The growing demand for efficient systems for the production of methane from biomass requires the developing of control strategies assuring the best performance and stability under a flexible operation. The biogas process is complex in which uncountable and relatively slow microbial reactions occur and still presents problems to be predicted and controlled it. In large-scale biogas plants the control of feeding to the digester is often done with a simple calculation after applying the trial and error method (Gaida et al., 2011). Although there have been also some attempts to determine the optimal operating conditions of the process using the ADM1 (Batstone et al., 2002), the model retains high rigidity and can only be applied with equal complex techniques. Therefore, the ADM1 seems to be too complex for its application with respect to the usual monitoring capabilities in biogas plants. An anaerobicdigestion model can associate all the processes and reactions within the system through mathematical equations, variables and parameters to achieve a real process. The models include the relevant information of process to realistically describe the system; such as the characterization of substrates, effects of dilution, temperature or hydraulic behavior among others. The new AM2 developed in this thesis, was written in Aspen Custom Modeler (ACM), which is a simulation program to easily execute customized models. The objective of this program is that it can be used as a tool in BGP’s and at the same time can be used to maintain the stability of the system.
Hydrothermal carbonization (HTC) is a promising technology to convert highly wet biogenic residues into a solid product similar to lignite. Most studies focus on maximizing the solid yield and on various solid product applications. However, as HTC takes place in water, intermediates dissolve in the liquid phase resulting in a carbon loss of the feedstock leaving behind a highly contaminated process liquor. This study investigated the use of HTC process liquor as feedstock for anaerobicdigestion (AD). HTC process liquor is rich in common AD metabolites like organic acids but can also contain inhibiting compounds like phenol. Therefore, the effect of varying HTC process parameters and the use of phenol as sole carbon source for AD were also investigated. The first AD experiments compared two different reactor types treating HTC process liquor for 13 weeks at mesophilic conditions. An anaerobic filter (AF) proved to be more stable than a conventional stirred-tank reactor. Based on these results, AD experiments continued using two larger AFs operated at different temperatures. The degradation of organic compounds was still effective when increasing the organic loading rate up to 5 g COD L − 1 d − 1 and similarly decreasing the hydraulic retention time to five days. Thermophilic AD at an increased temperature had no distinct effect on degradation efficiency nor on methane production. Using liquid condensate from a steam-derived HTC process was also very effective in both AFs. However, a longer start- up of 15 days was necessary and phenol degradation was negatively influenced by an increased digestion temperature.
One technology option that is suitable for Southeast Asian countries is anaerobicdigestion (AD) which produces gas that can be used for cooking, generating heat for drying, and electric power. Using rice straw for AD can produce from 60 to 180 l of methane per kg of dry rice straw ( Lubken et al. , 2010 ; Mussoline et al. , 2013 ). However, the lack of knowledge on rice straw supply chains and utilization options mean that farmers are limited in their capacity to utilize this biomass for energy production, and thus they often burn rice straw in the field. For this reason, the current study was conducted to focus on the energy balance analysis of the supply chain from harvesting to storage of rice straw for use in AD. This research aims to contribute knowledge that will improve the sustainability of rice production systems.
Propionic acid (PA) is one of the most important and commercially valuable volatile fatty acids (VFA), which is extensively utilized in many industrial sectors such as food, pharmaceutical, medical, cosmetics and detergents (Border et al., 1987; Martínez-Campos & de la Torre, 2002; Morales et al., 2006). It can be obtained from a variety of sources and production methods (Ahmadi et al., 2017) but to date is mainly derived from fossil sources. Nevertheless, PA-production could be achieved in a sustainable manner if accomplished using renewable resources or even biomass waste and biological processes (Chen et al., 2013a; Li et al., 2016). Among the available biological methods, anaerobicdigestion (AD) is relatively simple and was suggested to have a high potential for further development (Eryildiz et al., 2020; Esteban-Gutiérrez et al., 2018; Shi et al., 2019). However, the low productivity of PA produced through AD limit its commercialization as it competes with petrochemical production methods and entails increased costs of PA separation and recovery from the fermentation broth. However, several techniques for PA recovery from different aqueous solutions and fermentation broths have been proposed in the last years including reactive extraction, membrane systems, electrodialysis, adsorption, and distillation (Vidra & Németh, 2018).
