (Schnürer, et al., 2009; Carlsson, et al., 2009; Chen, et al., 2008). Equation 12. Calculation of the amount of free ammonia in the reactor solution. 16

26 Ammonia Ammonium 100% 80% 60% 0% 20% 40% 40% 60% 20% 80% 0% ph 100% Figure 7. Concentrations of ammonia and ammonium at different temperatures; 25 ᵒC ( ), 38 ᵒC ( ) and 55 ᵒC ( ), calculated according to Equation 12. Ammonia has been reported to inhibit different microorganisms, and the biogas process is no exception and especially acetoclastic methanogens seem to be affected first. According to Sprott et al, ammonia can diffuse across the membrane. Inside the cell, due to low ph, ammonia is converted to ammonium meaning that ammonia consumes protons. Due to the consumption of protons ph rises and the cell tries to maintain the ph. The cell pumps in protons into the cell and pump out potassium ions (Figure 8) causing lack of potassium inside the cell. Since different methanogens contain different amounts of potassium they are inhibited by ammonia at different concentrations. Acetoclastic methanogens are more sensitive to ammonia since they contain less potassium compared to hydrogenotrophic methanogens. During degradation of high content of protein rich substrate formation of ammonium carbonate from ammonium can occur and give the process increased buffer capacity, and thereby increased resistance against organic overloading (Sprott, et al., 1986; Salminen, et al., 2002; Salminen, et al., 2002; Schnürer, et al., 2009; Schnürer, et al., 1999; Carlsson, et al., 2009). 17