Martin Denecke

Cloning, sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC-Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.

Protein analysis as a measure of active biomass in activated sludge.

Conventional methods to determine the biomass in activated sludge are the measurement of total suspended solids (TSS) and volatile suspended solids (VSS). Such methods do not distinguish between active biomass and inactive organic material. In this study, biomass was determined with both conventional methods and also through measuring the protein content with the modified Lowry method. In order to investigate the relationship between activity and biomass concentration in terms of TSS, VSS and protein content, some starvation experiments were conducted. It was found that the protein fraction of VSS differs under different starvation conditions. The biological activity of the activated sludge was measured as oxygen uptake rate (OUR). The strongest correlation could be measured between protein and OUR under various conditions. The results show that protein is an appropriate parameter for the measurement of the biological activity.

Characterization of methane oxidation in a simulated landfill cover system by comparing molecular and stable isotope mass balances

Biological methane oxidation may be regarded as a method of aftercare treatment for landfills to reduce climate relevant methane emissions. It is of social and economic interest to estimate the behavior of bacterial methane oxidation in aged landfill covers due to an adequate long-term treatment of the gas emissions. Different approaches assessing methane oxidation in laboratory column studies have been investigated by other authors recently. However, this work represents the first study in which three independent approaches, ((i) mass balance, (ii) stable isotope analysis, and (iii) stoichiometric balance of product (CO2) and reactant (CH4) by CO2/CH4-ratio) have been compared for the estimation of the biodegradation by a robust statistical validation on a rectangular, wide soil column. Additionally, an evaluation by thermal imaging as a potential technique for the localization of the active zone of bacterial methane oxidation has been addressed in connection with stable isotope analysis and CO2/CH4-ratios. Although landfills can be considered as open systems the results for stable isotope analysis based on a closed system correlated better with the mass balance than calculations based on an open system. CO2/CH4-ratios were also in good agreement with mass balance. In general, highest values for biodegradation were determined from mass balance, followed by CO2/CH4-ratio, and stable isotope analysis. The investigated topsoil proved to be very suitable as a potential cover layer by removing up to 99% of methane for CH4 loads of 35-65gm-2d-1 that are typical in the aftercare phase of landfills. Finally, data from stable isotope analysis and the CO2/CH4-ratios were used to trace microbial activity within the reactor system. It was shown that methane consumption and temperature increase, as a cause of high microbial activity, correlated very well.

Assessment of molecular detection of anaerobic ammonium-oxidizing (anammox) bacteria in different environmental samples using PCR primers based on 16S rRNA and functional genes

Eleven published PCR primer sets for detecting genes encoding 16S ribosomal RNA (rRNA), hydrazine oxidoreductase (HZO), cytochrome cd1-containing nitrite reductase (NirS), and hydrazine synthase subunit A (HzsA) of anaerobic ammonium-oxidizing (anammox) bacteria were assessed for the diversity and abundance of anammox bacteria in samples of three environments: wastewater treatment plant (WWTP), wetland of Mai Po Nature Reserve (MP), and the South China Sea (SCS). Consistent phylogenetic results of three biomarkers (16S rRNA, hzo, and hzsA) of anammox bacteria were obtained from all samples. WWTP had the lowest diversity with Candidatus Kuenenia dominating while the SCS was dominated by Candidatus Scalindua. MP showed the highest diversity of anammox bacteria including C. Scalindua, C. Kuenenia, and Candidatus Brocadia. Comparing different primer sets, no significant differences in specificity for 16S rRNA gene could be distinguished. Primer set CL1 showed relatively high efficiency in detecting the anammox bacterium hzo gene from all samples, while CL2 showed greater selectivity for WWTP samples. The recently reported primer sets of the hzsA gene resulted in high efficiencies in detecting anammox bacteria while nirS primer sets were more selective for specific samples. Results collectively indicate that the distribution of anammox bacteria is niche-specific within different ecosystems and primer specificity may cause biases on the diversity detected.

In situ microscopy as a tool for the monitoring of filamentous bacteria: a case study in an industrial activated sludge system dominated by M. parvicella

The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.

Population Dynamic of Microbial Consortia in a Granular Activated Carbon-Assisted Biofilm Reactor: Lessons from Modelling

In this work, experimental and long-term mathematical modelling approaches were combined to investigate mechanisms and drivers influencing on microbial consortia dynamics in an anaerobic granular biofilm reactor whereby dominantly anaerobic ammonium oxidation (anammox) and heterotrophic denitrifying bacteria can attach and grow on granular activated carbon (GAC). For this mean, a novel biofilm model including soluble microbial products (SMP) and extracellular polymeric substance (EPS) was developed to explain kinetics and abundance of independent microbial groups in terms of relative biovolume fraction and the spatial localization of bacteria in biofilm layers. The model was calibrated, validated and the model accuracy was checked using measured total nitrogen concentration and microbial biovolume fractions. For estimation of biovolume fraction an innovative enhanced protocol for quantitative fluorescence in-situ hybridization (qFISH), in-situ microscopy and digital image analysis for the cultivation-independent microorganisms was established. The model with EPS kinetics fits better for the bacteria groups of anammox bacteria, AOB and NOB compared to the model without EPS. Real-time producing BAP and UAP is simulated and it was presumed that he growth and existence of heterotrophs in anammox biofilm systems increase due to considering the autotrophic production of SMP.