After ensiling, subsamples were taken from every barrel for dry matter (DM) and ash determination, as well as for elemental and fiber analysis. Remaining silage was treated with the IFBB technique. This involved mixing the biomass with warm water (40 ◦ C) at a 1:4 ratio (material/water, on a fresh matter basis) in a modified concrete mixer heated with natural gas burners and stirred for 15 min at constant temperature. Subsequently, the material was squeezed by a screw press (type AV, Anhydro Ltd., Kassel, Germany) with a pitch of 1:6 and a rotation speed of 6 rpm, resulting in a press cake and a press fluid. The cylindrical screen encapsulating the screw had a perforation of 1.5 mm. Press fluids were frozen at − 20 ◦ C for later anaerobicdigestion. Dry matter, ash, and element concentrations in the press cake were determined according to the procedure applied for silage.
Anaerobicdigestion is a source of renewable energy that operates by putting organic waste in to a tank and allowing microbes to break down the organic material. One of the outputs from this process is biogas, which contains a significant amount of energy and can be burnt in a CHP (Combined Heat & Power) unit for creating both electricity and heat. The use ofanaerobicdigestion is of further interest, because unlike solar and wind power sources, it is not dependent upon the weather. A typical anaerobicdigestion plant will have the ability to store gas for a short time, and furthermore, biogas production can be controlled by varying the feeding amounts and times. This allows the system to be used as a renewable energy source, over which the plant operator has control over the time at which biogas is produced. A CHP unit running on biogas could be operated in a similar way as the gas turbines used at the time of writing. This would allow anaerobicdigestion to provide a renewable energy source that is capable of contributing to the balancing of the energy grid. Furthermore, generally the weather can be predicted with a high degree of accuracy over a short time, and electricity consumption trends are largely predictable using historical data. It would be possible in the future that a biogas plant operator can anticipate an increase of demand a day in advance for example, and feed the biogas plant an increased amount ofbiomass before the electrical demand increases. This will produce additional biogas as it is required, and will allow the plant operator to generate more electricity and support other renewable energy sources.
Lignocellulosic biomass has great potential to be used as a substrate for biogas production, although there are still difficulties concerning the efficiency due to the high complexity of the material (Sawatdeenarunt et al., 2015). Lignocellulose, which is composed out of cellulose, hemicellulose and lignin, is present in all plant derived substrates, which are mainly energy crops, harvest residues and organic waste from private gardens. The proportion of garden residues in municipal household waste depends strongly on the type of housing of the collecting area and varies seasonally (Hanc et al., 2011; Illmer and Gstraunthaler, 2009). In urban settlements grass, leaves and wood make up minor parts of the bio-waste and the composition does not vary much throughout the year, whereas the composition of bio-waste from areas with individual family housing is affected by seasonal garden activities (Hanc et al., 2011). Plant dry substance contains in average 80 % of lignocellulose, which is made up out of cellulose, hemicellulose and lignin (Reineke and Schlömann, 2007). However, in an anaerobicdigestion process the two main components, which cause a low biodegradability, are microbial cells and lignocellulosic material (Carlsson et al., 2012). Lignin is water insoluble and shows a high resistance against microbial degradation and therefore substrate pre-treatment methods were developed to break up lignocellulose and increase the availability of the cellulose before the anaerobicdigestion (Carlsson et al., 2012; Hendriks and Zeeman, 2009).
