No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Model-based analysis of microbial consortia and microbial products in an anammox biofilm reactor
Abstract
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.
Supplementary resource (1)
... Granules for reactor 2 Granules for inoculating reactor 2 were from the activated carbon biofilm part of a landfill leachate treatment plant. 12,25 The initial information about process performance and microbial community structure of the seed sludge, biofilms and granules is provided in previous research works. 12,[25][26][27] Feeding, aeration, mixing, settling and discharging time and pH control were identical for both reactors throughout the operation and controlled using a programmable logic controller control system (IKS-Aquastar; IKS Systems, Karlsbad, Germany). ...
... 12,25 The initial information about process performance and microbial community structure of the seed sludge, biofilms and granules is provided in previous research works. 12,[25][26][27] Feeding, aeration, mixing, settling and discharging time and pH control were identical for both reactors throughout the operation and controlled using a programmable logic controller control system (IKS-Aquastar; IKS Systems, Karlsbad, Germany). Each primary cycle of the SBR lasted for 12 h, including filling, intermittent aeration, and anoxic mixing, settling and discharge. ...
... The procedure for FISH, including pretreatment of the sample, cell fixation, washing procedure, preparation of hybridization and washing buffer, application of probe and sample, hybridization, and washing procedure, is explained in supporting information S2. 25,35 For biomass visualization, epifluorescence microscopy (Axio Imager 2; Zeiss, Jena, Germany) and confocal scanning light microscopy (Zeiss LSM 510; Carl Zeiss, Jena, Germany) were used. ...
BACKGROUND
Two hybrid biofilm reactors, an integrated fixed‐film activated sludge–sequencing batch reactor (IFAS‐SBR) and a hybrid SBR with a mixture of activated sludge and granules, were used to reach enhanced nitrogen removal based on the anaerobic ammonium oxidation (anammox) process under intermittent aeration.
RESULTS
IFAS‐SBR reached a more stable performance after a rapid start‐up period of 25 days, and it showed a more reliable performance than the other reactor. The highest nitrogen removal efficiency (NRE) was up to 99% based on NH4⁺‐N concentration and 91% based on total nitrogen concentration (TIN). A nitrogen removal rate of 0.01 to 0.15 kgN m⁻³ d⁻¹ was achieved throughout the operation based on the TIN. The set‐point of maximum dissolved oxygen (DO) at the peak of each aeration cycle was maintained at 1.2 ± 0.1 mg L⁻¹. The optimum working range for daily average DO concentration was 0.2–0.7 mg L⁻¹. Cold temperatures caused a decline of NRE, but NRE > 50% for temperatures above 13 °C was achieved.
CONCLUSIONS
Results implied a higher abundance of Candidatus brocadia species of anammox bacteria in the biofilm samples and Denitratisoma genus in the flocs. The most substantial contribution of shared taxa among all samples at class level was Betaproteobacteria and Gammaproteobacteria, and the least abundant class was Nitrospira. Overall, results implied that IFAS‐SBR is a robust configuration for anammox‐based nitrogen removal in sidestream, and controlled intermittent aeration could suppress nitrite oxidation and help to regain system efficiency.
... reduction.Liu et al. (2016) studied multiple bacteria-substrate interactions through the exchange of SMPs in both 1-d and 2-d anammox biofilm models.Azari et al. (2018) compared two kinetic PN-A models, with and without SMP/EPS mechanisms, in a full-scale granular biofilm reactor. The model with SMP/EPS kinetics was found to better fit the relative biomass abundance.Chen et al. (2020b) evaluated the effects of heterotrophs growing on decay-released products on TN removal efficiency under different biom ...
