Expression, Purification, and Biochemical Characterization of Human Afamin.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Oxygen tolerance capacity of upflow anaerobic solid-state (UASS) with anaerobic filter (AF) system.

In order to investigate the oxygen tolerance capacity of upflow anaerobic solid-state (UASS) with anaerobic filter (AF) system, the effect of microaeration on thermophilic anaerobic digestion of maize straw was investigated under batch conditions and in the UASS with AF system. Aeration intensities of 0-431mL O2/gvs were conducted as pretreatment under batch conditions. Aeration pretreatment obviously enhanced anaerobic digestion and an aeration intensity of 431mL O2/gvs increased the methane yield by 82.2%. Aeration intensities of 0-355mL O2/gvs were conducted in the process liquor circulation of the UASS with AF system. Dissolved oxygen (DO) of UASS and AF reactors kept around 1.39±0.27 and 0.99±0.38mg/L, respectively. pH was relatively stable around 7.11±0.04. Volatile fatty acids and soluble chemical oxygen demand concentration in UASS reactor were higher than those in AF reactor. Methane yield of the whole system was almost stable at 85±7mL/gvs as aeration intensity increased step by step. The UASS with AF system showed good oxygen tolerance capacity.

Reorganisation of a mesophilic biogas microbiome as response to a stepwise increase of ammonium nitrogen induced by poultry manure supply.

An anaerobic digestion experiment was investigated to evaluate the impact of increasing amounts of ammonium nitrogen due to poultry manure addition on the reactor performance, especially on the microbiome response. The microbial community structure was assessed by using a 16S rRNA gene approach, which was further correlated with the prevalent environmental conditions by using statistical analyses. The addition of 50% poultry manure led to a process disturbance indicated by a high VFA content (almost 10 g(HAc-Eq) L(-1)) in combination with elevated concentrations of ammonium nitrogen (5.9 g NH4(+)-N kg(FM)(-1)) and free ammonia (0.5 g NH3 kg(FM)(-1)). Simultaneously the microbiome, changed from a Bacteroidetes-dominated to a Clostridiales-dominated community accompanied by a shift from the acetoclastic to the hydrogenotrophic pathway. The "new" microbial community was functional redundant as the overall process rates were similar to the former one. A further increase of poultry manure resulted in a complete process failure.

A dynamic model for calculating methane emissions from digestate based on co-digestion of animal manure and biogas crops in full scale German biogas plants.

The focus of this work is the development of a model for the estimation of methane emissions for storage tanks of biogas plants. Those can be estimated depending on (i) hydraulic retention time in the digester, (ii) an arbitrary removal rate of the digestate from the storage tank and (iii) arbitrary temperature conditions in the storage tank. Furthermore, the model is capable of considering an arbitrary mixture of manure and crops in the input material. The model was validated by data from 21 full scale biogas plants in Germany digesting cow manure and crops. A realistic scenario for the removal rate and temperature conditions in the storage tank was then investigated and special emphasis was given to the effect of hydraulic retention time and proportion of crops in the mixture on the input VS methane yield from the digester and the storage tank.

Performance of a novel two-phase continuously fed leach bed reactor for demand-based biogas production from maize silage.

This study investigated the potential of producing biogas on demand from maize silage using a novel two-phase continuously fed leach bed reactor (LBR) which is connected to an anaerobic filter (AF). Six different feeding patterns, each for 1week, were studied at a weekly average of a volatile solids (VS) loading rate of 4.5 g L(-1) d(-1) and a temperature of 38°C. Methane production from the LBR and AF responded directly proportional to the VS load from the different daily feeding and resulted in an increase up to 50-60% per day, compared to constant feeding each day. The feeding patterns had no impact on VS methane yield which corresponded on average to 330 L kg(-1). In spite of some daily shock loadings, carried out during the different feeding patterns study, the reactor performance was not affected. A robust and reliable biogas production from stalky biomass was demonstrated.

Computer Tomographic Illustration of the Development of the Pulmonary Function in Bovine Neonates until the Twenty-First Day Postnatum.

The aim of this study was to analyze the development of the lung in newborn calves. The sample consisted of 28 Holstein Friesians calves which were examined clinically, and their chest segment was measured with computed tomography. The tests were performed on the first, sixth, and twelfth hours of life and after the first, second, and third weeks. Also, blood gases and blood counts were determined. Besides Kolmogorov-Smirnov tests, analyses of variance, t-tests (on a significance level of P < 0.05), and correlation analyses were used. The most significant changes occurred between birth and the first hour. However, there were significant differences in the gas filling between cranial and caudal and between dorsal and ventral parenchyma segments. This difference remained over the entire study period. At the end of the first week between 85 and 93% were involved in gas exchange. Only after the completion of the second week of life, the air supply was achieved throughout the whole lung. The pO2, pCO2, and pH values confirmed this. This study shows that a healthy bovine neonate needs about 2 weeks before all lung units are integrated into the gas exchange. This explains why calves in unfavorable environments often suffer from pulmonary affections.

Mesophilic anaerobic co-digestion of cow manure and biogas crops in full scale German biogas plants: a model for calculating the effect of hydraulic retention time and VS crop proportion in the mixture on methane yield from digester and from digestate storage at different temperatures.