The microbial community is strongly influenced by parameters like substrate, inoculum and environmental growth conditions. The first stage is dominated by bacteria like Bacteroidetes, Firmicutes and Proteobacteria, however, changing process conditions and substrates promote the enrichment of various microorganisms [3,27]. Among them, anaerobic fungi like Neocalimastix, Piromyces and Orpinomyces play an important role in hydrolysis and fiber degradation due to a strong multi-enzyme system to degrade cellulosic material [3,28]. Moreover, aerobic fungi like white-rot fungi are common for digestionof lignocellulosic biomass . In the hydrolysis stage, different metabolic types of fermentation have been identified with their bacterial key players. Butyric type fermentation with high production of acetic acid, butyric acid and hydrogen with dominance of Clostridium is considered best for two-stage AD and can be influenced by process parameters as pH, organic loading rate (OLR), oxidation–reduction potential  and hydraulic retention time (HRT) [30,31]. The influence of OLR seems to vary with the substrate, but various groups found that pH > 5.0 favors butyric type fermentation, whereas a lower pH leads to dominance of Lactobacilli producing lactic acid or ethanol metabolism [27,29,32]. Ethanol-type fermentation is observed mostly at pH 4.0–4.5 and may feature high gas production [27,33]. More alkaline pH favors production of acetic acid while inhibiting growth of methanogens . Propionic acid is another mayor fermentation product. Depending on the substrate, it can reach a proportion of 20–40%, predominantly in AD of protein-rich waste as sunflower oil cake  or waste activated sludge . It has been found that in the AD of carbohydrate-rich substrate, acetate and propionate are dominant at mesophilic temperature and acidic pH, whereas the fermentation shifts to butyrate at thermophilic AD [34,37]. While the fermentation type is always substrate-dependent, the pH plays a significant role in selecting the fermentation type and the distribution of SCCA [27,34,38]. Since butyric type fermentation is considered best for two-stage AD, pH regulation towards pH 5.0–6.0 in the hydrolysis stage plays a significant role for total process performance.
Besides organic residues, biomass from aquatic plants and algae has also been reported as potential feedstocks for biogas production. These feedstocks, generally considered as advanced or third generation feedstock, due to high biomass yield potentials caused by rapid growth and high photosynthetic efficiency, high diversity, no need for fertile agricultural land for cultivation and thus, no direct competition with food production [28-30]. Also, these feedstocks have low lignin concentration which impedes microbial digestion in terrestrial feedstocks. The AD of water based feedstocks is still in infancy and faces many challenges such as economical production and process instability due to high protein, lipid, sulphur, polyphenol, halogen or saline concentrations [28, 31]. Furthermore, the chemical composition and thus the methane potential and optimal harvest time vary with season and location .
One 2.0 L semi-continuous stirred tank reactor (S-CSTR) with a working volume of 1.5 L, operated at 37°C with 20 days hydraulic retention time (HRT) was set up to study the anaerobicdigestionof freeze dried Spirulina. The overall experiment lasted 440 days which included a 33 days adaptation to Spirulina and a 71 days start-up period. The remaining 336 days were divided into 5 periods (P-I to P-V) in which the organic loading rate (OLR) was gradually increased from 1.0 g to 5.0 g Spirulina L -1 day -1 (dry weight). The inoculum was obtained from a local wastewater treatment plant (Heepen Klaerwerk, Bielefeld, Germany) and the substrate, freeze dried Spirulina, was acquired from Sonnenmacht GmbH (Germany). Biogas production was measured with an on-line Milligascounter MGC-1 equipped with the Rigamo software v3.0 (Ritter Engineering, Germany) and normalized to standard conditions (0 °C; 1.0 atm). pH and redox potential were monitored, but not controlled, with Mettler Toledo pH (HA405-DPA-SC-S8/225) and redox (Pt4805- DPA-SC-S8/225) probes (Mettler Toledo GmbH, Germany). Mesophilic conditions were obtained with a Pt-1000 temperature sensor and a heater. In order to avoid rupture of the bacterial granules, constant stirring was performed with a floating magnet (Fisher Scientific GmbH, Germany). Daily purge and feed were performed manually with a syringe. Before purging, the biomass was settled by stopping the stirring for at least 30 minutes. Periodically the purged sludge was sampled for analysis; in that case the stirring was not stopped. The medium used to dissolve the freeze dried Spirulina for dosing at the desired OLR was modified after Vidal et al.  excluding the NH 4 Cl.