Anammox-mediated systems have attracted considerable attention as alternative cost-effective technologies for sustainable nitrogen (N) removal from wastewater. This review comprehensively highlights the importance of understanding microbial metabolism in anammox-mediated systems under crucial operation parameters, indicating the potentially wide applications for the sustainable treatment of N-containing wastewater. The partial nitrification-anammox (PN-A), simultaneous PN-A and denitrification (SNAD) processes have demonstrated sustainable N removal from sidestream wastewater. The partial denitrification-anammox (PD-A) and denitrifying anaerobic methane oxidation-anammox (DAMO-A) processes have advanced sustainable N removal efficiency in mainstream wastewater treatment. Moreover, N2O production/emission hotspots are extensively discussed in anammox-based processes and are related to the dominant ammonia-oxidizing bacteria (AOB) and denitrifying heterotrophs. In contrast, N2O is not produced in the metabolism pathways of AnAOB and DAMO-archaea; Moreover, the actual contribution of N2O production by dissimilatory nitrate reduction to ammonium (DNRA) and DAMO-bacteria in their species remains uncertain. Thus, PD-A and DAMO-A processes would achieve reduction in greenhouse gas production, as well as energy consumption for the reliability of N removal efficiencies. In addition to reaction mechanisms, this review covers the mathematical models for simultaneous anammox, partial nitrification and/or denitrification (i.e., PN-A, PD-A, and SNAD). Promising NO3− reduction technologies by endogenous PD, sulfur-driven autotrophic denitrification, and DNRA by anammox are also discussed. In summary, this review provides a better understanding of sustainable N removal in anammox-mediated systems, thereby encouraging future investigation and exploration of the sustainable N bio-treatment from wastewater.
... On average 90.0% of the influent ammonium (758 mg/L) was removed and oxidized by the activated sludge systems; 24.8% of the inputted ammonium (76.6 mg/L), 98.5% of the inputted nitrite (45.8 mg/L), and 97.4% of the inputted nitrate (13.4 mg/L) were removed by the following biofilm system (Table S1). Previously, frequent sampling and independent analyses confirmed the enrichment of anammox bacteria in the activated carbon modules, and anammox biofilms were shaped as spherical granules with red color and different sizes (1 to 13 mm) (Azari et al., 2018(Azari et al., , 2017 (Fig. S1). ...
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.
... HB2 uses NO 2 − as an electron acceptor, while HB3 uses NO 3 − as electron acceptor total active biomass (Agrawal et al., 2017;Kindaichi et al., 2004;Ni et al., 2012). The implementation of extracellular polymeric substances (EPS) into the modeling has proved successful for better predicting the amount of heterotrophic biomass that occurs in autotrophic systems (Azari et al., 2018;Liu et al., 2016), however, the EPS production and all its related process were out of the scope of this study. ...
The behavior of heterotrophic bacteria growing in systems with low or no external supply of chemical oxygen demand (COD) has become more relevant within the wastewater context. Growth strategies help to clarify how bacteria behave and adapt to different environmental conditions. In the case of substrate limited conditions, research has been mainly focused on the k‐strategy, whereas another important strategy: the yield strategy has not been explored intensely. Some authors have, however, demonstrated the implications of bacteria pursuing the yield strategy when living in structured environments and facing low‐substrate concentrations. This study uses a one‐dimensional biofilm model to study the influence of the affinity constant, the maximum growth rate, and the growth yield on the heterotrophic formation of dinitrogen gas (N2) in a completely autotrophic partial nitritation anammox system. The effect of these parameters on the composition and the diversity of the heterotrophic community is also evaluated. In a first scenario, heterotrophic bacteria are allowed to grow only on the COD produced by biomass decay. In a second step, the competition with a second group of heterotrophs using external COD as electron donor is assessed. For both evaluated scenarios, the results suggest that the yield plays a crucial role in the heterotrophic biomass and dinitrogen gas formation. Moreover, in the case of the community diversity the yield seems to be the decisive parameter. Finally, we conceptually compared the K and the yield strategy and give some insight to the possibility of both either being closely related or even being the same strategy.
... One source of the difference between SS and VSS is also due to the boundEPS (non-soluble EPS) content in the biofilm solid matrix fixed on the carriers. This has been shown to contribute around 10 % of total solid content as it was discussed in the last chapter (Azari et al., 2018). ...
