Monitoring the anaerobic digestion process
Transcription
Monitoring the anaerobic digestion process
Logo_JacobsUniversity.svg 27.11.10 17:55 Monitoring the anaerobic digestion process by Harry Michael Falk file:///Users/harry/Dropbox/PhD/proposal/Logo_JacobsUniversity.svg Seite 1 von 1 A thesis submitted in partial fulfilment of the requirements for the degree Doctor of Philosophy in Biochemical Engineering Approved, Thesis Committee ................................................................................................................... Prof. Dr. Dr. h.c. Roland Benz ................................................................................................................... Prof. Dr. Volker C. Hass ................................................................................................................... Prof. Dr. Laurenz Thomsen ................................................................................................................... Prof. Dr. Mathias Winterhalter Date of Defense: December 13, 2011 School of Engineering and Science Declaration I hereby declare that my thesis entitled “Monitoring the anaerobic digestion process” is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials applied are listed and specified in the thesis. Furthermore, I verify that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Bremen, September 17, 2012 Harry Falk 2 Abstract In the anaerobic digestion process, microorganism produce methane and carbon dioxide from organic substrates, either organic waste or renewable primary products. Being a versatile biofuel, biogas can be combusted in a combined heat and power plant to produce electricity and heat or, after purification, fed directly into the natural gas grid as biomethane. Due to the Renewable Energy Sources Act introduced in 1991 in Germany, this process became economically advantageous and led to a boom of biogas plants being built in Germany. A major problem of operating a biogas plant is to monitor an unstable process over time. Parameters like pH or redox potential do not necessarily suffice to estimate the degree of fermentation. At present, the preferred indication parameter are the concentrations of process intermediates, particularly short chain volatile fatty acids. They can be quantified with different gas or liquid chromatographic methods, which requires in-depth knowledge and expensive hardware and is usually carried out by specialized laboratories. Periodically, the digestate is sampled and sent in for analysis. Knowing the absolute concentrations of the different volatile fatty acids can only give a hint about the current fermentation status, though what would be more meaningful would be elucidating the dynamics of generation and degradation of the respective short chain volatile fatty acids. A new online technique using attenuated total-reflectance Fourier-transformed infrared spectroscopy (ATR-MIR-FTIR) was developed, which allows an online monitoring of the concentrations of the different volatile fatty acids in situ. This can give an insight into the dynamics of the anaerobic digestion process. It was adapted to a laboratory scale one-stage biogas plant fed with typically renewable primary products to simulate an agricultural biogas plant. Chemometric models were developed using spiked samples and samples from a real fermentation for acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acid. The methods were evaluated by monitoring the startup phase of the anaerobic digestion of ground wheat in a 10 l continuous-stirred tank reactor. Sample preparation, recording and analysis of IR-spectra of digestate were fully automated. Predictions of the absolute concentration for acetic and propionic acid were reasonable, the existence of other volatile fatty acids could be detected. The developed anaerobic sensor system is able to determine their concentration dynamics and can thereby help to utilize unused potential in biogas plants. Another ascending problem are the substrates being used for the production of biogas. Renewable primary products are in direct rivalry with the agricultural and food industry. For a sustainable future, other biomass sources have to be made accessible for energy production. In contrast to energy crops, especially waste products can be used for energy generation without any concerns. The biogas potential of potential substrates is estimated with parallel running batch experiments. In the process, the minor gas flow rate of these lab-scale fermentations has to be monitored closely and accurately. Especially for this purpose, an easy to build and maintain automated biogas meter was developed to measure the biogas flow in anaerobic digestion experiments. The flow meter is built upon the open-source Arduino platform, and can therefore be easily enhanced or adapted to other environments. Recordings are sent via ethernet to a MySQL database, making the data widely accessible. By combining nine fermenters into an array, triplicate batch experiments according to VDI 4630 with negative control, positive control and the test substrate are monitored effortlessly. Concluding, this thesis focuses on optimizing the biogas production with the development of novel measurement instrumentation. The already well-established process of industrial digestion of energy crops is made transparent with an advanced online measurement method for the volatile fatty acids. Methane potential of novel biodegradable substrates can be monitored with the affordable low-cost gasUino biogas meter. 4 Contents 1. Introduction 1.1. Energy supply 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.2. Biogas as a renewable energy source . . . . . . . . . . . . . . . . . . . . 8 1.3. EEG 2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.4. Basics of anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.4.1. Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.4.2. Acidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.4.3. Acetogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.4.4. Methanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.5. Process parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.5.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.5.2. pH value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 1.5.3. Volatile Fatty Acids (VFA) . . . . . . . . . . . . . . . . . . . . . . . 18 1.5.4. Total Solids and Volatile solids . . . . . . . . . . . . . . . . . . . . 19 1.5.5. Biogas potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.5.6. Organic loading rate (OLR) . . . . . . . . . . . . . . . . . . . . . . 20 1.5.7. Hydraulic Retention Time (HRT) . . . . . . . . . . . . . . . . . . . 21 1.6. Process variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1.7. Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2. Materials and Methods 25 2.1. Biogas yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2. Total solids and volatile solids . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.3. Sample preparation and analysis . . . . . . . . . . . . . . . . . . . . . . . 27 2.4. High Performance Liquid Chromatography (HPLC) . . . . . . . . . . . . . 27 2.4.1. Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.4.2. Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5 Contents 2.5. IR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.5.1. Infrared radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.5.2. Michelson Interferometer . . . . . . . . . . . . . . . . . . . . . . . 30 2.5.3. Attenuated total reflectance . . . . . . . . . . . . . . . . . . . . . . 33 2.5.4. Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy 35 3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2.1. Lab-scale biogas plant . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2.2. Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2.3. Controlling software . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.2.4. PLS method development and validation approach . . . . . . . . . 40 3.3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.3.1. FTIR-MIR spectra of the digestate . . . . . . . . . . . . . . . . . . 42 3.3.2. Chemometric analyses . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.3. Test of the developed methods . . . . . . . . . . . . . . . . . . . . 47 4. gasUino - biogas flow meter 51 4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 4.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4.2.1. Flow meter design . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4.2.2. Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2.3. Database & Web interface . . . . . . . . . . . . . . . . . . . . . . . 55 4.2.4. Fermenter bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.2.5. batchLab fermenter array . . . . . . . . . . . . . . . . . . . . . . . 57 4.2.6. Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5. Conclusion 62 List of Tables 64 List of Figures 65 6 Contents Bibliography 68 A. Appendix 75 A.1. Arduino sketch sourcecode . . . . . . . . . . . . . . . . . . . . . . . . . . 75 A.2. SQL statements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 A.3. LabVIEW Virtual Instruments . . . . . . . . . . . . . . . . . . . . . . . . . 79 7 1. Introduction 1.1. Energy supply The industrialized society is almost exclusively based on the use of fossil energy carriers. Coal, oil and natural gas are the main boosters of innovation in the last 100 years. Since the 1950s, also nuclear power is used as a source of energy, but the difficulty of an ultimate disposal of the nuclear waste and recent nuclear disaster of Fukushima, Japan leads to rethinking the safety and environmental impact. In Germany, all remaining nuclear power plants will go off-grid in 2022 [25]. Unfortunately, all fossil fuels are limited too and in the near future, all natural gas reservoirs and oil fields will have been exploited. Furthermore, the release of carbon dioxide into the atmosphere accelerates global warming and a catastrophic climate change (see 1.1). A transition to renewable, CO2 - neutral energies is not only necessary, but the only way for sustainabil- ity. The energy market of the future will be a mixture of all available energy sources - wind energy, geothermal heat, hydro power, solar energy and heat and energy from biomass, through different processes like combustion, thermochemical transformation (carbonization, biochar), physical-chemical transformation and biochemical transformation to ethanol or biogas. Especially the field of anaerobic digestion has a great potential for further improvements in the future [33]. 1.2. Biogas as a renewable energy source Biogas is a mixture of methane (CH4 : 55 - 70 %), carbon dioxide (CO2 : 30 - 45 %) and trace gases such as ammonia (NH3 ) and hydrogen sulfide (H2 S).The energy content is between 6 - 6.5 kWh · m-3 , which equals 0.6 - 0.65 L of oil · m-3 [33]. A symbiosis of various bacteria degrade organic matter under anaerobic conditions to the energetically interesting final product methane. The first systematic studies were conducted in 1770 by the physicist Alessandro Volta. He recognized the combustibility of marsh gas. Faraday later identified the combustible part as a hydrocarbon, but only in 1821, Avogadro 8 1. Introduction concentrations.pdf 2006-08-23 16:37 C M Y CM MY CY CMY K Figure 1.1.: Past and future CO2 concentrations. Since pre-industrial times, the atmospheric concentration of greenhouse gases has grown significantly. Carbon dioxide concentration has increased by about 31 %, methane concentration by about 150 %, and nitrous oxide concentration by about 16 % [77]. The present level of carbon dioxide concentration (around 375 parts per million) is the highest for 420 000 years, and probably the highest for the past 20 million years. [56] 9 Fachverband Biogas e.V. Angerbrunnenstraße 12 85356 Freising Telefon +49(0)81 61/98 46 60 Telefax +49(0)81 61/98 46 70 E-Mail [email protected] Biogas Segment Statistics 2010 1. Introduction Development of the number of biogas plants and the total installed electric output in megawatt [MW] (as of 06/2011) 7.000 2500 5.905 6.000 Number of biogas plants 2.291 Installed electric output (MW) 2000 4.984 1.893 5.000 3.891 3.711 3.500 4.000 1500 1.377 1.271 3.000 2.000 1.000 1.100 2.680 450 274 370 139 159 186 617 850 2.050 1.750 1.600 1.300 1.050 50 65 182 256 333 1000 650 500 390 0 0 Figure 1.2.: Development of the number of biogas plants the total installed electric Biogas Sector Statistics at aand Glance output in megawatt [MW] (as of 06/2011) in Germany [19] End of 2010 Forecast for 2011 Number of plants 5.905 (45) 7.000 (60) (of these feeding biomethane) managed to determine the chemical formula for methane [61]. The agricultural use of Installed electric output biogas fermenters began in the second half of the 20th century, 2.728 but many biogas plants 2.291 in MW were decommissioned only a short time due to defective function and the boom of Net electricity after production cheap oil. in MWh per annum 14,8 Mio. 17,8 Mio. Homes supplied with 4,2 Mio. 5,1 Mio. A renaissance resulted as a consequence of the oil crisis 1972. A change in thinking biogas-based electricity took place for alternatives to fossil fuels and and a dependency on foreign countries was Proportion of electricity 2,46 3,1 consumption in % questioned. The biogas process was further developed in the following years. EspeTurnover in Germany 5,1the Mrd.amendment of the 5,9 Mrd. cially the Electricity Feed Act inof€ 1990 and Renewable Energies Jobs 2004, and the development 39.100 44.500 plants with better Act (EEG) in 2000 and of cogeneration in % 10 10 efficiency had aExport greatrate impact of the number of biogas plants. This led to an exponenSource: Fachverband Biogas e.V. tial increase in biogas plants in Germany to about 6000 plants in the last 20 years (see figure 1.2). Also in developing countries like China or India the utilization of biogas as an energy source has been established. Thousands of mini-scale biogas plants of various capacities are supplying a great number of households with electricity and gas for cooking purpose [43]. 10 1. Introduction 1.3. EEG 2012 The latest version of the "Erneuerbare Energien Gesetz (EEG)" in Germany, which translates to “Renewable Energy Sources Act”, becomes effective in January 2012 and includes great changes for biomass based renewable energy. Paragraph 1, clause 1 of the EEG lists the following points as the main goals [38]: 1. Sustainable development of energy supply 2. Reducing the economical costs of energy supply considering long-term external effects 3. Protection of nature and environment 4. Reduction of conflicts over fossil energy resources 5. Promote the development of technologies for the production of electricity from renewable energies The future plan is to increase the share of the renewable energies in the total energy market to 35 % until 2020, 50 % until 2030, 65 % until 2040 and 80 % until 2050 and to integrate this amount of electricity into the electric supply. In previous versions of the EEG, a fixed amount was paid for the kWh, depending on the plant size (between 7.79 and 11.67 cent). Furthermore, different bonuses were added, which increased the compensation. Most importantly was the "Nachwachsende Rohstoffe (NaWaRo) Bonus, which added a compensation between 7 cent and 11 cent per kWh when exclusively using non-processed renewable raw materials (e.g. grain, oil, wood, waste of landscape management). The usage of the waste heat was also rewarded monetarily with 3 cent per kWh, if the heat use was listed in the EEG (KWK bonus). Other bonuses were the formaldehyde-bonus, a compensation of 1 cent per kWh, if a certain concentration of formaldehyde in the exhaust gas was complied with. A compensation supplement of 2 cent per kWh was added for the use of innovative technologies, such as biogas upgrade to natural gas quality (technology bonus). Unfortunately, this led to an increasing demand of primary renewable products, which directly competed with the food industry and arable land. With the new EEG in 2012, the bonus system vanishes and is replaced with a more efficient compensation system based on 11 1. Introduction 3. Zu §§ 27, 27a und 27b EEG: Vergütungen für Strom aus Biomasse 3.1. Vergütungsstruktur für Strom aus Biomasse Vergütung für Biogasanlagen (ohne Bioabfall) und Festbrennstoffanlagen Bemessungs leistung Grundvergütung Einsatzstoff- Einsatzstoff- GasaufbereitungsBonus vergütungs vergütungs klasse (§ 27c Abs.2) klasse II 3) I 2) [kW el ] Bioabfallvergärungsanlagen 5) (§ 27a) Kleine GülleAnlagen (§ 27b) [ct/kWh] 75 4) 6) 25 700 Nm³/h: 3 150 500 14,3 12,3 6 750 11 5 5.000 11 4 20.000 6 - 8 1.000 Nm³/h: 2 16 1.400 Nm³/h: 1 8 / 6 4) 14 - 2) 3) 4) 5) Über 500 kW bis 5.000 kW nur 2,5 ct/kWh für Strom aus Rinde und Waldrestholz. Nur für ausgewählte, ökologisch wünschenswerte Einsatzstoffe. Über 500 kW bis 5.000 kW nur 6 ct/kWh für Strom aus Gülle (nur Nr. 3, 9, 11 bis 15 der Anlage 3 BiomasseV). Gilt ausschließlich für Biogasanlagen, die bestimmte Bioabfälle (nach § 27a Abs. 1) vergären und unmittelbar mit einer Einrichtung zur Nachrotte der festen Gärrückstände verbunden sind. Die nachgerotteten Gärrückstände müssen stofflich verwertet werden. Die Vergütung ist nur mit dem Gasaufbereitungs-Bonus kombinierbar. 