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 cd 1-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.

Image processing for identification and quantification of filamentous bacteria in in situ acquired images.

BACKGROUND: In the activated sludge process, problems of filamentous bulking and foaming can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy, commonly combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The in situ microscope appears as a suitable tool for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and eliminating sampling, preparation and transport of samples. In this context, a proper image processing algorithm is proposed for automated recognition and measurement of filamentous objects. METHODS: This work introduces a method for real-time evaluation of images without any staining, phase-contrast or dilution techniques, differently from studies present in the literature. Moreover, we introduce an algorithm which estimates the total extended filament length based on geodesic distance calculation. For a period of twelve months, samples from an industrial activated sludge plant were weekly collected and imaged without any prior conditioning, replicating real environment conditions. RESULTS: Trends of filament growth rate-the most important parameter for decision making-are correctly identified. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72 % of the filaments pixels, with a false positive rate of at most 14 %. An average execution time of 0.7 s per image was achieved. CONCLUSIONS: Experiments have shown that the designed algorithm provided a suitable quantification of filaments when compared with human perception and standard methods. The algorithm's average execution time proved its suitability for being optimally mapped into a computational architecture to provide real-time monitoring.

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.

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge.

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.

Performance evaluation and model-based optimization of the mainstream deammonification in an integrated fixed-film activated sludge reactor.

This study aimed to model and optimize mainstream deammonification in an integrated fixed-film activated sludge (IFAS) pilot plant under natural seasonal temperature variations. The effect of gradually decreasing temperature on the performance was evaluated during a winter season and a transition period to summer conditions, and the correlation of the performance parameters was investigated using principal component analysis (PCA). The optimization of intermittent aeration in the long-term (30 days) dynamic conditions with on/off ratio and dissolved oxygen (DO) set-point control was used to maximize the N-removal rate (NRR) and N-removal efficiency (NRE). Optimization results (DO set-point of 0.2-0.25 mgO2/L, and on/off ratio of 0.05) increased the NRE and NRR of total inorganic N (daily average) from 30% to > 50% and 15 gN/m3d to 25 gN/m3d, respectively. This novel long-term optimization strategy is a powerful tool for enhancing the efficiency in mainstream deammonification.

Discovery of a new genus of anaerobic ammonium oxidizing bacteria with a mechanism for oxygen tolerance.

In the past 20 years, there has been a major stride in understanding the core mechanism of anaerobic ammonium-oxidizing (anammox) bacteria, but there are still several discussion points on their survival strategies. Here, we discovered a new genus of anammox bacteria in a full-scale wastewater-treating biofilm system, tentatively named "Candidatus Loosdrechtia aerotolerans". Next to genes of all core anammox metabolisms, it encoded and transcribed genes involved in the dissimilatory nitrate reduction to ammonium (DNRA), which coupled to oxidation of small organic acids, could be used to replenish ammonium and sustain their metabolism. Surprisingly, it uniquely harbored a new ferredoxin-dependent nitrate reductase, which has not yet been found in any other anammox genome and might confer a selective advantage to it in nitrate assimilation. Similar to many other microorganisms, superoxide dismutase and catalase related to oxidative stress resistance were encoded and transcribed by "Ca. Loosdrechtia aerotolerans". Interestingly, bilirubin oxidase (BOD), likely involved in oxygen resistance of anammox bacteria under fluctuating oxygen concentrations, was identified in "Ca. Loosdrechtia aerotolerans" and four Ca. Brocadia genomes, and its activity was demonstrated using purified heterologously expressed proteins. A following survey of oxygen-active proteins in anammox bacteria revealed the presence of other previously undetected oxygen defense systems. The novel cbb3-type cytochrome c oxidase and bifunctional catalase-peroxidase may confer a selective advantage to Ca. Kuenenia and Ca. Scalindua that face frequent changes in oxygen concentrations. The discovery of this new genus significantly broadens our understanding of the ecophysiology of anammox bacteria. Furthermore, the diverse oxygen tolerance strategies employed by distinct anammox bacteria advance our understanding of their niche adaptability and provide valuable insight for the operation of anammox-based wastewater treatment systems.

Application of pyritic sludge with an anaerobic granule consortium for nitrate removal in low carbon systems.