The anaerobic ammonium oxidation (anammox) process has become popular as energy saving, and cost-effective biological nitrogen removal because it shortens the ammonia removal cycle, and directly converts ammonium (NH4+) to nitrogen gas using nitrite (NO2−) as an electron acceptor. During recent years, mathematical models explaining interactions between autotrophic and heterotrophic microorganisms including ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria and denitrifiers proved to be substantial for cost reduction, optimization of the system performance and operating conditions. In this work, experimental and mathematical modeling approaches were combined to investigate mechanisms influencing system performance and microbial consortia dynamics in granular and biofilm reactors.
In the first chapter, the 10 years performance of biological treatment for high ammonium removal from a full-scale landfill leachate has been showed. The plant was upgraded combining the activated sludge process followed by granular activated carbon (GAC) biofilm reactor. Based on a long-term analysis, the average total nitrogen removal efficiency of 94 % was achieved for wastewaters with a C: N ratio varying from 1 to 5 kgCOD kgN−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 GAC particles in the form of biofilm. 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 (FISH) was used for microbial diversity analysis on floccular and granular sludge samples. Anammox bacteria and nitrifiers were detected and Candidatus Scalindua was found in two forms of flocs and biofilms. Due to stochastic risk assessment based, the treatment criteria were achieved and the combination of GAC biofilm process and activated sludge can be a sought approach to better enrich anammox biomass for full-scale treatment applications to reduce operating costs and promote nutrient removal stability and efficiency.
Second, a mathematical model is proposed and validated for biological nitrogen removal in a granular system to describe independent short-term batch processes under anoxic conditions. The activated sludge model consists of anammox and heterotrophic bacteria using a novel stoichiometric matrix. Identifiability of sensitive biokinetic parameters of the model was
(iii)
assessed with regards to observed concentrations of ammonium-, nitrite-, and nitrate-nitrogen. The Chi-squared function was used for the error estimation and the R-squared index (R2) was used for the regression analysis. The results imply that the model can elucidate the interactions of nitrogen converting bacteria for various feeding characteristics. The calibration results showed satisfactory R2 equal to 0.95 and 0.97 for NH4-N and NO2-N respectively. For validation, model simulations were performed under three varying scenarios and R2 was more than 0.9 so that all forecasted values lied within the 95 % confidence interval. In addition, the estimated physiologic characterization of two dominant anammox species was discussed upon calibration and validation of the model. The maximum specific bacterial growth rates (μmax) for Candidatus Brocadia anammoxidans and Ca. Scalindua sp. were estimated at 0.0025 h−1 and 0.0048 h−1 respectively. Decay rate of Ca. Brocadia anammoxidans was estimated at 0.0003 h−1 which is 15 % higher than decay rate of species belonging to Ca. Scalindua.
In the next step, a comprehensive model explaining the roles and functions of soluble microbial products (SMP) and extracellular polymeric substance (EPS) was developed. Therefore, the model for a granular reactor for the leachate treatment discussed in the first chapter 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 anammox, nitrifying and heterotrophic denitrifying bacteria which can attach and grow on GAC particles. Two kinetic descriptions for the model were proposed: with and without SMP and EPS. The model accuracy was checked using recorded total inorganic nitrogen concentrations in the effluent and estimated relative abundance of active bacteria using quantitative FISH (qFISH). The models with and without EPS successfully simulated the relative abundance of the biomass in the biofilm solid matrix, with a high agreement with results obtained from qFISH. Nevertheless, the model was improved after the addition of EPS and SMP kinetics and the real-time production of EPS and SMP can be predicted as well. The models could predict the dynamics of nitrogen transformation well, and a linear relationship between predicted and measured total nitrogen was achieved (R2 = 0.66 for the model with EPS and R2 = 0.61 for the model without EPS). Averagely, the models predicted the relative abundance of 52 % for anammox, 5.2 % for AOB, 2.6 % for NOB and 22 % for heterotrophs. For the model with EPS, the EPS content contributed between 4 to 10 % of total biomass volume. Results suggested that the model with EPS fits better for active relative abundance (the standard error
(iv)
was less than 10 % of observation). The model with EPS and SMP also confirms that the growth and existence of heterotrophs in anammox biofilm systems slightly increases 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 (NSL) on the biofilm.