6) Sonderkategorie für Gülle-Biogasanlagen bis 75 kW installierter Leistung am Standort der Biogaserzeugungsanlage, nicht kombinierbar (d.h. keine zusätzliche Grund- oder Einsatzstoffvergütung bzw. Gasaufbereitungsbonus). 3.2. Grundvergütung für Anlagen zur Erzeugung von Strom aus Biomasse* Degression 7): 2,0 %; Vergütungszeitraum 20 Jahre Figure 1.3.: Compensation system EEG 2012 [26] I oder II Ohne einsatzstoffbezogene Zusatzvergütungen nach Einsatzstoffvergütungsklasse 5 MW el - 20 MW el the and the methane potential of certain substrates (biomass, which can be in ct/kWh 2012 The sole 14,30 11,00 to only 60 6,00 fermented). use of corn12,30 was also prohibited % by weight as a coun2013 14,01 12,05 10,78 5,88 termeasure Furthermore, the basic 2014 to monocultures. 13,73 11,81 10,56 compensation 5,76 was increased, but 2015 13,46 11,58 10,35 5,65 the usage of the waste heat is a requisite. Small manure plants up to 75 kW or digestion 2016 13,19 11,35 10,15 5,53 plants with organic waste received a separate compensation scheme between 2017 municipal 12,93 11,12 9,94 5,42 2018 12,67 10,90 9,74 5,32 16 cent and 25 cent per kWh. Figure 1.3 presents an overview of the new compensation 2019 12,41 10,68 9,55 5,21 2020Overall, the structure. positive idea of creating a9,36 new dynamic on 12,17 10,46 5,10the substrate market 2021 11,92 10,26 9,17 5,00 Jahr der Inbetriebamount nahme bis 150 kW el in ct/kWh 500 kW el - 5 MW el in ct/kWh 150 - 500 kW el in ct/kWh by widening the field of* Im possible substrate eligible for subsidization is being pursued. Sinne der Verordnung über die Erzeugung von Strom aus Biomasse in der crops ab 1. Januar geltenden Fassung This will hopefully (Biomasseverordnung help to shift the-BiomasseV) use of food as 2012 an exclusive substrate to more organic waste biomass (seeder figure 1.4). Gasaufbereitungsbonus (§ 27c,instead Abs. 2) unterliegen Degression von 2,0 % (§ 20, Abs. 2, Nr. 5). 7) Die Grundvergütung (§ 27, Abs. 1), die Vergütung für Bioabfallvergärungsanlagen (§ 27a), kleine Gülleanlagen (§ 27b) und der 6 12 1. Introduction Utilisable energy potential of Biogas Agricultural residues 13.7 PJ/a Energy crops (on 2 mio. hectares) 236 PJ/a FigureUtilizable 3: Utilisableenergy energy potential (Hartmann/Kaltsschmitt, 2002, reworked by FNR) Figure 1.4.: potential (Hartmann/Kaltsschmitt, 2002, reworked by FNR) [54] stainable production in European agriincreased utilisation of plant raw mateculture and forestry. One of the Federal rials and energy sources is however that Ministry of Agriculture’s (BMELV) main they are produced and used sustainably. 1.4. Basics of anaerobic funding domains is to test these approaSustainability, as defineddigestion in the 1987 ches through research projects and to Brundtland Report, means meeting the further develop them. Some of the stra-in which needs of the present generation without The anaerobic fermentation is a biological process involving many substeps, tegies that are being pursued are: compromising the ability of future genethe organic carbon is converted to its1).most form, the most 2 , and • Increasing theCO species diversity usedreduced rations to meet their own needs Su- oxidized in energy crop production; stainability therefore has an environform, CH4 . The process of biogas production can be divided in four stages, whereas • Breeding new varieties; mental, an economic and a social dimenthe first sion. and When the second as the third•and stage are linked closely with Newfourth production methods using lower applied as to well renewable raw of pesticidesdifferent and fertilisers as may be materials, this means utilisatieach other. Depending onthat thetheir degradability ofdoses the substrate, stages well as year-round vegetative cover on on needs to strike a balance between responsible their limitednecessary, decomposition whatfor is economically such as [33].fields; • The use of especially efficient converhigh and guaranteed biomass yields, sion processes; and what nature can be expected to tole1.4.1. Hydrolysis • The recycling of residues as fertiliser. rate. The social component refers among other things to people’s working condiThe BMELV’s task is to fundbacterial research in tions, new incomesubstrates opportunities a In this first step organic areand disassembled by extracellular enzymes an appropriate and consistent manner share of value-added processes. There into oligomers and monomers performed by facultative or obligatorily anaerobic bacterial so as to develop the most suitable meare many different approaches to su- such as Clostridium, Bacillus and Pseudomonas. Carbohydrate cleavage by cellulases, xylanases or amylases result in simple sugars and normally takes place within a few 6 hours; proteins are degraded by peptidases to single amino acids and (oligo-)peptides, and energy-rich and energy-rich lipids are decomposed by lipases into glycerol and fatty acids, normally within a few days. The biodegradation of other macro molecules like lignocellulose or lignin is very slow and incompletely [33]. 13 1. Introduction Table 1.1.: Examples of fermentation processes from glucose [72] Product Reaction Acetic acid C6 H12 O6 + 2 H2 O ! 2 CH3 COOH + 4 H2 + 2 CO2 Acetic, prop. acid C6 H12 O6 ! 4 CH3 CH2 COOH + 2 CH3 COOH + 2 CO2 + 2 H2 O Butyric Acid C6 H12 O6 ! CH3 CH2 CH2 COOH + 2 CO2 + 2 H2 Lactate C6 H12 O6 ! 2 CH3 CHOHCOOH Ethanol C6 H12 O6 ! 2 CH3 CH2 OH + 2 CO2 1.4.2. Acidogenesis The second phase of the first stage is the acidogenesis, named after the formation of volatile fatty acids. Monomers, the end products of the hydrolysis step, are taken up by different facultative and obligatorily anaerobic bacteria such as Bifidobacterium spp. Selenomonas spp. and Flavobacterium spp. and further degraded to acetic acid, propionic acid, butyric acid, alcohols, hydrogen and carbon dioxide. The formation of the end products is related with the partial hydrogen pressure; if the H2 partial pressure is increasing, fewer reduced compounds like acetate are formed [33]. 1.4.3. Acetogenesis In this step long chain fatty acids are reduced to acetic acid (C2 ) and hydrogen (H2 ) by acid-forming bacteria like Acetobacterium spp., Sporomusa spp. and Ruminococ- cus spp. Under standard conditions, these biochemical reaction are endergonic (see table 1.2). For the degradation propionic acid, G0 = 76.2 kJ/mol are needed. At very low concentrations of H2 , however, the acetate formation by oxidation of the long chain fatty acids is thermodynamically possible. The problem, that acetogenic bacteria are obligatory H2 producers on the other hand is solved by living with the methanogenic bacteria syntrophically, a process termed "interspecies hydrogen transfer" [24]. The methanogenic bacteria remove the H2 , which is formed by the acetogenic bacteria and therefore lowering the hydrogen partial pressure [33]. Figure 1.5 presents the influence of the hydrogen partial pressure on the free energy the bacteria can gain in the acetogenesis and methane formation from carbon dioxide and hydrogen. Clearly, all the reactions are thermodynamically favorable only in a small window. 14 1. Introduction 0 endergonic exergonic Butyrate –1 Methane 2 logp H (bar) –2 –3 –4 ideal hydrogen concentration Propionate –5 –6 –7 –8 80 40 0 –40 ∆ G at pH7;25 –80 –120 –160 °C(kJ/Reaction) Figure 1.5.: Thermodynamic window of the degradation of the volatile fatty acids [33] Table 1.2.: VFA degradation during acetogenesis; G0 ’; T = 25 °C, pH 7, pH2 10-5 atm, pCH4 0.7 atm, c(VFA) 1 mM, HCO3 - 0.1 mM[21, 72] Substrate Reaction G0 G0 ’ [kJ/mol] [kJ/mol] Propionate C3 + 2 H2 O ! CH3 COOH + 3 H2 + CO2 +76.2 -14.6 iso-Butyrate iC4 + 2 H2 O ! 2 C2 + 2 H2 +48.4 -25.9 Butyrate nC4 + 2 H2 O ! 2 C2 + 2 H2 +48.4 -25.9 iso-Valerate iC5 + 2 H2 O + CO2 ! 3 C2 + H2 +20.2 -36.8 Valerate nC5 + 2 H2 O ! C3 + C2 +48.8 -25.9 15 1. Introduction Substrate type CO2 type Table 1.3.: Methanogenic degradation [33] Chemical reaction 4 H2 + HCO3 - + H+ ! CH4 + 3 H2 O CO2 + 4 H2 ! CH4 + 2 H2 O CO2 4 HCOO- + 2 H2 O + H+ ! CH4 + 3 HCOOAcetate CH3 COO- + H2 O ! CH4 + HCO3 Methyl type 4 CH3 OH ! 3 CH4 + HCOO3 - + H+ + H2 O Methyl type CH3 OH + H2 ! CH4 + H2 O e.g. Methyl type:2 CH3 CH2 CH2 OH + CO2 ! CH4 + 2 CH3 COOH ethanol Gf [kJ/mol] -135.4 -131.0 -130.4 -30.9 -314.3 -113 -116.3 Methanogenic species All species Many species Some species One species 1.4.4. Methanogenesis The final step of reducing the organic intermediates to methane is the methanogenesis, an exergonic reaction, which only takes place under strictly anaerobic conditions. The responsible bacteria all belong to the archaea family, such as Methanococci spp., Methanobacteria spp. and Methanomicrobia spp. Central to the anaerobic fermentation and methane generation this methanogenic bacteria are very sensitive to all sort of process disturbances, especially pH-fluctuations and oxygen. Due to their very low growth rate, the whole process of anaerobic digestion is optimized for these bacteria. Not all methanogenic species can use all available substrates. These can be divided into three major groups and their methane forming reactions can be found in table 1.3: • CO2 type: CO2 , HCOO• Methyl-type: CH3 OH, CH3 NH3 , (CH3 )2 NH2 + ,(CH3 )3 NH+ , CH3 SH, (CH3 )2 S • Acetate type: CH3 COOThe energy yield for the bacteria is varying with the biochemical reactions. The direct reduction of CO2 and H2 gains up to - 136 kJ / mol and can be done by all methanogenic species. In contrast, the comproportionation of acetate only yields - 31 kJ / mol. Interestingly, only 27 % - 30 % of the methane arises from the reduction pathway, while 70 % are generated by the combined reduction and oxidation of acetate [33]. 16 1. Introduction 1.5. Process parameters The previous section clarified, that the different bacteria have a different optimum for different process parameters. The first stage of hydrolysis and acidogenesis has a pH optimum of 3 - 4 and can also have aerobic conditions, in contrast to methanogenesis, which has to be strictly anaerobic. It is important to inhibit fluctuations like rapid substrate changes or temperature shifts, because this can lead to a deficit of the gas production. With a two-stage plant, it is possible to optimize the two stages of the biochemical reactions. In a one-stage plant, the process must be optimized for the methanogenic bacteria, because of their low growth rate and higher sensitivity to environmental factors [33]. The following sections will give a short overview of the different parameters that can be either set to an optimal range or should be monitored closely for an early detection of problems during the biochemical breakdown of the biomass to methane. 1.5.1. Temperature For every biological process, the temperature is one of the most important factors. Dependent on the microorganisms optimum, different temperature levels are used for the fermentation process: 1. psyrchophilic at a temperature range between 15 °C and 25 °C 2. mesophilic at a temperature range between 25 °C and 45 °C 3. thermophilic at a temperature range between 45 °C and 70 °C At a higher temperature, the biogas yield is increased (thermophilic microorganisms), but meanwhile the susceptibility to errors is also heightened. If the process temperature is low, the biogas yield will decrease as well. Most biogas plants will therefore be run at a mesophilic temperature range with temperatures around 37 °C to 40 °C to ensure a balanced compromise between biogas yield and process stability. In two-stage systems, the first hydrolysis step and the methane digester can be operated at different temperatures, although this varies individually depending on the used substrate. 17 1. Introduction 1.5.2. pH value The pH optimum of the methanogenic bacteria is at pH = 6.7 - 7.5. If it decreases to pH < 6.5, a positive feedback leads to a further decrease, because the activity of the methanogenic bacteria is inhibited and thus the volatile fatty acids in the process cannot be oxidized. With rising concentrations, the pH sinks even more and the process will come to a halt. In the fermentation process, however, two buffer systems ensure a pH in the optimal range. A too strong acidification is avoided by the carbon dioxide / hydrogen carbonate / carbonate buffer system, which is created by the equilibrium between dissolved carbon dioxide and hydrocarbonic acid (pKa = 6.35): CO2 $ H2 CO3 $ H+ + HCO3 - $ 2 H+ + 2 CO3 2At pH 4, the buffer equilibrium is shifted to free carbon dioxide, at pH 13 all carbon dioxide is bound as carbonate in the system. For monitoring the process, a rise of the carbon dioxide percentage in the biogas can be an indicator for a process disturbance [33]. A second system, the ammonia-ammonium buffer inhibits a too weak acidification (pKa = 9.25): NH3 + H2 O $ NH4 + + OHNH3 + H+ $ NH4 + Although these buffer systems equilibrate the pH, both can still be overloaded with e.g. a too high organic load of easy degradable carbohydrates (starch powder, potato wastewater), which can lead to a rapid increase of the volatile fatty acids. 1.5.3. Volatile Fatty Acids (VFA) Besides the hydrogen concentration, the concentrations of the different volatile fatty acids serve as one of the best process indicators. They are either products of degradation steps from the hydrolysis and acidogenesis or serve as substrate for the acetogenesis and methanation. As the different stages are linked and in equilibrium, the concentration of the volatile fatty acids is naturally low. With a change of the environmental conditions (pH drop, inhibition of the degradation, substrate overload, temperature instabilities), the bacteria can be inhibited and the concentrations can increase. In the literature, different rules and inhibition limits can be found for the concentrations. As a 18 1. Introduction Table 1.4.: Overview of TS and VS percentages of common substrates [10] Substrate TS [%] VS [%] Methane yield [L / kg VS] Municipal organic waste 60 - 75 30 - 70 300 - 900 Fat (grease separator) 2 - 70 77 - 99 1300 Crop 84 - 88 95 600 - 800 Maize 40 - 42 95 - 97 390 - 400 Cattle manure 6 - 12 68 - 85 150 - 400 Green cut 12 - 42 87 - 93 450 - 750 rule of thumb, especially the increase of propionic acid and other short chain fatty acids can be problematic, because they themselves inhibit the degradation even more. But biochemical reactions are dependent on the temperature, the inoculum and especially the used substrates, every biogas plant can exhibit a unique acid spectrum. This should be monitored closely to detect any changes in the dynamics (see chapter 3). 