Granules recovered from a highly reduced anaerobic digester were capable of active nitrogen removal in the absence of exogenous electron donors, averaging 0.25 mg mgNO3--N /gVSS/d over 546 days of operation. Electron mass balance indicated that about half the influent nitrate was converted to ammonia via DNRA and another half denitrified. This capacity was associated with an onion-like structure of multiple layers enriched in reduced iron and sulfur, and a complex microbial community shown by metagenomic sequencing to consist of multiple physiological groups and associated activities, including methanogenesis, denitrification, dissimilatory nitrate reduction to ammonia (DNRA), iron oxidation and reduction, and sulfur reduction and oxidation. Nitrate reduction was supported by both entrained organic material and reduced iron and sulfur species, corresponding to 2.13 mg COD/gVSS/d. Batch incubations showed that approximately 15% of denitrified nitrate was coupled to the oxidation of sulfur derived from both sulfate respiration and granular material enriched in iron-sulfide. Inhibition of sulfate reduction resulted in redirection of electron flow to methanogenesis and, in combination with other batch tests, showed that these granules supported a complex microbial community in which cryptic redox cycles linked carbon, sulfur, and iron oxidation with nitrate, sulfate, iron, and carbon dioxide reduction. This system shows promise for treatment of nitrate contaminated ground water without addition of an external organic carbon source as well as wastewater treatment in combination with (granular) sludge elimination leading in a net reduction of solid treatment costs.

Activities and metabolic versatility of distinct anammox bacteria in a full-scale wastewater treatment system.

Anaerobic ammonium oxidation (anammox) is a key N2-producing process in the global nitrogen cycle. Major progress in understanding the core mechanism of anammox bacteria has been made, but our knowledge of the survival strategies of anammox bacteria in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Here, by combining metagenomics with in situ metatranscriptomics, complex anammox-driven nitrogen cycles in an anoxic tank and a granular activated carbon (GAC) biofilm module of a full-scale WWTP treating landfill leachate were constructed. Four distinct anammox metagenome-assembled genomes (MAGs), representing a new genus named Ca. Loosdrechtii, a new species in Ca. Kuenenia, a new species in Ca. Brocadia, and a new strain in "Ca. Kuenenia stuttgartiensis", were simultaneously retrieved from the GAC biofilm. Metabolic reconstruction revealed that all anammox organisms highly expressed the core metabolic enzymes and showed a high metabolic versatility. Pathways for dissimilatory nitrate reduction to ammonium (DNRA) coupled to volatile fatty acids (VFAs) oxidation likely assist anammox bacteria to survive unfavorable conditions and facilitate switches between lifestyles in oxygen fluctuating environments. The new Ca. Kuenenia species dominated the anammox community of the GAC biofilm, specifically may be enhanced by the uniquely encoded flexible ammonium and iron acquisition strategies. The new Ca. Brocadia species likely has an extensive niche distribution that is simultaneously established in the anoxic tank and the GAC biofilm, the two distinct niches. The highly diverse and impressive metabolic versatility of anammox bacteria revealed in this study advance our understanding of the survival and application of anammox bacteria in the full-scale wastewater treatment system.

Specific and effective detection of anammox bacteria using PCR primers targeting the 16S rRNA gene and functional genes.

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the nitrogen cycle by coupling ammonium and nitrite to produce dinitrogen gas (N2). Polymerase chain reaction (PCR) is a fast, simple, and sensitive method that is widely used to assess the diversity, abundance, and activity of the slow-growing bacteria. In this review, we summarize and evaluate the wide variety of PCR primers targeting the 16S rRNA gene and functional genes (hzo, nir, and hzs) of anammox bacteria for their effectiveness and efficiencies in detecting this group of bacteria in different sample types. Furthermore, the efficiencies of different universal high-throughput sequencing 16S rRNA gene primers in anammox bacteria investigations were also evaluated to provide a reference for primer selection. Based on our in silico evaluation results, none of the 16S rRNA gene primers could recover all of the known anammox bacteria, but multiple hzo and hzs gene primers could accomplish this task. However, uncertain copies (1-3 copies) of hzo genes were identified in the genomes, and the hydrazine oxidation reaction catalyzed by hydrazine oxidoreductases (HZOs) can also be catalyzed by other hydroxylamine oxidoreductases (HAOs) in anammox bacteria, which can potentially result in large deviations in hzo-based qPCR and RT-qPCR analyses and results. Therefore, the use of optimal primers targeting unique hzs genes are recommended, although the efficiencies of these newly designed primers need further verification in practical applications. This article provides comprehensive information for the effective and specific detection of anammox bacteria using specific primers targeting the 16S rRNA gene and functional genes and serves as a basis for future high-quality primer design.

