Model-based identification and testing of appropriate strategies to minimize N2O emissions from biofilm deammonification.

Based on a one-year pilot plant operation of a two-step biofilm nitritation-anammox pilot plant, N2O mitigation strategies were identified by applying a newly developed biofilm modeling approach. Due to adapted plant operation, the N2O emission could be diminished by 75% (8.8% → 2.3% of NH4-Noxidized_AOB). The results (measurement and simulation) confirm the huge importance of denitrification as an N2O source or N2O sink, depending on the boundary conditions. A significant reduction of N2O emissions could only be achieved with a one-step deammonification system, which is related to low nitrite and HNO2 concentrations. Increased oxygen concentrations in the bulk phase are not related to decreased emissions. N2O formation by ammonium-oxidizing bacteria (AOB) just shifts deeper into the biofilm; zones with low oxygen concentrations are not avoidable in biofilm systems. Low oxygen concentrations in the bulk phase, however, result in a reduction of the total net N2O formation due to increased activity of heterotrophic bacteria directly at the source of N2O formation (outer biofilm layer). For the model-based identification of mitigation strategies, the standard modeling approaches for biofilms were expanded by including the factor-based N2O formation and emission approach. The new model 'Biofilm/N2OISAH' was successfully validated using data from pilot-scale measurement campaigns. Altogether, the investigation confirms that the employed digital model can strongly support the development of N2O mitigation strategies without the need for specialized measurement inside the biofilm.

Model assisted identification of N2O mitigation strategies for full-scale reject water treatment plants.

In a 3-year research project, a new approach to forecast biological N2O formation and emission at high-strength reject water treatment has been developed (ASM3/1_N2OISAH). It was calibrated by extensive batch-tests and finally evaluated by long-term measurement campaigns realized at three wastewater treatment plants (WWTPs) with different process configurations for nitrogen removal of reject water. To enable a model application with common full-scale data, the nitritation-connected supplementary processes that are responsible for N2O formation are not depicted in the model. Instead, within the new model approach the N2O formation is linked to the NH4-N oxidation rate by defining specific formation factors [N2O-Nform/NH4-Nox], depending on the concentrations of NO2 and O2 as well as the NH4 load. A comparison between the measured and the modeled N2O concentrations in the liquid and gas phase at the full-scale treatment plants prove the ability of the proposed modelling approach to represent the observed trends of N2O formation, emission and reduction using the standard parameter set of kinetics and formation factors. Thus, enabling a reliable estimation of the N2O emissions for different operational conditions. The measurements indicate that a formation of N2O by AOB cannot completely be avoided. However, a considerable reduction of the formed N2O was observed in an anoxic environment. Applying the model, operational settings and mitigation strategies can now be identified without extensive measurement campaigns. For further enhancement of the model, first results for kinetics of N2O reduction kinetics by denitrification processes were determined in laboratory-scale batch tests.

Safety and feasibility of various fasting-mimicking diets among people with multiple sclerosis.

BACKGROUND: Fasting-mimicking diets have shown promise in experimental autoimmune encephalitis and are currently being investigated among people with multiple sclerosis (MS). Ensuring adherence to diet changes is critical to determining the efficacy of such interventions. OBJECTIVE: Our primary aim was to evaluate the safety and feasibility of several fasting-mimicking diets and investigate whether various levels of clinical support improve diet adherence among people with MS. Secondarily, this study evaluated the impact of fasting-mimicking diets on weight and patient-reported outcomes (PROs). METHODS: We conducted three pilot studies (two randomized controlled for 6 months; one randomized with transition to single arm) restricting either the amount or timing of calorie intake over 24 or 48 weeks. Interventions included calorie restriction (daily or intermittently) or time-restricted feeding. Adherence measures varied across studies but were collected at study visits along with weight and PRO data. RESULTS: A total of 90 participants enrolled; 70 completed the studies, with no serious adverse events reported. Overall adherence to the calorie restriction diets was poor. When participants were tasked with maintaining a diet in a pragmatic setting, neither previously completed intense clinical support and education, nor weekly electronic communication throughout the diet period appeared to improve diet adherence. Participants who were able to adhere to a calorie restriction diet predictably lost weight. In contrast to calorie restriction, adherence to a time-restricted feeding (TRF) diet was relatively good. No statistically significant changes in PROs were observed in an intention-to-treat analysis. CONCLUSION: The role diet may play in clinical outcomes in MS remains unknown, as class I evidence is lacking. Diet adherence remains a primary barrier to the feasible conduct of large, randomized controlled diet trials. Strict adherence to a TRF dietary change may be more feasible than calorie restriction and should be considered in future fasting-mimicking diet trials. ClinicalTrials.gov Registry:A Pilot Study of Intermittent Calorie Restriction in Multiple Sclerosis - NCT02647502. A Pragmatic Trial of Dietary Programs in People with Multiple Sclerosis (MS) - NCT02846558.