1.5.4. Total Solids and Volatile solids Substrates can have a great variation of their water content and the ratio of organic to inorganic fraction. Especially from liquid biomass like manure to corn silage, big differences can be observed (see table 1.4). It is important to estimate the contingents to establish a stable organic loading rate and hence continuous gas production in the anaerobic digestion process. The estimation is done with standard methods, see section 2.2. 1.5.5. Biogas potential For the estimation of the degradation efficiency of the substrates, the maximum biogas yield has to be identified. If the substrate composition and the organic content is known, the ideal biogas yield, according to Buswell and Mueller [28] can be calculated as (simplified): Cc Hh Oo ! ( c2 + h8 - o4 ) CH4 This formula can only be an approximation, because the volatile solids have to be separated into easy-degradable (carbohydrates, protein, lipids) and hard-degradable fractions (lignin, cellulose), known as FOM (content of fermentable organic matter) [78]: 19 1. Introduction FOM = VS - VSnon-degradable To estimate the objective biogas potential of the used substrates, batch fermentations have to be carried out. The VDI guideline 4630 [75] serves as the standard protocol for comparable batch fermentation studies. At least nine batch fermenters are needed for valid results (see also chapter 4): • a triplet of the sludge, without substrate, as the negative control to estimate the residual gas potential, • a triplet with a defined, 100 % degradable substrate as the positive control to estimate the activity of the sludge and to eliminate inhibition effects, • a triplet of the substrate tested. The amount of VS [%] of the inoculum sludge should be < 2 % and the ratio of of V S substrate 0.5 V S inoculum sludge An experiment is run for 28 days and the recorded biogas volume is furthermore corrected to standard conditions (T = 0 °C; p = 1013 hPa). 1.5.6. Organic loading rate (OLR) The organic loading rate refers to the daily feeding amount of fermentable biomass (VS), based on the digester volume. In a mesophilic operation, values between 3.5 and 5 kg VS · m-3 d-1 have proven to be successful [74]. Most of the biogas plants still run under load to minimize the possibility of process errors and therefore exhibit a lot of unused potential. It could be shown, that doubling the OLR from 2.11 to 4.25 kg VS m-3 d-1 can also double the plant capacity of 500 kW to 1000 kW without building any additional fermenters [49]. OLR[ g V S Substrate [g] ]= L·d V Fermenter [L] · d 20 Desulphurization 1. Introduction CHP Headspace Silage Heat storage Hydrolysis Plant Measurement Manure Control Center Gas Substrate Digestate Data Figure 1.6.: Two-stage agricultural biogas plant 1.5.7. Hydraulic Retention Time (HRT) Closely linked to the volumetric loading rate is the hydraulic retention time (HRT). It specifies the statistically average residence time of the substrate in the digester: HRT [d] = V Fermenter [L] V Feed [ Ld ] In conjunction with the digester temperature, the residence time is the decisive factor for the degree of conversion of biomass into biogas. With short residence times, only the easily degradable substances are methanized. At longer residence times of 20 or more days, also difficult degradable substrates can be converted to biogas. Hydraulic retention times of 30 to 40 days have been proven to be satisfactory [10, 74]. 21 1. Introduction 1.6. Process variants The digestion process only needs an anaerobic atmosphere, a stable temperature, degradable biomass and a suitable bacteria inoculum. Therefore, specifically engineered for the used substrate, different plant designs have been established. Primary renewable products usually get co-fermented with manure in a continuously stirred tank reactor in a one stage or two stage system (see figure 1.6). The first and the second stage are separated, providing optimal conditions for hydrolysis / acidogenesis (pH < 4) and acetogenesis / methanogenesis (T = 40 °C, pH 6.5 - 7.5). The methane fermenter is tempered and stirrers inhibit temperature drifts as well as administer the substrate evenly across the active volume. Generated biogas is stored under a membrane ceiling prior to desulfurization and combustion to generate electricity and heat. Being nutrient rich, the digestate is stored in a post-digestion tank and used to fertilize the soil. 22 1. Introduction 1.7. Aim In continuously operated industrialized plants, the different time constants for hydrolysis, acidogenesis, acetogenesis and methanogenesis can lead to low organic loading rates and long hydraulic retention times for plant operation to ensure a stable digestion process. This consequently leads to underloaded biogas plants with capacious fermentation tanks, which may exhibit a optimization potential. As a plant operator, informed decisions need to be made, wether to increase organic loading rates or decrease hydraulic retention times while maintaining a stable bioprocess. The intermediates in the breakdown from macromolecule to methane and carbon dioxide are the volatile fatty acids and generally favored as process indicators. If the digestion is balanced, the generated volatile fatty acids are catabolized instantly in the direction of methane. Conversely, increasing concentrations of these intermediates can be an indicator for process inhibition e.g. overfeeding, shortened retention time, ammonia inhibition due to protein rich substrates or presence of antibiotics in the process. Chromatographic methods are used to determine the concentrations of the volatile fatty acids on an irregular basis, which can only give a snap-shot of the current process status. Furthermore, every biogas plant exhibits a certain profile of volatile fatty acids in a stable process, depending on the bacteria involved and the substrate mixture fed. The anaerobic digestion of biomass to biogas is a complex process, with a lot of different biological, chemical, and physical reactions involved. It is difficult to narrow down the complex interactions between substrate and inoculum to one or two absolute parameters. Depending on the composition of the substrate, the velocity-determining step of the biochemical reactions will vary. Although the major components of all biomasses are carbohydrates, proteins and lipids, their chemical structures exhibit enormous variations and subsequently some substrates are easily degradable by microorganisms (e.g. starch), while others (e.g. lignin) are more difficult to catabolize and take time to be completely degraded. This makes it difficult to estimate the methane potential of novel biomass or waste products, purely based on a composition analysis. For meaningful results, standardized triplicate batch experiments of substrates are conducted and the resulting biogas yield is monitored closely. These tests are usually limited by high priced equipment to monitor minor gas flows. 23 1. Introduction The common denominator and a possible solution for both problems is the design and development of novel measurement instrumentation. Real time information about the concentration dynamics of the volatile fatty acids in continuous stirred tank reactors would enable plant operators to better understand the "black-box" process of anaerobic digestion and help to exploit unused fermentation potential. Furthermore, a good online measurement of the intermediates can help to improve algorithms for mathematical models of the process. An online sensor system, based on IR-spectroscopy was developed, which is able to evaluate the concentration dynamics of the individual volatile fatty acids. An infrared spectrometer was connected to an anaerobic digester and spectra of the digestate were recorded periodically. With reference analysis of the volatile fatty acids concentration, multivariate calibration allows to develop different chemometric models to predict the concentration dynamics in situ (see chapter 3). For the investigation of the methane potential of putative new substrates, a low-cost automated biogas flow meter was developed upon the open-hardware Arduino platform. Accompanying, a batch fermenter array with an integrated stirrer for three triplicate assays was designed and constructed. This enables upscaling of batch fermentation tests with comparatively minor monetary investments (see chapter 4). Both studies were done under the premise of developing new measurement instrumentation for the biogas process. Materials, instruments and common analytical methods used throughout the projects are introduced in the next chapter. 24 2. Materials and Methods The chemicals used in the experiments are summarized in 2.1, materials used are shown in 2.2 2.1. Biogas yield The produced biogas was measured with the gasUino, an in-house developed biogas flow meter. It was calibrated beforehand and the final biogas yield was corrected to standard conditions (T = 273.15 K, p = 1013 hPa) automatically. A detailed description is presented in chapter 4. 2.2. Total solids and volatile solids The determination of total solids of the sludge and the substrates is done according to DIN 12880 [30]. Samples are dried at 105 °C until a constant weight is achieved for 24 hours. To discern the portion of solid mass attributed to ash content from non-volatile organic content, the samples are furthermore ignited in a furnace at 550 °C until constant weight or for 24 hours, according to DIN 12879 [30]. Product name Microcrystalline cellulose Acetic Acid Propionic Acid Iso-butyric Acid Butyric Acid Iso-valeric Acid Valeric Acid Table 2.1.: Chemicals Manufacturer Avicel PH-10, Sigma Aldrich Carl Roth GmbH & Co. KG Merck Fluka AppliChem Fluka Fluka 25 Order Number 11365 3738.1 8.00605.0100 58360 A2582 59850 94530 2. Materials and Methods Instrument Annealing furnace Centrifuge pH meter pH electrode Thermostate HPLC system Autosampler UV Detector RI Detector HPLC Column Guard-column Compartment drier Micro scale Micro filter Fermenter Fermenter Communication Unit Gas collecting bags Multi-channel Peristaltic Pump Peristaltic Pump Peristaltic Pump Multiple Socket Outlet Spectrometer pH electrode Redox potential electrode Table 2.2.: Equipment Model name Brennofen U15 Mikroliter 2041 pH 523 Inlab Micro RM 6t 2707 2489 2414 Aminex HPX-87-H Micro-Guard Cation-H R 160P-* D1 Minisart RC 25 Biostat MD Biostat B Micro MFCS 50L, PETP/AL/PE Ismatec IPC XX 8000230 Ecoline VC SIS-PMS Tensor 27 405-DPAS-SC-K8S PT4805-DPA-SC 26 Manufacturer Uhlig, Germany Hettich, Germany WTW, Germany Mettler Toledo, Switzerland Lauda, Germany Waters, US Waters, US Waters, US Waters, US Bio-Rad Laboratories, US Bio-Rad Laboratories, US Heraeus Sartorius GmbH, Germany Sartorius GmbH Braun AG, Germany Braun AG, Germany Braun AG, Germany Tesseraux, Germany Ismatec, Germany Millipore, US Ismatec, Germany Gembird, Germany Bruker Optics, Germany Mettler Toledo, Switzerland Mettler Toledo, Switzerland 2. Materials and Methods 2.3. Sample preparation and analysis Sludge samples were extracted with a syringe and centrifuged at 13 000 rpm for 10 min to pelletize the solids. The supernatant was further filtered with a 0.45 µm micro filter (Minisart RC 25, Sartorius GmbH) into a chromatographic sample vial and stored at -20 °C. 2.4. High Performance Liquid Chromatography (HPLC) 2.4.1. Theory Liquid chromatography was discovered and named by the russian botanist Mikhail Semenovich Tswett at the beginning of the 20th century. With a column of calcium carbonate, he was able to separate chlorophyll a and b amongst other extracts from leave extracts [18]. Subsequently, other methods were invented like liquid-liquid chromatography or gas-liquid chromatography. A big leap forward was in the 1970s by Horváth et al. with the invention of High Pressure Liquid Chromatography (HPLC) systems [45]. A schematic overview of a isocratic HPLC System is given in figure 2.1. Separation of the mixture of compounds is achieved by moving a mobile phase (aceto nitril, methanol or water) through a densely packed column (stationary phase) with high pressure. After the injection of the sample, the analytes adsorp to the column and depending on the interactions, get eluted at specific retention times. With different chemical structures, a suitable detection mechanism is needed. Common methods are UV/Vis (for substances that absorp at a certain wavelength) and Refractive Index detection (e.g. for sugars). As a result, a chromatogram is recorded, where the different analytes are represented as peaks. For the quantitation, the peaks get integrated and with the help of a calibration curve, their concentrations can be estimated. 2.4.2. Protocol The samples were analyzed with a HPLC setup consisting of a binary HPLC Pump and an auto sampler (Model 2707, Waters, MA). To detect the volatile fatty acids, two successively connected detectors were used (UV/Vis detector 2489 at 210 nm and Refractive Index Detector 2414). An Aminex HPX-87-H column (Bio-Rad Laboratories, Richmond, 27 2. Materials and Methods Chromatogram HPLC Column Packing Material Computer Data Station Injector Auto Sampler Solvent (Mobile Phase) Sample Pump Solvent Manager Solvent Delivery System Detector Waste Figure 2.1.: Overview of a HPLC System (modified [14]) CA) combined with a suitable guard-column (Bio-Rad Micro-Guard Cation-H) was used as stationary phase. The column temperature was 40 °C. 5 mM sulfuric acid with a flow rate of 0.6 ml·min-1 served as mobile phase. The injection volume was 20 µl and the sample run time 60 minutes. For calibration, an external standard with varying concentrations for the different VFA was prepared (acetic acid 0 - 4 g/l, propionic acid 0 - 3 g/l, butyric, iso-butyric acid, butyric acid, iso-valeric acid and valeric acid 0-1 g/l). Integration and quantification of the recorded chromatograms were performed with the Breeze2 software package (Waters, MA). 2.5. IR Spectroscopy The infrared (IR) spectroscopy is a physical method of analysis which is used for the quantitative determination of substances. In general, it is the absorption measurement of different IR frequencies by a sample positioned in the IR beam. It can also be used for structure analysis, because different functional groups of molecules absorb character- 28 2. Materials and Methods istic frequencies. For the quantitative analysis, the relationship between the absorption of light and concentration of the substances is important, which is described in BeerLambert law: E = with ✓ lg I I0 ◆ =c ·↵ ·l • E = Extinction • I: Intensity of transmitted light • I0 : Intensity of irradiated light • c: Concentration of the analyte in the solution • ↵ : Molar extinction coefficient • l: Path length of light The concentration of the absorbing substance is therefore directly proportional to the extinction, if the layer thickness and wavelength are kept constant. In practice, the relationship between the two variables is described by calibrating. Each component of a mixture can thus quantitatively be determined, if a sufficiently intense absorption band can be found, which is not disturbed by the other analytes or by the solvent mixture. Using the multivariate calibration approach, also complicated multi-component analysis by specifying a broader spectral range are is possible. 