In the last chapter, feasibility of simultaneous nitrification, anammox and denitrification (SNAD) was tested using an integrated fixed-film activated sludge-sequencing batch reactor IFAS-SBR and another SBR mixing activated sludge with granules. During operation of both plants, a regular reduction of temperature was applied, and various aeration regimes were evaluated. IFAS-SBR reached to a stable performance after a short startup period of 25 days and it showed a higher nitrogen removal rate (NRR) compared to another reactor. Highest NRE was able to reach up to 99.9 % based on NH4+-N concentration and 91.2 % based on total inorganic nitrogen (TIN) concentration. Whereas for NRR, the highest values are 0.14 kgN m−3 d−1 based on NH4+-N concentration and 0.15 kgN m−3 d−1 based on TIN concentration. The optimized range for daily average dissolved oxygen (DO) concentration was determined to be between 0.2 – 0.7 mg L-1 and maximum real-time DO at the end of each aeration cycle was set between 1.0 to 1.2 mg L-1. Reduction of temperature caused an expected decline of NRE, although a good adaptation was achieved for temperature above 15 °C. For example, after 75 days of the operation, the NRE of 71.1 % was achieved despite the temperature was reduced to 20oC. For microbial identification, 16S ribosomal RNA (16S rRNA) gene sequence analysis and qFISH were applied and AOB was major bacteria group in flocs and anammox bacteria and AOB were found in biofilms attached to the carriers.
... Ratios of CO 2 fixation over ammonium oxidation have been described for nitrifying bacteria with molar ratios of 0.0236 to 0.0868 of mol bicarbonate taken up per mol ammonium oxidized (Belser, 1984). In a model-based analysis of microbial consortia and microbial products in an anammox biofilm reactor, a similar ratio of 0.0831 has been determined, recently (Azari et al., 2018). ...
Biotransformation of methane at landfill sites can be estimated by applying compound specific stable isotope analysis of methane from the anaerobic and the cover layer surface zone. Next to these two input parameters, merely the knowledge of the carbon isotopic fractionation of the bacterial methane oxidation in terms of the enrichment factor (ε) is required. However, many factors and conditions have been described to affect ε. These include temperature, the applied landfill cover, the type of expressed methane monooxygenase (MMO), and cell density. In this work we investigated the microbial methane oxidation with respect to temperature and type of methanotrophic enrichment culture. A newly designed setup was used to overcome potential CH4-substrate limitations such as diffusion that could affect the determined values of ε by improper and inhomogeneous mixing. The isotopic fractionation was determined based on the stable carbon isotope analysis of methane and carbon dioxide. The obtained value for isotopic fractionation was ε22°C = −0.0136 ± 0.0036. Also for the first time, bulk stable isotope analysis of bacterial cell mass was performed by flow injection analysis isotope ratio mass spectrometry.
In deammonification systems, nitrite-oxidizing bacteria (NOB) suppression and nitrous oxide (N2O) mitigation are two important operational objectives. To carry out this multivariable analysis of response, a comprehensive model for the N cycle was developed and evaluated against experimental data from a laboratory-scale deammonification granular sludge sequencing batch reactor. Different aeration strategies were tested, and the manipulated variables comprised the dissolved oxygen (DO) set point in the aerated phase, aeration on/off frequency (F), and the ratio (R) between the non-aerated and aerated phase durations. Experimental results showed that a high ammonium utilization rate (AUR) in relation to the low nitrate production rate (NPR) (NPR/AUR = 0.07–0.08) and limited N2O emissions (EN2O < 2%) could be achieved at the DO set point = 0.7 mg O2/L, R ratio = 2, and F frequency = 6–7 h–1. Under specific operational conditions (biomass concentration, NH4+-N loading rate, and temperature), simulation results confirmed the feasible aeration strategies for the trade-offs between the NOB suppression and N2O emission. The intermittent aeration regimes led to frequent shifts in the predominating N2O production pathways, that is, hydroxylamine (NH2OH) oxidation (aerated phase) versus autotrophic denitrification (non-aerated phase). The inclusion of the extracellular polymeric substance mechanism in the model explained the observed activity of heterotrophs, especially Anaerolineae, and granule formation.