Influence of operating conditions on nitrous oxide formation during nitritation and nitrification.

Nitrous oxide (N2O), a strong greenhouse gas, can be produced by ammonium-oxidizing bacteria (AOB) as a by-product of ammonium oxidation and can potentially be formed in all types of nitrification processes. However, partial nitritation has been reported to cause significantly higher N2O emissions than complete nitrification. In the study presented here, the mechanisms and factors that drive N2O formation by AOB were investigated with respect to different operational strategies to achieve nitrite accumulation base on combined evaluation of oxygen uptake rate (OUR) and N2O formation rate. On the one hand, N2O formation during partial nitritation and nitrification in a continuously stirred tank reactor (CSTR) with continuous aerobic conditions was observed. On the other hand, the effect of intermittent aeration on N2O formation during nitrification was investigated. The presence of nitrite, the extend of sludge-specific ammonium loading, low oxygen concentration, and transition from aerobic to anoxic conditions significantly increased N2O formation in this reactor independently from each other, indicating that different formation pathways, supposedly via nitrite or hydroxylamine, were active.

A new approach to energy-efficient treatment of wastewater produced by the fish industry in Vietnam.

Economic growth in Vietnam in the last few years has brought about an increasing demand for energy and has had a severe environmental impact. Fish processing is one of the fastest-growing industries that discharge organically-polluted wastewater. To counter these environmental problems, new technologies for energy-efficient treatment are needed. By coupling innovative nitrogen removal systems with anaerobic treatment processes, it is possible to realise such technologies. In the present project, a combined deammonification and anaerobic carbon removal system is presented. Special requirements to enable reliable treatment are discussed, taking industrial wastewater characteristics into consideration. To evaluate energetic efficiency, energy balance calculations based on data from a fish-processing factory are made. The determined specific energy consumption and production rates show that energy recovery is possible, even when COD and nitrogen removal efficiencies of over 90% are achieved. Depending on the pre-treatment employed, energy recovery rates ranging from 0.6 to 2.5 kWh/mt raw fish can be reached.

Comprehensive array CGH of normal karyotype myelodysplastic syndromes reveals hidden recurrent and individual genomic copy number alterations with prognostic relevance.

About 40% of patients with myelodysplastic syndromes (MDSs) present with a normal karyotype, and they are facing different courses of disease. To advance the biological understanding and to find molecular prognostic markers, we performed a high-resolution oligonucleotide array study of 107 MDS patients (French American British) with a normal karyotype and clinical follow-up through the Duesseldorf MDS registry. Recurrent hidden deletions overlapping with known cytogenetic aberrations or sites of known tumor-associated genes were identified in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 (RUNX1, 2x). One patient with a 7q22.1 deletion had an additional 5q31.2 deletion of the acute myeloid leukemia/MDS region, the smallest deletion identified so far and including the putative tumor suppressor (ts) genes, EGR1 and CTNNA1. One TET2 deletion was homozygous and one heterozygous, with a missense mutation in the remaining allele, further supporting its role as a ts gene. Besides these recurrent alterations, additional individual imbalances were found in 34 cases; in total, 42/107 (39%) cases had genomic imbalances. These patients had an inferior survival as compared with the rest of the patients (P=0.002). This study emphasizes the heterogeneity of MDS, but points to interesting genes that may have diagnostic and prognostic impact.

Determination of the nitrous oxide emission potential of deammonification under anoxic conditions.

Various studies have been performed to determine nitrous oxide (N2O) emissions from conventional biological nitrogen removal processes in wastewater treatment like nitrification and denitrification in the main stream. However, with respect to the overall emissions of a wastewater treatment plant, part-stream treatment for high-strength wastewater (e.g., sludge liquor) is also expected to hold a significant emission potential because of high concentrations and extreme boundary conditions. This paper presents results from a laboratory-scale study on nitrous oxide production by biomass from a deammonification process (nitritation + anammox) under anoxic conditions. It was discovered that N2O formation results from incomplete endogenous denitrification rather than anammox and is dependent on substrate availability. Based on direct measurements of the dissolved N2O concentrations in a sequencing batch reactor, the dynamic behavior of N2O production is characterized in more detail. The results show that, during anoxic conditions, the N2O emission potential of deammonification is significantly lower than from conventional denitrification.

Endolymphatic calcium supply for fish otolith growth takes place via the proximal portion of the otocyst.