2.5.1. Infrared radiation In the electromagnetic spectrum, infrared light takes its place between visible light and microwaves [0.78 - 1000 µm]. Like any electromagnetic wave, it is characterized by its wavelength [nm] and oscillation frequency f [Hz]. Of great importance in IR- spectroscopy is the wavenumber ⌫¯, that corresponds to the reciprocal of the wavelength with the unit cm-1 and is therefore directly proportional to the frequency as well as the energy of the absorption. A IR spectra is usually presented with the absorption intensity 29 2. Materials and Methods on the y-axis and the wavenumber on the x-axis. (see figure 3.4a) ⌫¯ = 1 1 · 104 = [cm] [µm] The stored energy in the wave can cause a transfer of energy - the energy of the wave can be absorbed by the molecular system and transferred into another energy form, e.g. thermal energy. However, this can only happen if it leads to a change of the dipole moment of the specific atom group. Therefore, diatomic molecules with identical atoms are IR inactive because they have no dipole moment. If a molecule is composed of different atoms, it can always interact with the infrared light, because wave motion leads to an anti-symmetrical shift of charge center and hence creates a dipole moment [42]. Dependent on the geometry of the molecule, different translational, vibrational, and rotational movements are possible. Upon irradiation with infrared light, the bonds in the molecules start to vibrate, which the major types of molecular vibrations being stretching and bending: Figure 2.2b presents the vibrational possibilities and their specific wavenumbers for these of a -CH2 group [62, 63]. Different absorption bands are therefore characteristic for different bonds (C-C, C-O, C-N, C=C, C=O, ...). Functional groups that have a strong dipole also show strong absorptions in the IR spectra. The specific wavenumbers and corresponding bands for the analysis of volatile fatty acids are discussed in chapter 3. 2.5.2. Michelson Interferometer An interferometer uses the effect of interference, the addition or subtraction of the amplitudes of superimposed waves to generate different wavelengths simultaneously. In figure 2.3, the effect of interference is exemplary presented with two superimposed sine waves with the same frequency and the same phase or 180° out of phase. If the waves are in phase, constructive interference produces a higher amplitude (A1 + A2 = 2A. Destructive interference occurs if the waves are out of phase by 180° and cancel themselves out. In the Michelson interferometer, main part of a Fourier-Transformed Infrared (FTIR) spectrometer (see figure 2.2), a wave is superimposed with itself to create an interferogram. Its general functionality is, that light is emitted from a light source and divided by a semi-transparent mirror into two beams. One half is reflected at the beam splitter 30 Figure 3.3: Three of six degrees of freedom of a two atom molecule: two rotational about x and z axis and one vibration along the y axis. dµ != 0 dx (3.2) There exist molecules having a dipole moment, but not fulfilling the above con- 2.dition, Materials and Methods for example the symmetric stretch of a CO2 molecule. Such molecules are either infrared inactive, or some of their modes, which do not fulfil the above condition, are not visible in an infrared spectrum. Examples of some basic infrared active modes, are 21 depicted in Figure 3.4. 3.1.1 Infrared Absorption C z H Rotation C H Symmetric Stretch H C H H Asymmetric Stretch H Scissors x n atio Vibr y H C C Rotation H H Rocking (a) Three of six degrees of freedom of a two atom H H C H Symmetric Bend dµ != 0 dx main classes of infrared induced vibrations [34, 19]. (3.2) There exist molecules having a dipole moment, but not fulfilling the above condition, for example the symmetric stretch of a CO2 molecule. Such molecules are either infrared inactive, or some of their modes, which do not fulfil the above con- Mirror dition, are not visible in an infrared spectrum. Examples of some basic infrared active modes, are depicted in Figure 3.4. C H Symmetric Stretch H Rocking C H H Asymmetric Stretch H C C H H x1 C H half-shivered mirror Scissors IR coherent light source H H Symmetric Bend H C H Asymmetric Bend (b) Major vibrational modes for a nonlinear group, Figure 3.3: molecule: Three of six two degrees of freedom of a x two atom molecule: twoExamples rotarotational about and z axis and CH4 [47]. Figure 3.4: of tional about xone andvibration z axis andalong one vibration along [47]. the y axis. the y axis. H H H y x2 H Asymmetric Bend Mirror Figure 3.4: Examples of main classes of infrared induced vibrations [34, 19]. Detector Figure 2.2.: Schematic setup of a Michelson Interferometer [79] 31 2. Materials and Methods (a) constructive interference (b) destructive interference Figure 2.3.: Interference of superimposed sinus waves to a rigidly mounted mirror and reflected back to the beam splitter, hence traveling the way of 2x1 As the beam-splitter is semi-transparent, the other portion gets transmitted to the second mirror and reflected likewise, hence passing the way of 2x2 . The key factor now is, that the second mirror is not mounted firmly but is movable, whereby the light actually passes a range of 2 · (x2 + y). Both partial beams get combined again at the beam-splitter and interference is occurring before striking the detector. This interference equals the distant of 2y. Constructive interference for a particular wavelength b is ob- tained exactly, if the path difference 2y is an integer multiple of this wavelength. For the other wavelengths from the broadband IR source destructive interference can be observed. Each position of the movable mirror thus corresponds to a certain wave number ⌫¯b . Before impinging on the detector, the modulated beam passes the sample. The registered signal of the detector is the interferogram, the intensity I(x) of the IR radiation as a function of the position of the movable mirror [42]. Fourier transformation converts this interferogram from the time domain into one spectral point on the frequency domain [63]. The ratio of this spectrum and a reference spectrum yields the desired spectrum with wavenumber on the x-axis, absorbance on the y-axis. A major advantage of FTIR spectroscopy is, that all emitted IR frequencies impinge on the detector simultaneously, thus enhancing the signal to noise ratio. This makes rapid measurements possible, which depend only on the time the mirror takes for moving across a particular path [42]. 32 2. Materials and Methods Sample n2 n1 dp n2 Figure 2.4.: Schematic representation of the attenuated total reflectance. n1 : Refractive Index of the crystal; n2 : Refractive Index of the sample; n2 < n1 ; dp = Penetration depth, [42] 2.5.3. Attenuated total reflectance The attenuated total reflectance (ATR) method makes it possible to record IR-spectra from solid or intransparent samples. For the measurement, the samples only have to stay in contact with the surface of the ATR crystal, which has to have a very high refractive index. On the boundary surface of two media with different refractive indices, the total reflected radiation creates an evanescent wave, which can penetrate the sample of about one wavelength (dp , c.f. fig. 2.4). The intensity of the wave is reduced (attenuated) by the sample in regions of the IR-spectrum, where the sample absorbs. Zinc selenide, thallium bromide-thallium iodide (KRS-5) or germanium are examples for crystals with a high refractive index [62]. 2.5.4. Quantitative Analysis To extract the concentration of the samples from the recorded spectrum, a calibration has to be done, which matches the varying spectra structure to a known concentration. In the classical univariate calibration, which is based on the Beer Lambert law (see 2.5), only one spectral data point is used. In contrast, the multivariate calibration uses the whole spectral structure for the calibration. This makes it possible to simultaneously estimate the concentration of different analytes in the sample mixture [31]. For the calibration, the method of Partial Least Squares (PLS) Regression, implemented in the QUANT2 Software Package (Bruker Optics, Germany) was used. For good PLS prediction models, the calibration set has to fulfill some principal requirements [47] : 33 2. Materials and Methods • the data set has to include all expected components which can lead to a variation in the spectra • for the analytes, the sample concentration should span the range of interest • the background signal should be the same for all samples; for biotechnologically produced products it is best to use spectra directly out of the process • the variation of the concentrations of the different analytes should be independent of each other To test the calibration method developed for accuracy, each model is assessed using a validation. Two validation methods are commonly used: the cross-validation and the test-set validation. In the cross-validation a sample is obtained prior to creating a model from the test set. The concentration of this sample is not known and therefore independent. With the remaining samples, a method is developed and used to predict the concentration of the prior skipped sample. Now, for this sample the reference concentration is known (e.g. analyzed with HPLC) as well as the predicted concentration. Subsequently, the sample is re-added to the test set and another sample is removed until all samples have been determined successively. To determine the accuracy now, the prediction error Root Mean Square of Cross Validation (RMSECV) is calculated. For a good model, this prediction error is low. The external test set validation is similar, but two real sets are formed: one for the calibration, and one as the test-set for the validation. Other variables which have to be taken into account are R2 of the prediction versus the reference analyses as well as the number of the internal latent variables, which the chemometric model is based on. Ideal are low numbers for the prediction errors and the internal latent variables and a high value for the correlation [31]. This section about IR spectroscopy was to give a very short overview of the principles and methods the next chapter is based upon. Detailed secondary literature can be found in the bibliography [31, 47, 62, 63]. 34 3. On-line mid infrared monitoring of the dynamics of the individual volatile fatty acids in the continuous fermentation of biogas 3.1. Introduction The degradation of biomass and the formation of methane is a complex biochemical process, which can be separated into four main phases: hydrolysis, acidogenesis, acetogenesis and methanation. A kinetic uncoupling between the syntrophic bacteria of acid producers and consumers in these different fermentation steps can be observed by an accumulation of the different volatile fatty acids [70]. Therefore, concentration and composition of the VFA are the best parameters to reflect the metabolic state of the biochemicalprocess [3, 23, 57]. Offline determinations of the VFAs are usually performed with chromatographic methods, including gas chromatography (GC) [1], Headspace GC [32] or high performance liquid chromatography (HPLC) [29]. On- and offline titrimetric methods can achieve a low cost and quick result of a VFA sum parameter, but ignore the individual composition of the specific VFA [48]. Pind et al. [57] concluded that measurements of all-individual VFAs are important for control purposes. The dynamics and the history should always be evaluated in close relationship to the conversion of other VFAs and the history of the reactor process. Few online methods for GC [20, 58, 64] and HPLC [81] have been applied as well. However, problems with sample preparation and biofouling make these methods not applicable for use in the field. FTIR-spectroscopy has been proven to be a real alternative without facing equivalent problems. This method is widely used in pharmaceutical, food, medical and bioprocess applications [46, 60]. In the field of anaerobic digestion Near Infrared Spectroscopy (NIR, 0.78 - 3 µm) was shown to be able to evaluate VFA content in glycerol-boosted anaerobic digestion processes [44] and in a hydrogen-producing bioreactor [80]. MIR-spectroscopy (Mid infrared spectroscopy, 3- 50 µm ) was already shown to be suitable for monitoring volatile fatty acid content, chemical oxygen demand, alkalinity, sulfate, ammonia and nitrate concentration in in- 35 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy dustrial wastewater treatment [68, 69]. In aerobic batch composting studies, FTIR spectroscopy was used to evaluate the different decomposition stages of bio waste [41, 65]. The aim of this work is the development of an online continuous sensor system based on MIR-ATR-FTIR-spectroscopy for measuring the individual concentrations of the different volatile fatty acids in the anaerobic fermentation of biogas for highly heterogeneous and viscous bio-slurries. 3.2. Materials and Methods 3.2.1. Lab-scale biogas plant An anaerobic continuous stirred-tank reactor (CSTR) with an active sludge volume of 10 l was operated at 40 °C and 75 rpm (Biostat MD, Braun, Melsungen). The inoculum sludge was taken from an operating biogas plant. The hydraulic retention time (HRT) was 42 d with a varying organic loading rate between 0 and 4 g l-1 d-1 VS. Different parameters (pH, redox potential) were monitored by electrodes (Mettler Toledo, Greifensee, Switzerland), the biogas flow was measured with a gasUino (see chapter 4). Sampling and feeding of the reactor were done manually once a day. The substrates used in the experiments were artificial substrates (corn starch powder and 10 % peptone) and ground wheat, respectively. 3.2.2. Analytical methods To estimate the concentrations of the volatile fatty acids, the samples were centrifuged and the supernatant was filtered and stored at -20 °C (see section 2.3). The protocol for the chromatographic analysis with HPLC can be found in section 2.4.2. MIR-ATR-FTIR equipment and data analysis The spectra were recorded with a TENSOR 27 (Bruker Optics, Ettlingen, Germany) FTIR spectrometer with an ATR-Cell (ZnSe) at room temperature (25 °C). Wavenumbers from 2800 cm-1 to 900 cm-1 were scanned at a resolution of 4 cm-1 . A background measurement with fresh water was done prior to measuring a digestate sample. The typical stable pH of the digestate samples is between 7 - 8, so no adjustment to a given 36 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy pH was done. Before sample-collection, the suspended solids were removed from the liquid phase by a raw mesh filter to achieve a homogeneous probe. This was either done manually for the calibration set or with a rotating filter unit for the continuous fermentation experiments (see figure 3.5). Each reference and sludge sample was scanned 256 times to get a well-balanced average spectra and to minimize temperature drifts or shifts due to sedimentation of solid particles. Both, sample and blank spectra were collected in absorbance mode. Partial least square (PLS-2) algorithm was used to create multivariate calibration models for acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid and valeric acid with the QUANT2 software package (Bruker Optics, Ettlingen). A separate model was set up for each chemical component. Principles of chemometrics and multivariate calibration are described elsewhere [51]. Process cycle of FTIR spectra recording during the continuous fermentation experiments The process of sludge extraction, spectra recording, and processing during the fermentation experiments was automated (c.f. 3.1). An acrylic glass chamber with two inputs and one output was constructed and mounted on top of the ZnSe crystal, see 3.2. The homogenous sludge filtrate is extracted from the inside of a rotating filter unit attached to the stirrer axis (modified, according to [58]). The filter has a pore size of approximately 1 mm2 and the rotation shearing force removes solids, which accumulate on the surface of the filter. A process cycle included the following steps (c.f. figure 3.1): 1. The acrylic glass chamber is flushed with fresh water by a peristaltic pump (2) (Millipore, USA) 2. A background spectrum of the water is recorded immediately 3. A multi-channel peristaltic pump (1) (Ismatec, Wertheim) transfers approximately 20 ml of the digestate from the filter unit to the flow chamber. The filling level of the fermenter is leveled out with fresh water, which results in a HRT of 42 days 4. IR-spectrum of the digestate is recorded and processed. 