Data from 24 full scale biogas plants in Germany digesting cow manure and crops were evaluated. Special emphasis was given to the effect of hydraulic retention time HRT and proportion of crops in the mixture (VS basis) p(VS,Crops)(Inp) on the methane yield from the digester [Formula: see text] and the storage tank [Formula: see text] at 37 and 22°C. The evaluation has shown model parameters for maximal methane yield of manure and crops [Formula: see text] at 270 and 420 Lkg(-1), respectively. For example, at HRT of 60days, maximum methane yield result to 249 and 388 Lkg(-1) for a crop proportion in the input of 0.0 and 1.0, respectively. The calculation of [Formula: see text] considers first order reaction rates and a temperature term f(T). Hence, at any arbitrary temperature in the range of 12°C

Do two-phase biogas plants separate anaerobic digestion phases? - a mathematical model for the distribution of anaerobic digestion phases among reactor stages.

In this article a mathematical model is introduced, which estimates the distribution of the four anaerobic digestion phases (hydrolysis, acidogenesis, acetogenesis and methanogenesis) that occur among the leach bed reactor and the anaerobic filter of a biogas plant. It is shown that only the hydrolysis takes place in the first stage (leach bed reactor), while all other anaerobic digestion phases take place in both reactor stages. It turns out that, besides the usually measured raw materials of the acetogenesis and the methanogenesis phases (organic acids), it is also necessary to analyze the process liquid for raw materials of the acidogenesis phase, i.e., sugars, fatty acids, amino acids, etc. The introduced model can be used to monitor the inhibition of the anaerobic digestion phases in reactor stages and can, thus, help to improve the control system of biogas plants.

Mathematical modeling of process liquid flow and acetoclastic methanogenesis under mesophilic conditions in a two-phase biogas reactor.

Acetoclastic methanogenesis in the second stage of a two-phase biogas reactor is investigated. A mathematical model coupling chemical reactions with transport of process liquid and with the variation of population of the microorganisms living on the plastic tower packing of the reactor is proposed. The evolution of the liquid is described by an advection-diffusion-reaction equation, while a monod-type kinetic is used for the reactions. Moreover, a new inhibition factor MO(max) is introduced, which hinders the growth of microorganisms when the plastic tower packing is overpopulated. After estimating the reaction parameters, the acetate outflow measured experimentally is in good agreement with that predicted by simulations. For coupling liquid transport with reaction processes, a spatial discretization of the reactor is performed. This yields essential information about the distribution of acetate and the production of methane in the reactor. This information allows for defining a measure of the effectiveness of the reactor.

Novel upflow anaerobic solid-state (UASS) reactor.

The aim of this work was to evaluate the feasibility of a novel upflow anaerobic solid-state (UASS) reactor equipped with liquor recirculation. The performance characteristics were obtained from a thermophilic UASS reactor (26.5L) operated for 68 days with a mixture of maize silage and straw. In order to prevent an accumulation of volatile fatty acids, the process liquor was continuously recirculated through anaerobic filters. By raising the volatile solids (VS) loading rate from 7.1 to 17 g(VS)L(-1)d(-1), the overall methane yield declined from 384 to 312 kg(VS)(-1), while the yield contribution of the anaerobic filters increased from 12% to 70%. The chemical oxygen demand (COD) of biodegradable particulate matter was removed by 86-93% with a maximum hydrolysis rate of 16.4 g(COD)L(-1)d(-1). Based on the results, we conclude that the UASS reactor is a promising solution for the digestion of various organic materials.

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass.

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.

Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate.

The bioconversion of renewable raw material to biogas by anaerobic microbial fermentation processes in completely stirred tank reactors (CSTR) is a valuable alternative resource of energy especially for rural areas. However, knowledge about the microorganisms involved in the degradation of plant biomass is still poor. In this study, a first analysis of the biogas-forming process within a CSTR fed continuously with fodder beet silage as mono-substrate is presented in the context of molecular data on the microbial community composition. As indicated by the conventional process parameters like pH value, content of volatile fatty acids, N:P ratio and the biogas yield, the biogas-forming process within the CSTR occurred with a stable and efficient performance. The average biogas yield based on volatile solids was 0.87m(3)kg(-1) at an organic loading rate of 1.2-2.3kgm(-3)d(-1). This amounts to 94% of the theoretical maximum. In order to identify microorganisms within the CSTR, a 16S rDNA clone library was constructed by PCR amplification applying a prokaryote-specific primer set. One hundred and forty seven clones were obtained and subsequently characterized by amplified rDNA restriction analysis (ARDRA). The sequences of 60 unique ARDRA patterns were estimated in a length of approximately 800-900bp each. Four of them were assigned to the domain Archaea and 56 to the domain Bacteria. Within the domain Archaea, all clones showed a close relationship to methanogenic species. Major bacterial groups represented in the clone library were the class Clostridia of the phylum Firmicutes (22% of all 16S rDNA clones), the class Deltaproteobacteria of the phylum Proteobacteria (24%), the class Bacilli of the phylum Firmicutes (22%) and members of the phylum Bacteroidetes (21%). Within these major groups, the highest biodiversity was found within the class Clostridia (35% of all operational taxonomic units). Members of the phyla Actinobacteria and Spirochaetes were represented only by 5 and 2 clonal sequences, respectively.