The aim of this study was to interpret the development of Anammox activity by a mathematical model in an UASB reactor -originally inoculated with methanogenic granules- at which Anammox progress has been also experimentally observed while treating chicken manure digestate. Since ammonium is derived from anaerobic degradation of nitrogenous compounds in chicken manure similar to any other nitrogen-rich organic wastes; the reactor was operated intentionally at favorable conditions [i.e.; with external nitrite source for NH4þ:NO2-ffi1.0] in order to make Anammox process to prevail as operation continued. Results indicated significant ammonia removals (60% on average) although influent concentration was gradually increased up to 200mg L-1. A modeling exercise has been undertaken to investigate the performance of the laboratory scale UASB reactor. In this scope, the experimental results were modeled by using Mantis2 model within GPSX 6.5 simulation software that included several built in libraries. Accordingly, effluent chemical oxygen demand (COD) and total ammonia nitrogen (TAN) concentrations could be predicted with reasonably good accuracy demonstrating successful calibration. The regression coefficient (R2) and mean relative absolute error (MRAE) parameters were found as 0.66 and 16% and 0.70 and 19%, respectively.
For eight months, a sequencing batch reactor (SBR) with integrated fixed-film activated sludge (IFAS) was operated in ambient temperature to study engineering and practical aspects of application of deammonification for mainstream conditions. For biofilm formation, K3 Kaldnes carriers were used, where the anaerobic ammonium oxidation (anammox) process can occur in deep layers of biofilm, while partial nitritation occurs in oxygen-rich outer layers. After the initial running phase of the reactor (Phase 1) to provide time for microorganisms to adapt, the COD: N ratio increased to around 2.6 in Phase 2 through reducing the ammonium concentration and increasing COD in synthetic wastewater to get closer to mainstream conditions. The total reaction time in each half-day batch cycle was kept 625 min throughout various phases, but the duration of intermittent aeration was regulated at 4 ± 1 min. While final nitrogen removal efficiency (NRE) for Phase 1 was 43%, at the end of Phase 2, it decreased to 37%. However, a maximum NRE at 90% was achieved during Phase 2. The identification of the responsible microorganisms was made through Fluorescence in situ hybridization (FISH), while Mixed Liquor Suspended Solid (MLSS) and Mixed Liquor Volatile Suspended Solid (MLVSS) was used to estimate the physical presence of bacteria. Ammonium oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AnAOB) were dominant bacteria, respectively. The adverse effects of a gradual increase of COD: N ratio from 0.17 to more than 2.0 caused a decline in NRE to around 15%.
Biological stability of treated wastewater is currently determined by methods such as biological oxygen demand, ATP-quantification, or flow-cytometric cell counting. However, the continuous increase in water reclamation for wastewater reuse requires new methods for quantifying degradation of biodegradable dissolved organic carbon (BDOC) ranging from very small to high concentrations of dissolved organic carbon (DOC). Furthermore, direct activity measures or absolute concentrations of BDOC are needed that produce comparable and reproducible results in all laboratories. Measuring carbon mineralization by CO 2 evolution presents a suitable approach for directly measuring the microbial degradation activity. In this work, we investigated the extent of BDOC in water samples from effluent of a wastewater treatment plant and after purification by ultrafiltration over 204 days. BDOC monitoring was performed with the recently introduced reverse stable isotope labeling (RIL) analysis using mid-infrared spectroscopy for the monitoring of microbial CO 2 production. Average BDOC degradation rates ranged from 0.11 to 0.32 mg L ⁻¹ d ⁻¹ for wastewater treatment plant effluent and from 0.03 to 0.22 mg L ⁻¹ d ⁻¹ after ultrafiltration. BDOC was degraded over >90 days indicating the long-term instability of the DOC. Degradation experiments over 88 days revealed first order kinetic rate constants for BDOC which corresponded to 12.7 · 10 ⁻³ d ⁻¹ for wastewater treatment plant effluent and 2.7 · 10 ⁻³ d ⁻¹ after ultrafiltration, respectively. A thorough sensitivity analysis of the RIL showed that the method is very accurate and sensitive with method detection limits down to 10 μg· L ⁻¹ of measured CO 2 .