The presence of calcium within the utricle of larval cichlid fish Oreochromis mossambicus was analysed by means of energy-filtering transmission electron microscopy. Electron-spectroscopic imaging and electron energy loss spectra revealed discrete calcium precipitations that were more numerous in the proximal endolymph than in the distal endolymph, clearly indicating a decreasing proximo-distal gradient. This decreasing proximo-distal gradient was also present within the proximal endolymph between the sensory epithelium and the otolith. Further calcium particles covered the peripheral proteinaceous layer of the otolith. They were especially pronounced at the proximal surface of the otolith indicating that otolithic calcium incorporation takes place here. Other calcium precipitates accumulated at the macular junctions clearly supporting an earlier assumption according to which the endolymph is supplied with calcium via a paracellular pathway. The present results clearly show that the apical region of the macular epithelium is involved in the release of calcium and that the calcium supply of the otoliths takes place via the proximal endolymph.

Effect of hypergravity on carboanhydrase reactivity in inner ear ionocytes of developing cichlid fish.

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish. Otolith growth in terms of mineralization mainly depends on the enzyme carboanhydrase (CA), which is responsible for the provision of the pH-value necessary for calcium carbonate deposition. Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (3 g, hg; 6 h) during development and separated into normally and kinetotically swimming individuals following the transfer to 1 g (i.e., stopping the centrifuge; kinetotically behaving fish performed spinning movements). Subsequently, CA was histochemically demonstrated in inner ear ionocytes (cells involved in the endolymphatic ion exchange) and enzyme reactivity was determined densitometrically. It was found that both the total macular CA-reactivity as well as the difference in reactivities between the left and the right maculae (asymmetry) were significantly lower (1) in experimental animals as compared to the 1 g controls and (2) in normally swimming hg-animals as compared to the kinetotically behaving hg-fish. The results are in complete agreement with earlier studies, according to which hypergravity induces a decrease of otolith growth and the otolithic calcium incorporation (visualized using the calcium-tracer alizarin complexone) of kinetotically swimming hg-fish was higher as compared to normally behaving hyper-g animals. The present study thus strongly supports the concept that a regulatory mechanism, which adjusts otolith size and asymmetry as well as otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

Calcium-tracers disclose the site of biomineralization in inner ear otoliths of fish.

Since changing gravity (concerning direction and amplitude) strongly affects inner ear otolith growth and otolithic calcium incorporation in developing fish, it was the aim of the present study to locate the site of mineralization in order to gain cues and insights into the provenance of the otoliths inorganic compounds. Therefore, larval cichlid fish (Oreochromis mossambicus) were incubated in the calcium-tracer alizarin complexone (AC; red fluorescence). After maintenance in aquarium water for various periods (1, 2, 3, 6, 9 and 12 h; 1, 2, 3, 5, 6, 7, 15, 29, 36 and 87 d), the animals were incubated in the calcium-tracer calcein (CAL; green fluorescence). AC thus labeled calcium being incorporated at the beginning of the experiment and would subsequently accompany calcium in the course of a possible dislocation, whereas CAL visualized calcium being deposited right at the end of the test. Subsequently, the otoliths were analyzed using a laser scanning microscope and it was shown that the initial site of calcium incorporation was located directly adjacent to the sensory epithelium and the otolithic membrane. Later, calcium deposits were also found on further regions of the otoliths' surface area, where they had been shifted to in the course of dislocation. This finding strongly indicates that the sensory epithelium plays a prominent role in otolithic biomineralization, which is in full agreement with an own electron microscopical study [ELGRA News 23 (2003) 63].

Geniom technology--the benchtop array facility.

Febit AG develops an integrated benchtop instrument for in situ microarrays preparation, hybridization, readout and data analysis.

Exploring nature's plasticity with a flexible probing tool, and finding new ways for its electronic distribution.

Concepts and results are described for the use of a single, but extremely flexible, probing tool to address a wide variety of genomic questions. This is achieved by transforming genomic questions into a software file that is used as the design scheme for potentially any genomic assay in a microarray format. Microarray fabrication takes place in three-dimensional microchannel reaction carriers by in situ synthesis based on spatial light modulation. This set-up allows for maximum flexibility in design and realization of genomic assays. Flexibility is achieved at the molecular, genomic and assay levels. We have applied this technology to expression profiling and genotyping experiments.

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi.

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.

Neuronal regulation of otolith growth and kinetotic behaviour.

Inner ear stones (otoliths) of larval cichlid fish were labelled with the calcium-tracer alizarin-complexone (AC) before animals were subjected to hypergravity (hg; 3 g). After the experiment, the otoliths' area between the two AC-labellings was measured. Growth of hg-otoliths was significantly slowed down as compared to 1 g-control specimens. In the course of a second experiment, the vestibular nerve was unilaterally transacted in neonate swordtail fish which were subsequently incubated in AC. Incorporation of AC was considerably lower in the otoliths of the transacted side. The results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector. Since the otolithic calcium incorporation ceased on the transected head sides, it is concluded that the regulation of otolith growth is based on the central nervous efferent vestibular system.