5. The flow chamber is rinsed again and the cycle restarts after two hours. 37 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy Figure 3.1.: Piping and instrumentation scheme of the anaerobic digester and the IRspectrometer. A sample is generated by a rotating filter unit mounted on the stirrer axis. Pump 1 transfers the digestate to the measurement chamber, fitted on top of the ZnSe crystal. Simultaneously, fresh water is transferred to the fermenter to balance the filling level. After the spectra recording is finished, crystal and chamber are cleaned with fresh water via pump 2. 38 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy (a) Top View (b) Side View Figure 3.2.: Acrylic glass chamber mounted on top of the ZnSe crystal Twelve spectra per day are recorded. It was chosen to flush the acrylic glass chamber before and after each recording of the sludge sample to ensure a minimized biofouling. Furthermore, the ZnSe crystal was periodically cleaned with paper tissue. 3.2.3. Controlling software The biogas lab plant is automated with the software LabVIEW 8.5 (National Instruments, Austin, TX). Different available or developed Virtual Instruments (VI) are used. Biostat MD and Biostat B are connected to the PC via a MFCS (RS-422) to the serial port. They can be addressed individually by defining their address in the input string, transmitted to the MFCS (1: Biostat MD, 2: Biostat B, see A.5, A.4). The answer string is separated by colons and converted to the respective values (temperature, jacket temperature, stirrer rpm, pH and redox potential). A gasUino was connected to a second serial port, which returns a comma-separated answer string with the number of clicks, the room temperature and the barometric pressure (see A.3d). After gathering all the data from the different devices, an INSERT string was assembled and the data was saved in a MySQL database with the help of LabSQL [7] (see A.7), as well as plotted on the front panel (c.f. A.2). An independent block structure was developed to transfer the sludge to the ZnSe crystal, record the IR spectra, and rinse the crystal (see A.3). Therefore, one inlet of the acrylic glass chamber was connected to the fermenter with a peristaltic pump, the second one to a water tank. Both pumps have to be set to on for cleaning 39 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy the crystal, because the high velocity of the cleaning led to a leak of digestate into the sample chamber after stopping the cleaning pump. To inhibit this effect, the revolution of the digestate pump was set to counter clockwise with a little flow rate during the cleaning. It could be controlled with a VI provided by the manufacturer [15] and was therefore connected to a third serial port. The cleaning pump had no interface for the PC and was controlled with a USB power socket (SIS-PMS, Gembird) by incorporating a command line program (SisPmCtlWin, [12]) into another VI (c.f. A.6). The OPUS Software Package and LabVIEW both support the protocol Dynamic Data Exchange (DDE) for interprocess communication. This allows LabVIEW to send commands to OPUS, if both software packages are running simultaneously (fig. A.8). The VI set the protocol file for the spectra recording and the developed QUANT2 methods as input parameters, which resulted in the estimated concentrations of the volatile fatty acids as the output parameters. These were furthermore saved to the database and presented as plots on the frontpanel. 3.2.4. PLS method development and validation approach The approach of developing and validating the different PLS methods to predict the concentration of the VFA is illustrated in figure 3.3. For the method development (see figure 3.3a), spiked samples of digestate with varying concentrations of the VFA and the samples from an anaerobic digestion, where a systematic overloading occurred, were used. The IR-spectra were recorded and HPLC measurements were done as reference analyzes. In total 147 samples of digestate were collected. Calibration set - part A (see figure 3.3a) Similar to Udén et al. (2009) [73], which estimated the VFA concentration in rumen samples by MIR-FTIR, a calibration set with spiked samples was used to develop the PLS methods for the different VFA. Therefore, the feeding of the biogas plant was halted until the concentrations of the VFA had decreased below the detection level of the HPLC. This VFA-free digestate was then used as the matrix for the spiked samples. Analytical grade acids were added to 5 ml of digestate in different concentrations, according to a developed scheme, where the variations of the concentrations of VFA were independent of each other (R2 of acetic acid vs. propionic acid low, same for all other combinations 40 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy CSTR lab-scale Biogas plant 10 L + VFA in different concentrations spiked samples FTIR record spectra HPLC check prepared samples Calibration Set Part A CSTR lab-scale Biogas plant 10 L stepwise increase of OLR stepwise increase of OLR concentrations of VFA increase FTIR automated spectra recording every 2h, 12 per day feedback loop +- feeding VFA-free digestate b) 40 °C, HRT 42d feedback loop +- feeding a) feeding halted CSTR lab-scale Biogas plant 10 L Prediction of the VFA concentrations with developed methods rotating filter / pump FTIR automated spectra recording HPLC every 2h, 1 sample 12 per day per day HPLC reference analysis: 1 sample per day Calibration Set Part B (a) Two-step method development with the manually(b) Test-set validation with a second continuous ferprepared calibration set (a) and the gathering of mentation experiment real process spectra (b). Figure 3.3.: Flow chart diagram of the chosen approach developing and validating the different methods 41 stepwise increase of OLR FTIR automated spectra recording every 2h, 12 per day Prediction of the VFA concentrations with developed methods from 1.1 HPLC reference analysis: 1 sample per day 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy of volatile fatty acids). MIR-spectra were recorded and HPLC controls were prepared, respectively. Calibration set - part B (c.f. fig. 3.3b) In addition to the manually prepared sample set, a continuous fermentation was set up to obtain the process dynamics of the VFA in a real bioprocess. The recordings of the spectra were automated and one spectrum per day as well as the corresponding HPLC results were added to the calibration set. Based on the calibration set, PLS methods to predict the VFA concentrations in the digestate of an anaerobic digestion were developed. Test of the methods The developed PLS prediction methods were then tested with a second continuous anaerobic digestion experiment. Here, the recorded dynamics of the volatile fatty acids served as a decision criterion when to increase the organic loading rate: If the feeding rate was raised and a reaction of the VFA could be observed, the feeding rate was not further increased until the system was successfully adapted to the new organic loading rate. If no reaction of the VFA could be monitored, the timespan between the increases of the OLR was abbreviated. 3.3. Results and Discussion 3.3.1. FTIR-MIR spectra of the digestate IR-spectra taken at various time points (day 6, day 16, day 26, day 37, day 47,day 57) during the continuous fermentation (c.f. 3.5) experiment are presented in figure 3.4a. The main absorbance region was found between 1800 cm-1 and 900 cm-1 , which represents the absorption region for early decomposition products like aldehydes, ketones, esters and short chain carboxylic acids. A typical IR band is present at 1640 cm-1 , which reflects the aromatic C=C bond and C=O group absorption of amides (Amid I band) or carboxylates. A second band of protein origin, an indicator for protein rich components, the Amid II band is located around 1570 and 1540 cm-1 , due to the N-H 42 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy (a) Relevant bands and spectra at different points in time of the continuous fermentation experiment (day 6, day 16, day 26, day 37, day 47, day 57). Spectra are shifted for clarity on the y-axis. (b) Spectra of the digestate at different points in time, z-axis = time 43 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy in-plane bend vibration and can be assigned to secondary amides [41, 65, 66]. The shoulder at 1425 cm-1 is due to the COO- stretch of carboxylates and the C-O stretch of carbonyls. Another characteristic region for the VFA is between 1265 cm-1 and 1240 cm-1 , where the C-O vibration can be observed. A significant peak can be seen between 1070 cm-1 and 1000 cm-1 . This region is assigned to the C-O stretching of polysaccharides and polysaccharide-like substances, Si-O of silicate impurities and clay minerals possibly in a complex with humic acids [41]. Figure 3.4b presents spectra from an actual fermentation in a 3D environment, where the change of the spectra over time and the increasing peak at 1551 cm-1 is evident. The heterogeneous and complex matrix of the sludge led to an elevated background noise. For method development, all spectra were smoothed and pre-processed with the first or second derivative to remove this background signal [46]. 3.3.2. Chemometric analyses The combined calibration set formed the basis for the chemometric analyses to circumvent two prior observed problems (data not shown): developed models only based on the artificial calibration set lacked the ability to predict the absolute concentrations of the different VFA in a real process in a satisfying range. If only spectra from an anaerobic digestion were used for the model development, it was not possible to differentiate the signals of the different VFA, because the correlation amongst each other (R2 ) was too high. The models, which had the best agreement between a low number of internal latent variables and a low root mean square prediction error (RMSPE), were chosen. Table 3.1 summarizes the properties for each chemometric model and table 3.2 presents the frequency ranges used in the methods. Acetic acid is one of the direct precursors for the methanogenesis. In contrast to the other VFA, an increased concentration of acetic acid (< 3000 mg/l) is not inhibiting the biochemical degradation processes at a pH > 7.5 [33]. Therefore, an equal distribution across the concentration range up to 3 g/l can be observed. The predictability is good, with a R2 of 0.94 and a RMSPE of 0.16 g/l. Inhibition of the process coincides with the increase of the propionic acid concentration [17, 22, 34, 59], making it a perfect indicator for reactor imbalances. The developed method spans a concentration range of 0 - 3 g/l with a RMSPE of 0.26 g/l and R2 of 0.88, with equal distributed values across the concentration range. These standard errors of predictions are consistent with the 44 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy Figure 3.4.: Regression plots from the PLS-Models for the different volatile fatty acids. PLS predicted concentrations were plotted against the HPLC reference analyses. All spectra were smoothed and pre-processed with the first or second derivative and different frequency ranges were chosen, respectively (cf. 3.1,3.2). 45 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy 1st derivative 2nd derivative 1st derivative 1st derivative 1st derivative 1st derivative 0 - 2.75 0 - 3.33 0 - 0.26 0 - 0.33 0 - 0.25 0 - 0.26 7 7 2 6 3 6 0.94 0.88 0.83 0.75 0.59 0.9 RMSPE [g/l] R2 internal latent variables 69 66 41 44 39 44 Concentration range [g/l] 71 67 42 44 40 44 Data pretreatment No. test spectra Acetic acid Propionic acid Iso-butyric acid Butyric acid Iso-valeric acid Valeric acid No. calibration spectra Component Table 3.1.: Summary of the properties of the developed methods for the different volatile fatty acids. 147 samples were collected. 0.156 0.235 0.0318 0.0472 0.0496 0.0191 Table 3.2.: Frequency ranges [cm-1 ] of the different developed methods. Compound Frequency Ranges Acetic acid 1801-1641 1589 - 1535 1429 - 1375 1323 - 1269 Propionic acid 1801 - 1747 1535 - 1269 Iso-butyric acid 1811 - 1622 1496 - 1433 1371 - 1182 Butyric acid 1492 - 1178 Iso-valeric acid 1716 - 1550 1389 - 1346 Valeric acid 1717 - 1661 1390.6 - 1335 12823 - 1173 46 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy findings of other studies with NIR (acetic acid: 0.28 - 0.57 g/l, propionic acid: 0.53 g/l) or MIR (acetic acid: 0.1 - 0.9 g/l) application [46]. Similar to propionic acid, the appearance or increasing concentrations of butyric acid, valeric acid and their iso-forms are also a sign of process disturbances [3, 52]. Their ability to inhibit the process is inverse to their concentration (inhibition at concentrations of 50 mg/l undissociated fatty acids without prior adaption for iso-butyric and iso-valeric acid [33]). It was therefore decided to use a smaller concentration range from 0 to a maximum of 300 mg/l to account this factor. All models exhibit a clustering of values around 0 g/l, due to their absence in the normal fermentation process. Samples with increased concentration are due to the calibration set part A and did also appear after the process was significantly overloaded. This shows the difficulty to create valid models for these higher chain fatty acids by monitoring a real process alone. The R2 of these methods are 0.83, 0.75, 0.59, 0.90 for iso-butyrate,butyrate, iso-valerate, and valerate, respectively. 