Bacterial communities are closely interrelated systems consisting of numerous species making it challenging to analyze their structure and relations. At present, there are several experimental techniques providing heterogeneous data, concerning various aspects of this research object. The recent avalanche of available metagenomic data challenges not only biostatisticians but also biomodelers, since these data are essential for improving the modeling quality, while simulation methods are useful for understanding the evolution of microbial communities and their function in the ecosystem. An outlook on the existing modeling and simulation approaches based on different types of experimental data in the field of microbial ecology and environmental microbiology is presented. A number of approaches focused on the description of microbial community aspects such as trophic structure, metabolic and population dynamics, genetic diversity, as well as spatial heterogeneity and expansion dynamics, are considered. We also propose a classification of the existing software designed for the simulation of microbial communities. It has been shown that, in spite of the prevailing trend for using multiscale/hybrid models, the integration between models concerning different levels of biological organization of communities still remains a problem to be solved. The multiaspect nature of integration approaches used for modeling microbial communities is based on the necessity of taking into account the heterogeneous data obtained from various sources by applying high-throughput genome investigation methods.
Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic toot in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The law quality of experimental data has so far hampered the comparison and validation of the different growth models proposed and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For. example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in K-s for glucose from some 5 mg liter(-1) to ca. 30 mu g liter(-1). The data suggest that a dilemma exists, namely, that either "intrinsic" K-s (under substrate-controlled conditions in chemostat culture) or mu(max) (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously tin particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally the relevance of the kinetic principles obtained from defined culture systems with single mixed, or. multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered established and exploited.
In this study, the reclamation of a calcareous sodic soil with the exchangeable sodium percentage (ESP) value of 26.6 % was investigated using the cheap and readily available chemical and organic materials including natural bentonite and zeolite saturated with calcium (Ca2+), waste calcite, three metal oxide nanoparticles functionalized with an acidic extract of potato residues, and potato residues. Chemical amendments were added to the soil at a rate of 2 %, while potato residues were applied at the rates of 2 and 4 % by weight. The ESP in the amended soils was reduced in the range of 0.9–4.9 % compared to the control soil, and the smallest and the largest decline was respectively observed in treatments containing waste calcite and 4 % of potato residues. Despite the reduction in ESP, the values of this parameter were not below 15 % at the end of a 40-day incubation period. So, the effect of solutions of varying sodium adsorption ratio (SAR) values of 0, 5, 10, 20, 30, 40, and 50 on sodium (Na+) exchange equilibria was evaluated in batch systems. The empirical models (simple linear, Temkin, and Dubinin-Radushkevich) fitted well to experimental data. The relations of quantity to intensity (Q/I) revealed that the potential buffering capacity for Na+ (PBCNa) varied from 0.275 to 0.337 ((cmolc kg−1) (mmol L−1)−1/2) in the control soil and amended soils. The relationship between exchangeable sodium ratio (ESR) and SAR was individually determined for the control soil and amended soils. The values of Gapon selectivity coefficient (K
G) of Na+ differed from the value suggested by U.S. Salinity Laboratory (USSL). The PHREEQC, a geochemical computer program, was applied to simulate Na+ exchange isotherms by using the mechanistic cation exchange model (CEM) along with Gaines-Thomas selectivity coefficients. The simulation results indicated that Na+ exchange isotherms and Q/I and ESR-SAR relations were influenced by the type of counter anions. The values of K
G increased in the presence of bicarbonate, sulfate, and phosphate in comparison with the presence of chloride, and the largest value was obtained in the presence of phosphate. So, it can be concluded that the presence of chloride anion is more favorable to reduce ESP compared to other anions, while the presence of phosphate anion makes the reclamation process more difficult. Furthermore, it is possible to reclaim sodic soils using inexpensive and readily available compounds such as potato residues and water management.