Influence of hypergravity on fish inner ear otoliths: I. Developmental growth profile.

Inner ear stones (otoliths) of larval cichlid fish Oreochromis mossambicus were marked with the calcium-tracer alizarin-complexone (AC) at 1 g earth gravity before and after a 3, 7, 14 or 21 days stay of the animals at hypergravity conditions (hg; 3 g, centrifuge). After the experiment, the otoliths' area between the two AC-labellings was measured with regard to size and asymmetry (size difference between the left and the right stones). Both utricular and saccular otoliths (lapilli and sagittae, respectively) continued growing in a linear way at hg, but growth was significantly slowed down as compared to parallely raised 1 g-control specimens. In case of bilateral asymmetry between the corresponding otoliths its formation in hg-animals became reduced as compared to the 1 g controls. The reduction of asymmetry was much more pronounced in the sagittae than in the lapilli. The latter result supports an earlier hypothesis, according to which especially a low sagittal asymmetry has a functional advantage. In general, the results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector.

Influence of hypergravity on fish inner ear otoliths: II. Incorporation of calcium and kinetotic behaviour.

Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (hg; 3 g, 14 days) during development. Following the transfer to 1 g (i.e., stopping the centrifuge) they were separated into normally and kinetotically swimming individuals (the latter performed spinning movements). During hg, the animals were maintained in aquarium water containing alizarin-complexone (AC), a fluorescent calcium tracer. Densitometric measurements of AC uptake into inner ear otoliths (optical density of AC/micrometers2) revealed that the kinetotic individuals had incorporated significantly more AC/calcium than the normally behaving fish. Since the amount of otolithic calcium can be taken as an approximation for otolith weight, the present results indicate that the otoliths of kinetotically swimming samples were heavier than those of the normally behaving larvae, thus exhibiting a higher absolute weight asymmetry of the otoliths between the right vs. the left side of the body. This supports an earlier concept according to which otolith (or statolith) asymmetry is the cause for kinetoses such as human static space sickness.

A 2-step non-surgical procedure and systemic antibiotics in the treatment of rapidly progressive periodontitis.

BACKGROUND: In the last few years knowledge about periodontal infections has increased enormously, nevertheless practitioners are still seeking guidelines for suitable treatment concepts. METHODS: The aim of this study was to examine the effect of doxycycline, metronidazole, and clindamycin used adjunctively in a 2-step nonsurgical procedure in patients with rapidly progressive periodontitis (RPP). The first step included scaling, root planing, and polishing (SRP) in each quadrant using 4 to 5 visits. The second step included full-mouth enhanced root planing (RP) and wound dressing in 1 or 2 visits after SRP and the beginning of antibiotic therapy. Forty-eight patients (mean age 32.4 years) with generalized RPP, with an average of 16 sites with probing depths (PD) deeper than 8 mm, and high counts of Porphyromonas gingivalis were randomly assigned to 4 different groups: group 1 (doxycycline) n = 12, group 2 (metronidazole) n = 15, group 3 (clindamycin) n = 11, and group 4 (control group; no antibiotic treatment) n = 10. Clinical evaluations, including plaque index (PI), sulcus bleeding index (SBI), probing depth (PD), clinical attachment level (CAL), and bacteriological and crevicular cell sampling, were done at baseline (BL), 3 weeks after SRP, and 6 and 24 months after RP. RESULTS: After the first step (SRP), we observed an improvement of PI and SBI in all 4 groups, but did not see any statistically significant PD reduction 3 weeks after SRP compared to baseline. However, 6 and 24 months after the second step (RP) we observed a significantly greater reduction of PD in groups 2 and 3 and a significantly greater CAL gain in comparison to groups 1 and 4. After 24 months, the attachment level gain in group 1 and group 4 was less than 1.5 mm, and less than 1.0 mm in PD site categories 6 to 9 mm and >9 mm. PI showed no significant difference between the groups throughout the period after SRP until 24 months, compared to 3 weeks after SRP. SBI decreased most in the metronidazole and clindamycin groups. P. gingivalis and Actinobacillus actinomycetemcomitans were almost completely eradicated in these 2 groups 24 months after RP. In addition, the phagocytotic capacity of crevicular polymorphonuclear neutrophils was increased in groups 2 and 3 after the second step. CONCLUSIONS: The present results show that metronidazole and clindamycin are effective antibiotics when used adjunctively in a 2-step nonsurgical procedure of scaling and root planing in RPP patients.

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.