3.3.3. Test of the developed methods In the next step, the performance of the developed methods was evaluated in a second continuous fermentation. In this experiment, the HPLC analysis served as a reference and was not included in the chemometric model. This approach of using two independent methods for estimating the VFA content was chosen, to evaluate if the models were able to predict the concentrations reliably. In contrast to the first continuous fermentation experiment, the PLS predicted concentrations of the VFA were also used as a decision criterion when to increase the organic loading rate. Figure 3.5 presents the reactions of pH, biogas generation and the volatile fatty acid content. The feeding started at an organic loading rate of 0.5 g l-1 d-1 at day 4 with a mixture of starch powder and peptone. Due to the rapid drop in pH and the increase of the organic acid concentrations, the feeding was stopped at day 11 until the concentrations of the volatile fatty acids had recovered. It was restarted day 17 with ground wheat as substrate at the same organic loading rate of 0.5 g l-1 d-1 . On day 24, the OLR was raised to 1 g l-1 d-1 . These increments led to increased levels of acetate, butyrate and valerate. Therefore, the OLR was kept steady until day 37 (1 g l-1 d-1 ), where all acids had been degraded again and afterwards increased to 2 g l-1 d-1 on day 37, furthermore to 3 g l-1 d-1 on day 42 and then to 4 g l-1 d-1 on day 48. This rapid perturbation induced reactions of all monitored volatile fatty acids, especially the concentrations of butyrate and valerate increased. The absolute 47 2.5 2 1.5 1 0.5 0 2.5 2 1.5 1 0.5 0 0.75 0.75 0.5 0.5 0.25 0.25 pH Acetic acid 1.0 2.0 3.0 4.0 3.0 7.6 7.4 7.2 Propionic acid 0.5 pH L 0 0.2 0.2 0.1 0.1 g/l Iso-butyric acid 0 0 0.2 0.2 0.1 0.1 g/l Butyric acid 0 0 0 0.2 0.2 0.1 0.1 g/l Iso-valeric acid g/l 7 700 600 500 400 300 200 100 0 0.5 g/l 8 7.8 L 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy 0 0 10 20 30 40 50 60 70 0 Time [d] Figure 3.5.: Methods did not include the corresponding HPLC results. a) straight line: pH, dashed line: cumulated biogas volume b) - e) dots: PLS-predicted concentrations. line: smoothed average of PLS-predicted values, points: HPLC reference analysis. Only prediction results are plotted, which passed the internal QUANT2 validity test. The shaded areas highlight the increasing organic loading rate. 48 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy reference concentration of the HPLC results is not matched by the PLS prediction. For acetate, a maximum deviance of about 0.5 g/l can be observed. Similar behavior can be seen for the other VFA. Comparing the PLS predicted values with the reference, propionate also shows a deviance of about 0.175 g/l. The first increase of propionate level from day 4 to day 13 fits the prediction well; the complete degradation is not matched precisely. The changing dynamics from day 50 to 70, however, are recognized. The dynamics of iso-butyrate and iso-valerate get detected in the same manner. As soon as they appear during the fermentation process,from day 30 to day 35 and again beginning from day 50, they can be detected via MIR spectroscopy. Butyrate seems not to be detectable in this experiment. All the spectra from day 0 to day 52 failed the internal validity tests of the QUANT2 software package and are therefore not plotted. Additionally the evaluation of the gathered chromatograms caused problems for butyrate, because no defined peak could be integrated. Therefore, neither of the two methods served valid concentration values, but the HPLC, as well as the chemometric model gives a hint about the existence of butyrate after day 45. The pH dropped from an initial value great than pH 8 to 7.3 +/- 0.2 and was stabilizing after 30 d in the optimal range between 6.7 and 7.5 [33], but did not show any reactions to the introduced perturbations due to the carbonate and ammonia buffer systems in the reactor. The experiment demonstrates the problem of controlling the anaerobic fermentation based on the pH and clearly shows the importance of monitoring the dynamics of the volatile fatty acids closely. The developed FTIR sensor makes it possible to monitor the dynamics of the volatile fatty acids. In this experiment, this information was already used to decide the best point in time to increase the OLR. After increasing the OLR from 0.5 g l-1 d-1 to 1 g l-1 d-1 on day 24, especially acetic acid, but also iso-butyric and iso-valeric acid exhibited an increase in their concentrations. Therefore, the next increase to 2.0 g l-1 d-1 happened only after the VFA were almost completely degraded again (day 38). This strategy also explains the following short time between the next to steps, to 3.0 g l-1 d-1 and 4.0 g l-1 d-1 . Only the reference analysis shows elevated levels of butyric acid, which hints at a process failure - but this was not detectable with the anaerobic sensor system. The concentrations of the higher chain fatty acids were stable, which served as an indicator for raising the OLR. Clearly, the short time of only one week between the increase of the OLR had a major impact on the bioprocesses, where usually hydraulic retention times great than 30 days can be found. Just 2 days after the last step to 4.0 g l-1 d-1 , the concentrations 49 3. Online monitoring of the volatile fatty acid dynamics with MIR spectroscopy of all volatile fatty acids increased again, the biogas production slowed down and the process did not recover afterwards. The results indicate, that a successful control algorithm for the anaerobic digestion has to be based on all available parameters. Even if the dynamics of the volatile fatty acids are the best indicator for process failures at the moment, they cannot be used out of context for decisions regarding alterations of the biogas fermentation process. 50 4. gasUino - a low-cost biogas flow meter based on the open-source electronics prototyping platform Arduino 4.1. Introduction For a sustainable future, the energy supply has to shift from using fossil fuels to renewable energies. The transformation of biomass to biogas or bioethanol contributes to a mixture of renewable energies like solar power, wind power or geothermal energy. To maximize the biogas output and degradation of the organic substrates, their biogas yield potential has to be identified. This can have a great variation in quality between different substrates like corn, animal manure or organic wastes depending on the composition of carbohydrates, protein and lipids as well as the structure of the biomass. The estimation is usually carried out by triplicate batch fermentation tests in laboratory scale according to VDI 4630 [75]. Al- though the preparation of the tests is sim- Figure 4.1.: gasUino with controlling unit in the front ple, the correct measurement of the produced biogas can be time consuming and error-prone. The easiest method, the water displacement principle with a graduated cylinder has to be maintained regularly and can have a great variability in the measurements. A special glass apparatus (Eudiometer) is the standard method for estimating the biogas potential of municipal waste. Unfortunately, this is also operated manually. To 51 4. gasUino - biogas flow meter address these problems, researchers started developing laboratory biogas flow meters, based on different designs, which can handle the minor gas flows and are able to record the gathered data to a personal computer [2, 36, 39, 50, 53, 55, 67, 71]. The electronic hardware needed for these devices is normally an in-house development of the different institutes, which makes it difficult to reproduce for other working groups and lab environments. Therefore, we developed the gasUino - a low cost biogas meter based on the open-source electronics platform Arduino, which addresses these problems. The biogas flow meter is based on the liquid displacement principle like a U-shaped manometer, with the main focus of making it easy to assemble and operate, affordable and robust. According to the open hardware spirit, it will be released under an open license. 4.2. Materials and Methods A complete overview of the used materials is given in the Appendix, see table A.1 and table A.2. 4.2.1. Flow meter design The cell for the measurement of the biogas volume is formed by an U-shaped tube (see figure 4.4a). Two serological pipettes (Stripette, 25 ml, Corning, MA) are connected by a PVC tube and filled with a sealing liquid of 75 % NaCl (pH 2) to enhance the conductivity and decrease the gas solubility according to Walker et al. [76]. A 3/2 way rocker solenoid valve (Typ 6606, 12V, bürkert, Ingelfingen, Germany) connects the U-shaped tube with the anaerobic digester and a gas collecting bag (PETP/AL/PE, 20 l, tesseraux, Bürstadt, Germany). In between bioreactor and valve, a gas-washing bottle filled with silica gel removes the water vapor. Two physically separated open graphite electrodes are used as contacts for the switch-like detection mechanism: 1. The valve is not activated and the gas can freely flow from the digester to the Ushaped tube. Due to the developing pressure of the biogas, the liquid column is lifted until the sensor gets in contact with the sealing liquid. 2. Bridging the contacts triggers a 10 second activation of the valve ("click"), which inverts the inlet and outlet of the valve. The weight of the liquid column pushes the biogas into the gas-collecting bag, until the liquid column is balanced again. 52 4. gasUino - biogas flow meter 3. The valve switches back to the start position and the next cycle begins. Depending on the filling level of the sealing liquid in the pipets, the corresponding volume of a click is determined by pushing air into the system with a graduated syringe. The calibration volume is calculated by V calib = V number of clicks clicks The sensor (temperature and air pressure) values and clicks get sent to a web server in a preset interval where the standard volume (0 °C, 1013 hPa) is calculated by Vstandar d [ml] = Vcalib [ml] · clicks · pambient [hP a] · 273.15[K]) 1013[hP a] · (Tambient [ C] + 273.15[K]) and stored in a database for further analyzing and plotting. All components of the biogas flow meter are embedded in a custom designed laser-cut case of acrylic glass (see A.9, Fig. 4.2). 4.2.2. Electronics The gasUino biogas flow meter is built upon the Arduino - an open source electronics prototyping platform based on flexible, easy-to-use hardware and software [4]. Different circuit boards (shields) are available and can be used to extend the possibilities of the Arduino, by means of stacking them together. For ethernet access, the ethernet shield [5] was incorporated. For the simultaneous operation of nine gasUinos, two separate circuit boards were designed and connected with a 40-Pin ribbon cable: 1. The connection shield (see figure 4.3a) provides the power supply (12 V), which drives the solenoid rocker valves and supplies 9 V to the Arduino via a voltage regulator (LM340, STMicroelectronics, Geneva, Switzerland). Up to nine gasUinos can be connected to the board via terminal blocks. 10k⌦ pull-up resistors are used for the sensing mechanism; NPN transistors (TIP102, ON Semiconductors, Phoenix, AZ, US) switch the valves. 2. A LCD Display (162C, Displaytech, Hong Kong), a real time clock (DS1307, Maxim, Sunnyvale, CA, US), the environmental sensors for temperature (LM35, National 53 4. gasUino - biogas flow meter (a) Picture of the assembled gasUino (without electronics) (b) Exploded View Figure 4.2.: gasUino 54 4. gasUino - biogas flow meter (a) batchLab connection shield. Nine flow meters can(b) batchLab mega. A developed shield stacked bebe connected. tween the Arduino mega and the Ethernet shield. Incorporates a temperature and pressure sensor, a LCD Display and tactile buttons. Figure 4.3.: Board layouts for the developed shields. Corresponding circuit diagrams can be found in the Appendix, see A.11 and A.10. Semiconductor, Santa Clara, CA, US) and pressure (MPX4115, Freescale Semiconductor, Austin, TX, US) and three freely programmable tactile switches are soldered on this shield, which is stacked between the Arduino Mega 2560 and the Ethernet shield (see figure 4.3). 4.2.3. Database & Web interface The data acquisition and storage is based on standard technologies. A web application, developed with the PHP framework CodeIgniter [9] is running on an Apache2 web server [6] and MySQL [11] as the relational database. It can be accessed at http://biogas.jacobs-university.de (see fig. 4.4b). In a given interval, the Arduino connects to the server and submits the recorded values with a HTTP-GET command. On the server, a PHP [8] script parses and validates the request and stores the data with a timestamp in the database. With the help of PHP and Gnuplot [40], the data can be interactively retrieved, plotted and exported into a comma separated values file (CSV) for further data processing. For the calculation of the correct standard volume, the calibrated volume for each gasUino is also stored in the database and accessible via the web interface. 55 4. gasUino - biogas flow meter (a) U-shaped tube, sensor head and 3/2 way rocker valve (b) Web frontend for the gasUino / batchLab Figure 4.4. 56 4. gasUino - biogas flow meter The detailed and commented gasUino sketch can be found in the Appendix under section A.1 as well as the SQL statements to create the table structure (A.1). The source code of the PHP application would go beyond the scope of the thesis and is therefore available online for further studying, see [13]. 4.2.4. Fermenter bottle The fermenter is composed of a 2 L glass bottle with a gas-washing bottle fixture. One gas outlet is connected via the gas washing bottle with silica gel to the flow meter. The second outlet forms the inlet for the flexible stirring mechanism. A Norprene Tubing is routed trough the glass tube into the active sludge. It is a very flexible but still strong material to overcome the small radius of the 90º angle (c.f 4.5b). For achieve homogenous conditions via stirring, the end of the tube was folded like an 8 and secured with cable straps (see fig. 4.5a). Due to the flexibility, the stirrer can be removed with the bottle lid for feeding and cleaning. 4.2.5. batchLab fermenter array A heating bath tank with 10 mm wall thickness was built with acrylic glass. The tank can host a thermostat and nine fermenter bottles. On the backside, nine 12V DC motors with 60 rpm are mounted, which power the flexible stirrer inside the bottle. On the front side, a hole was kept to feed the biogas outlet tube through the wall. Floating plastic balls (Ritter Chemie, Ritterhude, Germany) on the surface reduce the evaporation of the water. The flexible stirrer is connected to the motor with a cutting ring fitting. A flexible stirrer axis, which also allows a quick dismantling is formed by three hooks. The motors are powered by a small PCB with terminal blocks and a 12 V connector (c.f. fig. 4.5) and are rotating counter-clockwise to ensure a gas tight connection to the fermenter. 4.2.6. Performance The biogas flow meter setup was tested in a stress test. It was connected to a membrane pump with a high steady flow of air (90 l / d) and the clicks were recorded for 15 days. 57 4. gasUino - biogas flow meter (a) Fermenter array heating bath tank, 10 mm acrylic glass 58 (b) Detail of the mounted motor and the flexible stirring system 4. gasUino - biogas flow meter (a) Fermenter array heating bath tank (b) batchLab fermenter array (a) Circuit diagram (b) Board layout Figure 4.5.: Stirrer Power 59 4. gasUino - biogas flow meter 4.3. Results and Discussion The detection mechanism of the gasUino dictates, that the calibrated volume must not change over time, otherwise the error of the measurement will increase with every click. Although the used sealing liquid of 75% NaCl has a even higher boiling point of 104.9 °C compared to the standard sealing liquid of 0.5 M sulfuric acid [35], evaporation could be observed, resulting in a slight increase of the graduated volume and therefore in a smaller overall biogas yield. This error correlates to the elapsed time as well as the number of detected clicks, and hence the initially calibrated volume. Considering an overall idealized biogas yield of carbohydrates of 0.746 l / g [27], after 28 days, the detected biogas volume for 10 g of microcrystalline cellulose (MCC, positive control) is 7.46 l. With a calibrated volume of 5 ml, 1492 clicks should be detectable during the experiment duration. In the worst case (5.6 ml as the actual volume) this would result in only 1332 clicks, which would equal 6.66 l of biogas and and an underestimation of 10 %. For a calibrated volume of 20 ml, however, the error will decrease (20 ml = 373 clicks, 20.6 ml = 362 clicks; deviation of 3 %). As the loss of sealing liquid is time dependent and the highest activity of biomass degradation takes place in the first 10 - 14 days, this error will even be smaller in a real application. For a comparative study with triplicates according to the VDI 4630 guideline, it is suggested to calibrate the flow meters with a volume greater than 15 ml and furthermore, use the same volume in all parallel flow meters. A stress test over a period of 15 days was done to estimate this error development (see figure 4.6). With a steady flow of around 90 l / day, more than 220,000 clicks could be detected during the experiment time. If the flow of the membrane pump is constant and without evaporation, a straight line was expected. For this, an extrapolation of the first two days was plotted as the reference. Evaporation or other loss of the sealing liquid leads to an increase of the volume, and therefore an expanded time between the clicks, which leads to a truncation of the line. This effect can be seen starting at day 8, after 100,000 clicks have been registered. As mentioned previously, this does not resemble a real fermentation experiment, where dependent on the volume a maximum of 2,000 clicks can be estimated. Overall, the deviance is small. Another fact which has to be taken into consideration is the kinetics of an actual batch fermentation. In figure 4.6b, the biogas development of microcrystalline cellulose, which served as the positive 60 4. gasUino - biogas flow meter 2 105 900 Measurement First 2 days extrapolated 2 105 800 1 105 700 5 600 1 105 500 clicks ml/VS 1 10 A B C D E F G 8 10 4 400 6 10 4 300 4 104 200 4 100 2 10 0 100 0 0 2 4 6 8 time [d] 10 12 (a) Stress test 14 0 2 4 6 8 time [d] 10 12 14 (b) Exemplary results of digestion of microcrystalline cellulose, positive control in batch fermentation tests Figure 4.6.: Stress test of the biogas flow meter and the result of the positive controls with mcc in different batch setups control in all batch fermentations (A - G) is presented. All curves express a sigmoidal form, the greatest changes in the slope can be observed in the first 15 days, where the evaporation as an error source is irrelevant. On a technical side, the stress test also proved that the sensing and valve switching mechanism is robust. The gasUino was furthermore tested in actual fermentation experiments. All biogas volume recordings in this thesis were done with different versions of the biogas counter. It was successfully implemented into a continuous operated biogas fermenter (see chapter 3), a dry batch fermentation setup [37] and standard batch methane potential tests with different novel substrates [16]. 