sample alignments when Multicorer cores were used and heterogeneity of the sediment (Treude et al., 2003), because the variation of the three parallel samples did not reflect such a heterogeneity on a centimeter scale as to explain the differences in rates. It has also been proposed that the methane loss during coring is responsible for an underestimation of in situ methane concentrations (Abegg and Anderson, 1997; Niewöhner et al., 1998) and this can potentially lead to an underestimation of methane fluxes and AOM. Such a loss is mainly observed at high methane concentrations and depends on the time between coring and taking individual samples (Abegg and Anderson, 1997). However, the methane concentration in the STMZ of the Skagerrak cores was well below saturation concentration at atmospheric pressure and is unlikely to have gassed out to such an extent as to create a 15 times lower AOM rate. Moreover, rough profiles sampled right after retrieval of the core at the top of each core section showed no significant loss of methane in the SMTZ but only in the bottom part of the core, where concentrations where higher than saturation at atmospheric pressure. Another possibility is that methanogenesis rates taking place in the AOM zone could counterbalance AOM rates. At station S10 methanogenesis was present in the SMTZ, but rates were only ~ 10 % of the AOM rates, so that this does not seem to be a likely explanation for either the difference between measured rates and calculated methane fluxes (Iversen and Jørgensen, 1985) or the low AOM rates compared to SRR.
a clinical setting to confirm the relevance for patients. In 1992, Burks and coauthors reported a 100-fold and 10-fold reduced IgE binding capacity of peanut and soybean allergens, respectively, after exposure to enzymes mimicking human digestion [ 110 ]. The different outcome for major food allergen sources was underlined by a study performed more than 10 years later using codfish as a model antigen [ 111 ]. After digestion with simulated gastric fluid, the IgE binding capacity of codfish proteins was reduced more than 10,000-fold. This was shown in a reduced histamine release activity from basophil of healthy donors, which were passively sensitized with sera from codfish allergic patients. Also in a clinical setting, the impact of gastric enzymes on fish allergenicity was confirmed [ 94 ]. The diameter of positive skin test reactions was significantly reduced after pre-digestionof allergens. Furthermore, the lowest observed adverse effect level in double-blind, placebo controlled food challenges (DBPCFC) was significantly higher. The pre-digestion was performed with gastric enzyme tablets clinically used for patients with reduced gastric acid secretion. Also for celery allergens, the influence of gastric enzymatic digestion on allergenicity could be confirmed in celery allergic patients with a mean age of 72 years [ 112 ]. Even in this age group, skin test reactivity was significantly altered when test allergens were pre-incubated with digestive enzymes, highlighting the impact of gastric digestion on food allergenicity.
Thus the digestionof ribonuclease A by protein ase K proceeds in a way very similar or even identi cal with the subtilisin or elastase [4 ]. To prove identity we isolated RNase K following the recom mended procedures used for RNase S purification which were also successfully applied for RNase E isolation of ribonuclease digested with elastase [4, 7, 8 ]. A 250 mg sample of RNase A was digested at 2 °C with proteinase K and the whole digestion mixture was applied on an ion exchange column (Amberlite CG 50 I I ). The trypsin sensitive peak (pool II ) was lyophilized and desalted on Sephadex G 25. 80% of the initial activity was recovered by this procedure and the product — RNase K — was used for final characterization (Table I ). Chroma tography of RNase K on Sephadex G 75 using 30% acetic acid as eluant resulted in the separation of K-protein and K-peptide (recovery: 75% K-protein and 12% K-peptide of the applied RNase K weight bases).