Model-based analysis of microbial consortia and microbial products in an anammox biofilm reactor

A mathematical model for a granular biofilm reactor for leachate treatment was validated by long-term measured data to investigate the mechanisms and drivers influencing biological nitrogen removal and microbial consortia dynamics. The proposed model, based on Activated Sludge Model (ASM1), included anaerobic ammonium oxidation (anammox), nitrifying and heterotrophic denitrifying bacteria which can attach and grow on granular activated carbon (GAC) particles. Two kinetic descriptions for the model were proposed: with and without soluble microbial products (SMP) and extracellular polymeric substance (EPS). The model accuracy was checked using recorded total inorganic nitrogen concentrations in the effluent and estimated relative abundance of active bacteria using quantitative fluorescence in-situ hybridization (qFISH). Results suggested that the model with EPS kinetics fits better for the relative abundance of anammox bacteria and nitrifying bacteria compared to the model without EPS. The model with EPS and SMP confirms that the growth and existence of heterotrophs in anammox biofilm systems slightly increased due to including the kinetics of SMP production in the model. During the one-year simulation period, the fractions of autotrophs and EPS in the biomass were almost stable but the fraction of heterotrophs decreased which is correlated with the reduction in nitrogen surface loading on the biofilm.

Illumina sequencing for the identification of filamentous bulking and foaming bacteria in industrial activated sludge plants

In this study, Illumina sequencing was used for the identification of bulking and foaming bacteria in industrial wastewater treatment plants. The reliable identification of bulking and foaming bacteria represents the first step in developing effective and specific control strategies to avoid disturbances in activated sludge systems. Illumina sequencing revealed 432 16S rRNA operational taxonomic units, representing phylotypes and including 21 bulking and foaming bacteria in the two investigated industrial wastewater treatment plants. Foaming represents the most severe problem in the cascade biology system. Up to 22.5% of all sequencing reads are bulking and foaming bacteria, including Chryseobacterium, Candidatus Microthrix parvicella and Gordonia sp. as the dominant bulking and foaming bacteria which are known for foam formation. Moreover, Illumina sequencing revealed an increase in Candidatus Microthrix parvicella and Gordonia sp. reads from activated sludge to foam and scum samples, indicating a preferred flotation and/or growth advantages in the foam and scum layers. Analyses of the taxonomic assignment and distribution showed that the phylum Actinobacteria is the most dominant phylum, underlining the key role of Actinobacteria in bulking and foaming. Multivariate data analysis was applied, revealing that the dominant bulking and foaming bacteria are positively correlated with the sludge age and influent flow and negatively correlated with the dissolved oxygen level and the temperature. In terms of developing a specific control strategy, the positive linear relationships to the fatty acid and surfactant sludge loadings are highlighted and the removal of lipid compounds from the wastewater influent could avoid an overgrowth of bulking and foaming bacteria.

Water re‐use in practice

Closing water loops in industry and the reuse of wastewater for irrigation purposes should not only be practiced in industrialised countries but it is extremely important in arid or semi‐arid regions. This paper shows, on the basis of three case studies, the possibilities of environmental protection on the one hand, and the achievement of financial benefits on the other hand by introducing membrane technology. The case studies are about three different fields, in order to describe that the application of membrane technology leads to an effective water treatment, which makes it possible – depending on the application – to recover valuable materials or to provide suitable service water. In the case of combining biological wastewater treatment and membrane technology the potential of reusing municipal wastewater for irrigation purposes will fit the increasing demands in this sector.

Das 3A-Verfahren: Eine innovative Kombination aus Biogaserzeugung und Abfallbehandlung

Bei der Vergarung konnen im Gegensatz zu der Kompostierung pro Tonne eingesetztem Bioabfall zusatzlich ca. 80–140 m3 Biogas erzeugt werden [1, 2]. Die Vergarung hat weiterhin den Vorteil, das auch strukturarme und flussige Abfalle, die bei der Kompostierung problematisch sind behandelt werden konnen. Aus den genannten Grunden findet die Vergarung in zunehmendem Mase Eingang in die Behandlung biogen-organischer Abfallstoffe, zumal die Investitionskosten fur Vergarungsanlagen denen von gekapselten Kompostierungsanlagen gleichen [3]. Da das Endprodukt der Vergarung den gleichen Qualitatsanspruchen wie dem “konventionell”; unter aeroben Bedingungen erzeugten Kompost entsprechen und mit ihnen konkurrieren mus (Rottegrad, Geruchsfreiheit, Hygienisierung etc.), wird der Vergarung meist noch eine Kompostierung angeschlossen [4, 5]. Um die Vorteile beider Prozesse in einem Verfahren zu nutzen, wurde von der DR. -ING STEFFEN Ingenieurgesellschaft mbH mit Hilfe einer halbtechnischen Versuchsanlage ein kombiniertes Kompostierungs-und Vergarungsverfahren -das 3A-Verfahren- entwickelt.