61 5. Conclusion The aim of this work was to develop methods and instruments to decipher the "blackbox" process of anaerobic digestion. This problem was approached from two different directions. As presented in chapter 3, an online sensor system for the volatile fatty acids in the digestate was developed. It confirmed, that ATR-MIR-FTIR-spectroscopy proves to be a good method to monitor the process of anaerobic digestion. The developed PLS models can predict the dynamics of the different volatile acids and their concentration in a satisfying concentration range. The fully automatic evaluation makes it possible to use the system as an alarm to detect arising problems of the bioprocess in an early stage. It can give time to counter steer possible process breakdowns. Likewise, the technology may optimize biogas plants by utilizing unused potentials, due to e.g. low organic loading rates. This is especially interesting for plants operated in thermophilic mode, because a higher process temperature also accelerates biochemical reactions. A continuing study of the application of the sensor at a full scale biogas plant digesting municipal organic waste is carried out at the moment. Despite the obvious advantages for biogas plant operators, it can also help to improve the available mathematical models for the anaerobic digestion process. Integrated in a biogas simulator, these could be used to train plant personnel, prior operating the real plant. In current models, the volatile fatty acids are summarized in one variable, which gets calculated from other input values (feed, pH, gas production). The possibility to measure the concentration of the volatile fatty acids in detail may vastly aid in the optimization of the prediction capabilities of these adapted models. A very simple calculation to estimate the performance of a fermentation is the Buswell Equation. Based on the different fractions of lipids, proteins and carbohydrates, an idealized maximum biogas volume can be calculated. As different substrates exhibit different chemical structures with different degradation constants, a real fermentation has to be performed to really estimate the biogas potential of substrates. In the second project (chapter 4), a biogas meter was developed upon the open hardware platform Arduino, which makes it possible to monitor these little gas flows in laboratory environments. It is not specialized to only monitor the volume flow 62 5. Conclusion in biogas potential tests or continuous setups. Other applications can be the estimation of the residual gas potential of municipal waste before storing it in landfills, or monitoring the carbon dioxide volume of the anaerobic phase of ethanol production due to its adjustable resolution. In contrast to other commercially available products, it is open sourced and low-cost. Two major advantages can be achieved with this approach. It can enable workgroups in the field of anaerobic digestion to upscale their biogas potential tests, due to the low price, and the availability of source code and design files makes it easily possible to adapt the equipment to the specific laboratory needs. In conclusion, this work shows that for the optimization of the anaerobic digestion process, a lot more parameters and their interaction have to be monitored more closely, although this does not necessarily require high-tech equipment to do so. 63 List of Tables 1.1. Examples of fermentation processes from glucose [72] . . . . . . . . . . . 14 1.2. VFA degradation during acetogenesis; G0 ’; T = 25 °C, pH 7, pH2 10-5 atm, pCH4 0.7 atm, c(VFA) 1 mM, HCO3 - 0.1 mM[21, 72] . . . . . . . . . 15 1.3. Methanogenic degradation [33] . . . . . . . . . . . . . . . . . . . . . . . . 16 1.4. Overview of TS and VS percentages of common substrates [10] . . . . . 19 2.1. Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2. Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.1. Summary of the properties of the developed methods for the different volatile fatty acids. 147 samples were collected. 3.2. Frequency ranges [cm-1 ] . . . . . . . . . . . . . . 46 of the different developed methods. . . . . . . . 46 A.1. batchLab materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 A.2. batchLab electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 64 List of Figures 1.1. Past and future CO2 concentrations. Since pre-industrial times, the atmospheric concentration of greenhouse gases has grown significantly. Car- bon dioxide concentration has increased by about 31 %, methane concentration by about 150 %, and nitrous oxide concentration by about 16 % [77]. The present level of carbon dioxide concentration (around 375 parts per million) is the highest for 420 000 years, and probably the highest for the past 20 million years. [56] . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.2. Development of the number of biogas plants and the total installed electric output in megawatt [MW] (as of 06/2011) in Germany [19] . . . . . . . . . 10 1.3. Compensation system EEG 2012 [26] . . . . . . . . . . . . . . . . . . . . 12 1.4. Utilizable energy potential (Hartmann/Kaltsschmitt, 2002, reworked by FNR) [54] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.5. Thermodynamic window of the degradation of the volatile fatty acids [33] . 15 1.6. Two-stage agricultural biogas plant . . . . . . . . . . . . . . . . . . . . . . 21 2.1. Overview of a HPLC System (modified [14]) . . . . . . . . . . . . . . . . . 28 2.2. Schematic setup of a Michelson Interferometer [79] . . . . . . . . . . . . . 31 2.3. Interference of superimposed sinus waves . . . . . . . . . . . . . . . . . . 32 2.4. Schematic representation of the attenuated total reflectance. n1 : Refractive Index of the crystal; n2 : Refractive Index of the sample; n2 < n1 ; dp = Penetration depth, [42] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 65 List of Figures 3.1. Piping and instrumentation scheme of the anaerobic digester and the IRspectrometer. A sample is generated by a rotating filter unit mounted on the stirrer axis. Pump 1 transfers the digestate to the measurement chamber, fitted on top of the ZnSe crystal. Simultaneously, fresh water is transferred to the fermenter to balance the filling level. After the spectra recording is finished, crystal and chamber are cleaned with fresh water via pump 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2. Acrylic glass chamber mounted on top of the ZnSe crystal . . . . . . . . . 39 3.3. Flow chart diagram of the chosen approach developing and validating the different methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.4. Regression plots from the PLS-Models for the different volatile fatty acids. PLS predicted concentrations were plotted against the HPLC reference analyses. All spectra were smoothed and pre-processed with the first or second derivative and different frequency ranges were chosen, respectively (cf. 3.1,3.2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.5. Methods did not include the corresponding HPLC results. a) straight line: pH, dashed line: cumulated biogas volume b) - e) dots: PLS-predicted concentrations. line: smoothed average of PLS-predicted values, points: HPLC reference analysis. Only prediction results are plotted, which passed the internal QUANT2 validity test. The shaded areas highlight the increasing organic loading rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.1. gasUino with controlling unit in the front . . . . . . . . . . . . . . . . . . . 51 4.2. gasUino . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4.3. Board layouts for the developed shields. Corresponding circuit diagrams can be found in the Appendix, see A.11 and A.10. . . . . . . . . . . . . . 55 4.4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.5. Stirrer Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4.6. Stress test of the biogas flow meter and the result of the positive controls with mcc in different batch setups . . . . . . . . . . . . . . . . . . . . . . . 61 A.1. Main block diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 A.2. Frontpanel A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 A.3. Frontpanel B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 66 List of Figures A.3. Loop for continuous data retrieval from Biostat MD, B and gasUino . . . . 84 A.3. Loop for automatic sample measurement with the FTIR . . . . . . . . . . 86 A.4. Data retrieval from Biostat B . . . . . . . . . . . . . . . . . . . . . . . . . . 87 A.5. 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Gas production potential of forage and cereal crops in biogas production. Landtechnik 64, 317–321 (Jan. 2009). 79. Wikipedia. Michelson-Interferometer — Wikipedia, Die freie Enzyklopädie [Online; Stand 20. November 2011]. 2011. <http://de.wikipedia.org/w/index.php? title=Michelson-Interferometer&oldid=93057041>. 80. Zhang, M.-L. et al. Rapid and accurate determination of VFAs and ethanol in the effluent of an anaerobic H2-producing bioreactor using near-infrared spectroscopy. Water Research (Jan. 2009). 81. Zumbusch, P., Meyer-Jens, T., Brunner, G. & Märkl, H. On-line monitoring of organic substances with high-pressure liquid chromatography (HPLC) during the anaerobic fermentation of waste-water. Applied Microbiology and Biotechnology 42, 140–146 (1994). 74 A. Appendix A.1. Arduino sketch sourcecode #include <Ethernet.h> #include <EthernetDHCP.h> #define interval 10000 //modify = noOfCounters //Ethernet// int noOfCounters = 9; byte mac[] = { int counter[9]; 0x90, 0xA2, 0xDA, 0x00, 0x35, 0x9D }; //printed on Ethernet Shield byte server[] = { //Sensors 212,201,46,122 }; // biogas.jacobs-university.de int gasPins[]={ 49,45,25,24,28,32,40,44,48}; const char* ip_to_str(const uint8_t*); //Voltage for sensors //LCD// int voltagePins[]={ #include <LiquidCrystal.h> 41,22,33}; // initialize the library with the numbers of the interface pins LiquidCrystal lcd(12, 11, 9, 8, 7, 6); char buf[6]; long unsigned displayTime; #define lcdInterval 3000 Client client(server, 80); //DS1307// #include <WProgram.h> // clock #include <Wire.h> //clock #include <DS1307.h> int dateTime[6]; //Sensors// #define refVoltage 5 #define tempPin 10 #define pressurePin 8 float pressure; float temperature; //Transistor pins int transistors[]={ 47,29,23,26,30,34,42,46,27}; //Transistor on/off int OnOff[]={ 0,0,0,0,0,0,0,0,0}; long unsigned milliDelay[9]={ 1,1,1,1,1,1,1,1,1}; //buttons int buttons[3]={ 14,15,16}; void setup() { EthernetDHCP.begin(mac, 1); Serial.begin(9600); delay(2000); RTC.stop(); RTC.set(DS1307_SEC,00); RTC.set(DS1307_MIN,46); RTC.set(DS1307_HR,9); RTC.set(DS1307_DOW,3); RTC.set(DS1307_DATE,20); RTC.set(DS1307_MTH,7); RTC.set(DS1307_YR,11); RTC.start(); //token=which batchlab/gasUino array #define token "batchLabB" // Serial String serialString; long lastConnectionTime = 0; boolean lastConnected = false; #define postingInterval 60000 #define deltaTInterval 1000 //set the voltagePins to Output and HIGH = 5V for (int i=0; i<3; i++) { pinMode(buttons[i], INPUT); pinMode(voltagePins[i], OUTPUT); digitalWrite(voltagePins[i], HIGH); } long unsigned last_tm[9]; long unsigned delta_Interval =0; 75 A. Appendix //init LCD lcd.begin(16,2); lcd.print("batchLab mega 2.0"); delay(500); lcd.scrollDisplayLeft(); delay(500); displayTime = millis(); milliDelay[i]=millis(); switch_transistor(i);// increase_counter(i); if (digitalRead(gasPins[i]) == LOW) { OnOff[i]=1; last_tm[i]=millis(); } } } void loop() { static DhcpState prevState = DhcpStateNone; static unsigned long prevTime = 0; updateDisplay(); DhcpState state = EthernetDHCP.poll(); if(!client.connected() && (millis() - lastConnectionTime > postingInterval)) { updateDB(); } lastConnected = client.connected(); if (prevState != state) { Serial.println(); } switch (state) { case DhcpStateDiscovering: Serial.print("Discovering servers."); break; case DhcpStateRequesting: Serial.print("Requesting lease."); break; case DhcpStateRenewing: Serial.print("Renewing lease."); break; case DhcpStateLeased: { const byte* ipAddr = EthernetDHCP.ipAddress(); const byte* gatewayAddr = EthernetDHCP.gatewayIpAddress(); const byte* dnsAddr = EthernetDHCP.dnsIpAddress(); lcd.clear(); lcd.setCursor(0,0); lcd.print("My IP address is "); lcd.setCursor(0,1); lcd.println(ip_to_str(ipAddr)); delay(2000); lcd.setCursor(0,0); lcd.print("Gateway IP Adress: "); lcd.setCursor(0,1); lcd.println(ip_to_str(gatewayAddr)); delay(2000); Serial.print("DNS IP address is "); lcd.setCursor(0,0); lcd.print("DNS IP Adress: "); lcd.setCursor(0,1); lcd.println(ip_to_str(dnsAddr)); delay(2000); break; } void reset_gas(int i){ if (digitalRead(buttons[0]) == HIGH) { counter[i]=0; } } void switch_transistor(int i){ if (OnOff[i] == 1 && millis() - last_tm[i] <= interval){ digitalWrite(transistors[i], HIGH); } else if (OnOff[i] == 1 && millis() - last_tm[i] >= interval){ digitalWrite(transistors[i], LOW); OnOff[i]= 0; milliDelay[i]= 0; } } void increase_counter(int i){ if (milliDelay[i] == 0){ counter[i]++; } } float get_temperature() { int span = 25; int averageLM35 = 0; float temperature = 0; for (int i = 0; i < span; i++) { //loop to get average of 20 readings averageLM35 = averageLM35 + analogRead(tempPin); } averageLM35 = averageLM35 / span; //convert the analog data to a temperature temperature = (refVoltage * averageLM35 * 100.0)/1024.0; return temperature; } } } prevState = state; float get_pressure() { float pressure; pressure = ((analogRead(pressurePin)/1024.0 + 0.095))/0.009; return pressure; } for (int i=0; i<noOfCounters; i++) { void updateDisplay(){ 76 A. Appendix int dateTime[6]={ RTC.get(DS1307_YR,true), RTC.get(DS1307_MTH,false), RTC.get(DS1307_DATE,false), RTC.get(DS1307_HR,false), RTC.get(DS1307_MIN,false), RTC.get(DS1307_SEC,false) }; else if (millis() - displayTime > (lcdInterval*4) && millis() - displayTime < (lcdInterval*5)) { lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 4: "); lcd.print(counter[3]); lcd.setCursor(0,1); } // Loop to rotate date, sensors and gasUino results on display if (millis() - displayTime < lcdInterval){ lcd.clear(); lcd.setCursor(0,0); if (dateTime[3] < 10) { lcd.print("0"); } lcd.print(dateTime[3]); lcd.print(":"); if (dateTime[4] < 10) { lcd.print("0"); } lcd.print(dateTime[4]); lcd.print(":"); if (dateTime[5] < 10) { lcd.print("0"); } lcd.print(dateTime[5]); else if (millis() - displayTime > (lcdInterval*5) && millis() - displayTime < (lcdInterval*6)) { lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 5: "); lcd.print(counter[4]); lcd.setCursor(0,1); } else if (millis() - displayTime > (lcdInterval*6) && millis() - displayTime < (lcdInterval*7)) { // lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 6: "); lcd.print(counter[5]); lcd.setCursor(0,1); } lcd.setCursor(0,1); lcd.print(get_temperature()); lcd.write(223); lcd.print("C"); lcd.print(" "); lcd.print((get_pressure() * 10)); lcd.print(" hPa"); lcd.setCursor(0,0); } else if (millis() - displayTime > (lcdInterval) && millis() - displayTime < (lcdInterval*2)) { lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 1: "); lcd.print(counter[0]); lcd.setCursor(0,1); } else if (millis() - displayTime (lcdInterval*2) && millis() lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 2: "); lcd.print(counter[1]); lcd.setCursor(0,1); } else if (millis() - displayTime (lcdInterval*3) && millis() lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 3: "); lcd.print(counter[2]); lcd.setCursor(0,1); } > displayTime < (lcdInterval*3)) { else if (millis() - displayTime (lcdInterval*7) && millis() lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 7: "); lcd.print(counter[6]); lcd.setCursor(0,1); } else if (millis() - displayTime (lcdInterval*8) && millis() lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 8: "); lcd.print(counter[7]); lcd.setCursor(0,1); } else if (millis() - displayTime (lcdInterval*9) && millis() lcd.clear(); lcd.setCursor(0,0); lcd.print("gUo 9: "); lcd.print(counter[8]); lcd.setCursor(0,1); } else { displayTime = millis(); } > displayTime < (lcdInterval*8)) { > displayTime < (lcdInterval*9)) { > displayTime < (lcdInterval*10)) { } > int process_values(){ displayTime < (lcdInterval*4)) { char ch = ’0’; if (Serial.available() > 0) { char ch = Serial.read(); if (ch == ’v’) //pv = process values { Serial.print(get_temperature()); Serial.print(","); Serial.print(get_pressure()*10); 77 A. Appendix Serial.print(","); client.print(counter[5]); client.print("&f7="); client.print(counter[6]); client.print("&f8="); client.print(counter[7]); client.print("&f9="); client.print(counter[8]); client.print(" "); client.println("HTTP/1.0\r\n"); client.println("HOST: 212.201.46.122"); lastConnectionTime = millis(); client.stop(); for (int i=0; i<noOfCounters; i++) { Serial.print(counter[i]); Serial.print(","); } Serial.println(""); } } } const char* ip_to_str(const uint8_t* ipAddr) { } static char buf[16]; else { sprintf(buf, "%d.