4.4. Enzyme Assays
All activity experiments were performed in the absence of oxygen. Cells of wild-type C. defragrans grown anaerobically on α-pinene were resuspended in 25 mM Tris-Cl, pH 8.0 and disrupted by two passages through a French-Press cell disrupter (SLM Aminco, Rochester, NY, USA) at 8.6 MPa. The crude cell lysate, as well as the soluble and pelleted fractions obtained after ultracentrifugation (230,000 × g for 30 min at 4 ◦ C), were dialyzed in anoxic 25 mM Tris-Cl, pH 8.0 (1:10 6 ) and tested for enzyme activity. A 500 µL assay contained 2.5–5 mg protein, 2 mM dithiothreitol, 5 mM Mg 2+ , 10 mM Mn 2+ , 10 mM ATP, 0.5% Tween 20 and 60 mM of a bicyclic monoterpene. When indicated, divalent cations were replaced for 10 mM Ca 2+ supplied as CaCl 2 . The reactions were initiated by monoterpene addition and incubated for 16–18 h at 28 ◦ C. Hydrophobic metabolites were extracted with 100 µL n-hexane and analyzed by GC-FID as aforementioned. Retention times shown in Figures 1 and 2 are shifted due to column shortening during equipment maintenance. Monoterpene identity was confirmed by analysis with internal standards and GC-MS analysis.
The first aspect of the thesis was the investigation of MOW properties, especially the evaluation of MOW quality with regard to treatment and recycling of process residues onto arable lands. Results show that MOW composition is rather heterogenic. This is indicated by a high variance of biogas yields and degradability as well as digestate properties. MOW composition also determines the availability and behavior of elements during biochemical conversion. The heterogeneity of nutrient composition (mean SD = 16%) is much lower than the heterogeneity of heavy metal contents (mean SD = 53%, see results section 6 Table 13). High heavy metal loads in MOW are related to high impurity contamination. A positive correlation (R ≥ 0.7) was found between impurity contents and the concentration of Cd, Pb and Zn in the investigated MOW samples. Bilitewski and Härdtle (2013) confirm that especially plastics contribute to Cd, Pb and Zn contents in MOW. The impurity content of investigated MOW samples ranges from 0% to 12% proving the heterogenic results of heavy metal analysis. In comparison with other studies, heavy metal contents of MOW in this study seem to be generally higher. This leads to the assumption, that regional peculiarities such as geological background exposure could be another reason for high heavy metal contamination of MOW. Either way, results make clear that impurities and heavy metal contamination of MOW can become problematic when related products are supposed to be used as soil amendments. A few of the investigated MOW samples already exceed thresholds of BioAbfV for Cd, Pb and Zn. Therefore, besides nutrients the behavior of contaminants throughout the biological treatment process has been addressed by this thesis.
penetration over time as it was gray-greenish, i.e. oxidized in the top 4 cm during all investigations. However, the decrease in AOM in the top 4 cm in late September, when the sediment was strongly resuspended by wind-induced currents, indicates an increase in oxygen penetration compared to early September which inhibited AOM archaea (Zehnder and Brock, 1980). The aerobic methanotrophic community, on the other hand, probably reacted very slowly to the change from anoxic to oxic conditions. During potential rate measurements we observed a lag-phase between 50 and 1000 hrs in aerobic MO (this study and Eppelin 2002). This was the time, surface slurries of Eckernförde Bay sediments needed to show elevated activity of aerobic MO after the addition of oxygen and methane. The induction of aerobic MO is associated with the general ability of aerobic methanotrophs to form resting stages (exospores and cysts) during unfavorable periods (Heyer 1990).Therefore, an oxygen penetration into the sediment in late September could have inhibited AOM archaea whereas aerobic methanotrophs have yet been in a resting stage. This would explain the decrease of methane oxidation in late September compared to early September and March. In March, when aerobic methanotrophs had presumably already germinated and exhibited growth, aerobic MO rates clearly exceeded AOM in the surface sediment.