Foundations of the participatory approach in the Mekong River basin management.

Integrated Water Resource Management (IWRM) was acknowledged as a leading concept in the water management for the last two decades by academia, political decision-makers and experts. It strongly promotes holistic management and participatory approaches. The flexibility and adaptability of IWRM concept are especially important for large, transboundary river basins - e.g. the Mekong river basin - where natural processes and hazards, as well as, human-made "disasters" are demanding for a comprehensive approach. In the Mekong river basin, the development and especially the enforcement of one common strategy has always been a struggle. The past holds some unsuccessful experiences. In 2016 Mekong River Commission published IWRM-based Basin Development Strategy 2016-2020 and The Mekong River Commission Strategic Plan 2016-2020. They should be the main guiding document for the Mekong river development in the near future. This study analyzes how the concept of public participation resembles the original IWRM participatory approach in these documents. Therefore, IWRM criteria for public participation in international literature and official documents from the Mekong river basin are compared. As there is often a difference between "de jure" and "de facto" implementation of public participation in management concepts, the perception of local stakeholders was assessed in addition. The results of social survey give an insight if local people are aware of Mekong river basin development and present their dominant attitudes about the issue. The findings enable recommendations how to mitigate obstacles in the implementation of common development strategy.

More than a decade of experience of landfill leachate treatment with a full-scale anammox plant combining activated sludge and activated carbon biofilm.

The performance of biological treatment for high ammonium removal from landfill leachate has been demonstrated. The plant was upgraded combining the activated sludge process followed by activated carbon reactor. Based on a long-term analysis of data collected from 2006 to 2015, the average total nitrogen removal efficiency of 94% was achieved for wastewaters with a C: N ratio varying from 1 to 5 kg-COD kg-TN-1. But without the presence of activated carbon reactor, the average of biological removal efficiency for total nitrogen was only 82% ± 6% for the activated sludge stage. It means that up to 20% of the nitrogen in the influent can only be eliminated by microorganisms attached to granular activated carbon. After upgrades of the plant, the energy efficiency showed a reduction in the specific energy demand from 1.6 to less than 0.2 kWh m-3. Methanol consumption and sludge production was reduced by 91% and 96%, respectively. Fluorescent in situ Hybridization was used for microbial diversity analysis on floccular sludge and granular biofilm samples. Anaerobic ammonium oxidation (anammox) bacteria and nitrifiers were detected and Candidatus Scalindua was found in two forms of flocs and biofilms. Due to stochastic risk assessment based on the long-term data analysis given in this research, the treatment criteria were achieved and the combination of granular activated carbon biofilm process and activated sludge can be a novel and sought approach to better enrich anammox biomass for full-scale treatment applications to reduce operating costs and promote nutrient removal stability and efficiency.

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.

Evaluating the influence of wastewater composition on the growth of Microthrix parvicella by GCxGC/qMS and real-time PCR.

This study underlines the significance of long chain fatty acid (LCFA) content in wastewater influents as an influencing factor promoting the growth of Candidatus 'Microthrix parvicella' (M. parvicella), the most common filamentous bacteria causing foam in activated sludge systems worldwide. Quantification of M. parvicella by real-time polymerase chain reaction (real-time PCR) and analysis of LCFAs by means of two-dimensional gas chromatography coupled with mass spectrometry (GCxGC/qMS), involving solid phase micro-extraction (SPME) to enhance sensitivity, were combined for the first time as a monitoring tool. The results indicate a highly significant correlation between the abundance of M. parvicella and the total LCFA loading (r = 0.96) and linolenic acid C18:3 (r = 0.98) in particular. Additionally, comparison of slope values for the direct correlations of all significant LCFAs found in the analyses showed that the influence of LCFAs on M. parvicella growth increases with an increasing degree of unsaturation of carbon chains. These findings suggest that by removing lipid compounds from the incoming waters, substrate availability would be limited for M. parvicella.