%d.%d.%d\0", ipAddr[0], ipAddr[1], ipAddr[2], ipAddr[3]); lcd.println("connection failed"); return buf; } } } void updateDB (){ if (client.connect()) { client.print("GET /index.php/batchlab/insert?& key=qwertzui123456!&token="); client.print(token); client.print("&temperature="); client.print(get_temperature()); client.print("&pressure="); client.print(get_pressure()*10); client.print("&f1="); client.print(counter[0]); client.print("&f2="); client.print(counter[1]); client.print("&f3="); client.print(counter[2]); client.print("&f4="); client.print(counter[3]); client.print("&f5="); client.print(counter[4]); client.print("&f6="); A.2. SQL statements -- ---------------------------------------------------------- Table structure for table -- ‘configuration‘ CREATE TABLE IF NOT EXISTS ‘configuration‘ ( ‘id‘ int(11) NOT NULL AUTO_INCREMENT, ‘token_id‘ char(12) COLLATE utf8_unicode_ci NOT NULL, ‘fermenter_id‘ int(11) NOT NULL, ‘experiment_id‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘volume_calib‘ float NOT NULL, ‘description‘ tinytext COLLATE utf8_unicode_ci NOT NULL, PRIMARY KEY (‘id‘) ) ENGINE=MyISAM DEFAULT CHARSET=utf8 COLLATE=utf8_unicode_ci AUTO_INCREMENT=40 ; -- -------------------------------------------------------- 78 A. Appendix --- Table structure for table -- ‘sensor‘ CREATE TABLE IF NOT EXISTS ‘sensor‘ ( ‘id‘ int(11) NOT NULL AUTO_INCREMENT, ‘timestamp‘ timestamp NOT NULL DEFAULT CURRENT_TIMESTAMP, ‘token_id‘ char(12) COLLATE utf8_unicode_ci NOT NULL, ‘temperature‘ float NOT NULL, ‘pressure‘ float NOT NULL, PRIMARY KEY (‘id‘) ) ENGINE=MyISAM DEFAULT CHARSET=utf8 COLLATE=utf8_unicode_ci AUTO_INCREMENT=1352224 ; -- ---------------------------------------------------------- Table structure for table -- ‘users‘ CREATE TABLE IF NOT EXISTS ‘users‘ ( ‘id‘ int(11) NOT NULL AUTO_INCREMENT, ‘email‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘password‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘salt‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘hash‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, PRIMARY KEY (‘id‘) ) ENGINE=MyISAM DEFAULT CHARSET=utf8 COLLATE=utf8_unicode_ci AUTO_INCREMENT=3 ; -- ---------------------------------------------------------- Table structure for table -- ‘volume‘ CREATE TABLE IF NOT EXISTS ‘volume‘ ( ‘id‘ int(11) NOT NULL AUTO_INCREMENT, ‘sensor_id‘ int(255) NOT NULL, ‘timestamp‘ timestamp NOT NULL DEFAULT CURRENT_TIMESTAMP, ‘fermenter_id‘ int(11) NOT NULL, ‘experiment_id‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘token_id‘ varchar(255) COLLATE utf8_unicode_ci NOT NULL, ‘clicks‘ int(11) NOT NULL, ‘volume_nl‘ float NOT NULL, PRIMARY KEY (‘id‘) ) ENGINE=MyISAM DEFAULT CHARSET=utf8 COLLATE=utf8_unicode_ci AUTO_INCREMENT=740467 ; A.3. LabVIEW Virtual Instruments 79 A. Appendix Figure A.1.: Main block diagram 80 A. Appendix Figure A.2.:81 Frontpanel A A. Appendix Figure A.3.: Frontpanel B 82 A. Appendix (a) Request data from Biostat MD (c.f. A.5) (b) 500 ms wait 83 (c) Retrieve data from Biostat B (c.f. A.4) A. Appendix (d) Get data from gasUino and correct to standard conditions (e) Insert the data in the SQL-Database 84 (f) Update the plots on the front panel (c.f. A.2) Figure A.3.: Loop for continuous data retrieval from Biostat MD, B and gasUino A. Appendix (a) Reverse the multichannel pump, set speed to 50 (b) Start the cleaning of the crystal by starting the cleaning pump, setting power socket to ON (c) Start multichannel pump, wait for 20 sec (d) Stop the cleaning pump, setting power socket to OFF (e) Stop the multichannel pump (f) Wait until the next measurement begins 85 (g) Start the cleaning of the crystal by starting the cleaning pump, setting power socket to ON (h) Start multichannel pump, wait for 20 sec A. Appendix (i) Stop the cleaning pump, setting power socket to OFF (j) Stop the multichannel pump (k) Start the background measurement (l) Set the multichannel pump to a slower velocity and change direction (m) Transfer a sample from the fermenter onto the crystal, start the multichannel pump for 3 minutes (n) Stop the multichannel pump 86 (o) Start the IR-measurement and analyzing of the spectra (c.f. A.8) (p) Update the plots on front panel A (c.f. A.2) Figure A.3.: Loop for automatic sample measurement with the FTIR A. Appendix Figure A.4.: Data retrieval from Biostat B 87 A. Appendix Figure A.5.: Data retrieval from Biostat MD Figure A.6.: Control of the USB Power Socket 88 A. Appendix Figure A.7.: Assembly of the SQL input string 89 A. Appendix Figure A.8.: Control of the OPUS Software; Measuring and Analyzing of the IR-spectra 90 Name Acrylic Glass Tank Custom designed CostarÆ Stripette-Pipette Flask, 2000ml Floating plastic balls Gas collection bags, 20l Gas wash bottle Gas wash bottle gasUino Acrylic Glass Housing Lab-Ring, open Norprene Tube 3.2x1.6 PVC Tubing 3mm x 1mm Reducing Adapter, 3-5 / 6-10 mm, 55 mm length Silicagel, colour indicator orange Silicon Tube 4mm x 1.5mm Slotted cheesehead steel screw M2.5x20mm Spacer 8x3mm round Stirring Motor 12V / 60 rpm Thermostate Julabo ED Zinc plated steel hexagon full nut, M2.5 Sinker Stirrer Tubes for gasUino Connecting Fermenter / Gas collecting bag Gas drying Input for second electrode Mounting PCB and Stirring Motors Mounting PCB and Stirring Motors Stirrer Waterbath Heating Mounting PCB and Stirring Motors Gas drying Function Heating bath and Stirrer Mound U-Tube Fermenter reduce evaporation Manufacturer CD Thalau Corning Simax Ritter Chemie tesseraux Lenz Laborglasinstrumente Lenz Laborglasinstrumente formulor Kleinfeld Labortechnik Tygon DEUTSCH & NEUMANN Brand Kraemer and Martin DEUTSCH & NEUMANN RS Components RS Components Philips Julabo RS Components Table A.1.: batchLab materials Distributor CD Thalau Omnilab Omnilab Omnilab tesseraux Omnilab Omnilab formulor Omnilab Carl Roth Omnilab Omnilab Omnilab Omnilab RS Components RS Components RS compontents Omnilab RS Components 5424053 AH62.1 5205358 9207336 5042552 5205266 546-6146 102-6205 336-315 5422645 560-287 Order Number 11/025 5380583 5072336 5003390 35005000600 6073489 / 5530058 6331669/5530158 A. Appendix 91 A. Appendix Figure A.9.: Laser-cut holder for the gasUino, 5 mm acrylic glass; Dimensioning in mm. 92 Figure A.10.: Circuit diagram: batchLab connection shield A. Appendix 93 A. Appendix Figure A.11.: Circuit diagram: batchLab connection shield 94 Name 16x2 reflective STN yel/grn, 80x36 3310Y conductive plastic pot,10K lin 9mm 34 way IDC strain relief 34way polarised skt w/o strain relief 36way header, 5.7mm, 7.6mm,size3 3mm Easy Break Terminal Strip 4 conductor screened cable, 30m 6x6mm tactile switch, 5mm H 1.6N Arduino Ethernet Shield Arduino Mega 2560 Arduino Stackable Header Kit batchlab connection shield 4.3a batchlab mega 4.3 batchlab stirrer power 4.5 Cable 2/20 Cable 2/20 Capacitor 1uF Carbon Resistor, 0.25W ,5%, 10k Carbon Resistor, 0.25W ,5%, 1k Ceramic Capacitor 470 pF Cover Nut, M3 CR2032 Lithium Coin Cell 3V Crystal 32.768KHz 3x8mm Cutting ring fitting, 6mm -> 4mm Diode 1N4004-E3 IC LM35 DZ Intos Patchcable Cat 5e 10m Magnetic Valve 3/2 way, 12V, Typ 6606 MPX5114A NPN darlington transistor, TIP102G 8A PCB mount DC power socket 2.1mm 1A 12V Power Supply 12V, 2.08A, 25W Radial Ceramic capacitor 330nF 50Vdc Radial Z5U ceramic cap, 100nF 50V 5mm Real time clock, DS1307 56B RAM DIP8 Ribbon Cable 34way Slotted cheesehead steel screw M2.5x20mm Slotted cheesehead steel screw M3x16mm Spacer 8x3mm round Surface mount PCB coin cell holder,20mm Terminal Block MKDS 1/12-3.81 Terminal Block MKDS 1/6-3.81 Threaded Bar M3 Voltage Regulator 9V 2A TO220 Zinc plated steel hexagon full nut, M2.5 Zinc plated steel hexagon full nut, M3 95 Mount PCB 12V –> 9V Mounting PCB and Display Mounting PCB and Display Mounting PCB and Display Mounting PCB and Display Mounting PCB and Display Battery Holder Connecting the wires Voltage Regulator Voltage Regulator Clock Pressure Sensor Switching Valves Temperature Sensor LAN Switch Ethernet Connectivity Central Processing Board Connecting PCBs PCB PCB PCB Sensor Sensor for pressure sensor Sensor for Transistors for pressure sensor Mount PCB Buffer for Clock Clock Connecting Stirrer to Motor Connecting PCBs Function Display Adjusting LCD Brightness Vishay National Semiconductor Intos bürkert Freescale ON Semiconductor RS Components Mean Well Murata Murata Maxim Speedbloc RS Components RS Components RS Components Keystone Phoenix Contact Phoenix Contact Graupner STMicroelectronics RS Components RS Components Alpha Wire Alpha Wire RS Components RS Components RS Components Epcos Toolcraft RS Components Fox Electronics Brand Displaytech Bourns TE Connectivity TE Connectivity RS Components RS Components Alpha Wire TE Connectivity Arduino.cc Arduino.cc Sparkfun Distributor RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components TinkerSoup RS Components TinkerSoup Q-Print electronics Q-Print electronics Q-Print electronics RS Components RS Components RS Components RS Components RS Components RS Components Conrad Electronics RS Components RS Components Rauh Hydraulik RS Components RS Components amazon bürkert RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components RS Components Conrad Electronics RS Components RS Components RS Components Table A.2.: batchLab electronics 111-9183 111-9183 521-2315* 707-7745 707-7666 211-4729* 521643-62 597-201 547-6985 170272 628-9029 533-5907 B000AGETB0 137-782 2509678637* 545-0494 448-382 721-2269 721-5278 652-9995 540-2726 289-9931 546-6146 560-782 102-6205 219-7954 648-7891 220-4377 261494-62 686-9751 560-287 560-293 Order Number 532-6408 522-0625 454-2390 454-2413 251-8339 716-7346 111-9048 479-1413 131 / DEV-09026 715-4084 370 / PRT-10007 A. Appendix Nomenclature ⌫¯ Wavenumber [cm-1 ] Wavelength µl mikro Liter AL Aluminum Arduino Arduino is a tool for making computers that can sense and control more of the physical world than your desktop computer. It is an open-source physical computing platform based on a simple micro controller board, and a development environment for writing software for the board [4] ATR Attenuated Total Reflectance batchLab Nine fermenters and gasUinos combined in one array C2 Acetic acid C3 Propionic acid CH4 Methane CHP Combined heat- and power plant click The sensors of the gasUino get in contact with the sealing NaCl solution. The conductivity is sensed and the valve is activated, which reverts inlet and outlet. The weight of the liquid column pushes the biogas into the gasbag, until the liquid column is balanced again. CO2 Carbon dioxide CSTR Continuous Stirred Tank Reactor 96 A. Appendix CSV Comma separated values; A file format used as a portable representation of a database. Each line is one entry or record; the fields in the record are separated by commas. This format is often used to import data into spreadsheet software. DDE Dynamic Data Exchange EEG Erneuerbare Energien Gesetz - Renewable Energy Law Ethernet A system for connecting a number of computer systems to form a local area network, with protocols to control the passing of information and to avoid simultaneous transmission by two or more systems FOM Content of fermentable organic matter FTIR Fourier-Transformed Infra-Red gasUino biogas flow meter based on the open-source electronics prototyping platform Arduino GC Gas chromatography H2 S Hydrogen sulfide HRT Hydraulic Retention Time [d] HTTP Hypertext Transfer (or Transport) Protocol, the data transfer protocol used on the World Wide Web HTTP-GET Requests a representation of the specified resource iC4 Iso-butyric acid iC5 Iso-valeric acid KWK Kraft-Wärme-Kopplung MIR Mid Infra Red NaCl Sodium Chloride NaWaRo Nachwachsende Rohstoffe - primary renewable products 97 A. Appendix nC4 Butyric acid nC5 Valeric acid NH3 Ammonia OLR Organic Loading Rate [g VS l-1 d-1 ] PE Polyethylen PETP Polyethylene terephthalate pH potentia Hydrogenii PHP Hypertext Preprocessor PLS Partial Least Squares PVC Poly Vinyl Chloride RMSECV Root Mean Square of Cross Validation RMSPE Root Mean Square Prediction Error Shield Shields are boards that can be plugged on top of the Arduino PCB extending its capabilities. The different shields follow the same philosophy as the original toolkit: they are easy to mount, and cheap to produce. Sketch A sketch is the name that Arduino uses for a program. It’s the unit of code that is uploaded to and run on an Arduino board SQL Structured Query Language Substrate Biomass, which can be digested to biogas substrate biomass, which can be fermented TS Total solids (after drying at 105 °C for 24 h) U-shaped tube Two serological pipettes are connected by a PVC tube VDI Verein Deutscher Ingenieure 98 A. Appendix VFA Volatile Fatty Acids VI Virtual Instrument VS Volatile solids (after combustion at 550 °C for 24 h) ZnSe Zinc Selenide 99 Acknowledgements I would like to thank ... ... Prof. Dr. Dr. hc. Roland Benz for giving me the opportunity to do my PhD thesis under his kind supervision and for all the support throughout the last years. all remaining members of my PhD committee, Prof. Dr.-Ing. Volker C. Hass, Prof. Dr. Laurenz Thomsen and Prof. Dr. Mathias Winterhalter. ... my diploma students Tobias Dörr and Yann Barbot for the exceptional collaboration. ... Dr. Peter Reichling and Dr. Christian Andersen for introducing me into the scientific work and their enduring interest in "our" work. ... the technicians in Würzburg, Albert Gessner, Marcus Behringer and Willi Bauer, the technicians from the Bremen, Maik Dreßel, Michael Hofbauer, Stefan Baltrusch, Rene Popp and CD Thalau. ... my former colleagues in Würzburg and our current workgroup in Bremen. ... the Internet, especially all developers and makers behind the Arduino Platform. ... my family and friends. ... my Grandmother. ... Sebastian. 100