Many thanks to all colleagues in- and outside the institute who contributed to bring this work to completion. I am especially thankful to colleagues at the Department of Microbiology for such a nice working atmosphere. Thanks to Jens Harder, who always encouraged me to ask by saying “es gibt keine dummen Frage”; Christina Probian for original protocols from “Widdel´s Schule”; Cathrin Wawer for the “fight” with hydrogenases; Olaf Kniemayer for a lot of tips in doing experiments and the radio “Bremen Vier”; Martin Krüger, Katja Nauhaus and Christine Flies for providing sediment samples, and all other colleagues who kept me in good hands with whatever I need. Special thanks to Katja Nauhaus, Olav Grundmann and Simon Kühner for the friendly atmosphere in the office and, of course, for the proof-reading of my thesis. I am grateful to former members of our research group, in particular Jan Küver for such a nice cooperation in “growing” phylogenetic trees, Karsten Zengler for teaching anaerobic techniques and Stefan Sievert for introducing the DGGE tool. Thanks to FISH experts Marc Mußmann and Annelie Pernthaler (Molecular Ecology Department) for their help in analysis of bacteria on corroding surface. I am thankful to Dr. Achim W. Hassel (MPI Düsseldorf) for wonderful iron samples and productive collaboration; Dr. Cam Ha (IBT Hanoi) for giving me a chance to work with sulfate-reducing bacteria and latter to fall in love with them.
members: the cyclohexane-1,2-diol degrading strain Lin22 (91.4%), the propylbenzene de- grading strain PbN1 (90.9%) and to Dechloromonas aromatica strain RCB (91.1%). From the Thauera group the closest relative is the toluene degrader, Thauera aromatica K172. Other ﬁve phylotypes were found, three related to Denitratisoma oestradiolicum (84.4 to 94%), one to Chlorobium phaeobacteroides DSM 266 (80% identity) and one to candidate division OP11 sequences from a river estuary mangrove (89.8% identity) (Figure SM 12.5). To resolve the relative dominance of the organisms in the enrichments, we applied FISH with phylum- to group-speciﬁc oligonucleotide probes (Figure 12.4). All bacteria were de- tected with a DNA stain (4’.6-diamidino) 2-phenylindol (DAPI). We determined the relative percentage of probe targeted cells in relation to the number of DAPI stained cells. The general Bacteria probe (Eub-338 I-III) hybridized 97% of the total cells in both enrichment cultures (Figure 12.3A). The class speciﬁc Betaproteobacteria probe (Bet-42a), hybridized to more than 93% cells in both enrichments (Figure 12.3B). Newly designed probes (pxyn- 440 and pxyn-644) speciﬁc for the Denitratisoma-related phylotype which dominated both clone libraries, targeted more than 91% of cells in both enrichment cultures (Figure 12.3C). Within the Betaproteobacteria class, Azoarcus and Thauera clade, comprises all de- scribed denitrifying alkylbenzene degraders (Widdel and Rabus, 2001). In contrast, the dominant microorganism in our denitrifying cultures was only distantly related to Azoarcus and Thauera species. The next relatives at the 16S rRNA level are denitrifying microorgan- isms capable of steroid degradation, the 17-estradiol degrader D. oestradiolicum (Fahrbach et al., 2006), and two cholesterol degrading isolates, S. denitriﬁcans and strain 72Chol (Harder and Probian, 1997; Tarlera and Denner, 2003). In another study the dominant phy- lotype of denitrifying benzene degrading enrichment cultures, was also related to a steroid degrader, Sterolibacterium denitriﬁcans (Ulrich and Edwards, 2003). Until now, the ability of steroid degraders to use monocyclic aromatics is unknown. Our enrichments were capable to grow on two alkylbenzenes (toluene, p-xylene) and two polar monoaromatics (benzoate,
Each enzyme has many biophysical parameters, of which values are partly not known in advance. Likewise the substrate has to be modeled as a geometrical object with a complex structure and should enclose multiple physical properties, like crystallinity, degree of polymerization and active surface. The initialization has to be performed with data provided by experimentalists. Updates during ongoing simulation have to be guaranteed to be at least feasible from physical, biological and chemical points of view. The simulated system is coupled via the substrate in a nonlinear manner. Additionally, the interaction between enzymes should not be excluded a priori, which adds another nonlinear coupling. Due to these circumstances, the possible phase space has a high dimension of up to 11 continuous variables combined with up to 8 discrete and categorical degrees of freedom, making the system an interesting research object.
assess geo-information on different temporal and spatial scales. Satellite technology is currently changing from an experimental status towards operational systems with a long-term perspective of delivering data and information products. A typical measuring set-up is shown in figure 1 using the reflectance spectrum of vegetation to extract information about green biomass.