Activated sludge models (ASMs) have been widely used for process design, operation and optimization in wastewater treatment plants. However, it is still a challenge to achieve an efficient calibration for reliable application by using the conventional approaches. Hereby, we propose a novel calibration protocol, i.e. Numerical Optimal Approaching Procedure (NOAP), for the systematic calibration of ASMs. The NOAP consists of three key steps in an iterative scheme flow: i) global factors sensitivity analysis for factors fixing; ii) pseudo-global parameter correlation analysis for non-identifiable factors detection; and iii) formation of a parameter subset through an estimation by using genetic algorithm. The validity and applicability are confirmed using experimental data obtained from two independent wastewater treatment systems, including a sequencing batch reactor and a continuous stirred-tank reactor. The results indicate that the NOAP can effectively determine the optimal parameter subset and successfully perform model calibration and validation for these two different systems. The proposed NOAP is expected to use for automatic calibration of ASMs and be applied potentially to other ordinary differential equations models.
Partial nitritation/anammox (PN/A) has been one of the most innovative developments in biological wastewater treatment in recent years. With its discovery in the 1990s a completely new way of ammonium removal from wastewater became available. Over the past decade many technologies have been developed and studied for their applicability to the PN/A concept and several have made it into full-scale. With the perspective of reaching 100 full-scale installations in operation worldwide by 2014 this work presents a summary of PN/A technologies that have been successfully developed, implemented and optimized for high-strength ammonium wastewaters with low C:N ratios and elevated temperatures. The data revealed that more than 50% of all PN/A installations are sequencing batch reactors, 88% of all plants being operated as single-stage systems, and 75% for sidestream treatment of municipal wastewater. Additionally an in-depth survey of 14 full-scale installations was conducted to evaluate practical experiences and report on operational control and troubleshooting. Incoming solids, aeration control and nitrate built up were revealed as the main operational difficulties. The information provided gives a unique/new perspective throughout all the major technologies and discusses the remaining obstacles.
Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.
Anaerobic ammonium oxidation (Anammox) is a cost-effective new process to treat high-strength nitrogenous wastewater. In this work, the microbial interactions of anaerobic ammonium oxidizers and heterotrophs through the exchange of soluble microbial products (SMP) in Anammox biofilm and the affecting factors were evaluated with both experimental and modeling approaches. Fluorescent in situ hybridization (FISH) analysis illustrated that Anammox bacteria and heterotrophs accounted for 77% and 23% of the total bacteria, respectively, even without addition of an external carbon source. Experimental results showed the heterotrophs could grow both on SMP and decay released substrate from the metabolism of the Anammox bacteria. However, heterotrophic growth in Anammox biofilm (23%) was significantly lower than that of nitrifying biofilm (30-50%). The model predictions matched well with the experimental observations of the bacterial distribution, as well as the nitrogenous transformations in batch and continuous experiments. The modeling results showed that low nitrogen surface loading resulted in a lower availability of SMP leading to low heterotrophic growth in Anammox biofilm, but high nitrogen surface loading would lead to relative stable biomass fractions although the absolute heterotrophic growth increased. Meanwhile, increasing biofilm thickness increased heterotrophic growth but has little influence on the relative biomass fractions.
Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.
Volume 62, no. 6, p. 2157, Table 1: the sequence for probe Nso1225, 5(prm1)-CGCGATTGTATTACGTGTGA-3(prm1), should read 5(prm1)-CGCCATTGTATTACGTGTGA-3(prm1). [This corrects the article on p. 2156 in vol. 62.].
Abstract A mathematical model is proposed and validated for biological nitrogen removal in a granular biofilm reactor to describe independent batch processes under anoxic conditions. The activated sludge model consists of anammox and heterotrophic bacteria using a novel stoichiometric matrix. Identifiability of sensitive biokinetic parameters of the model was assessed with regards to observed concentrations of ammonium-, nitrite-, and nitrate-nitrogen. The Chi-squared function was used for the error estimation and the R-squared index (R2) was used for the regression analysis. The results imply that the model can elucidate the interactions of nitrogen converting bacteria in a biofilm system for various feeding characteristics. The calibration results showed satisfactory R2 equal to 0.95, 0.97 and 0.67 for NH4-N, NO2-N and NO3-N respectively. For validation, model simulations were performed under three varying scenarios and R2 was more than 0.9 so that all forecasted values lied within the 95% confidence interval. In addition, the estimated physiologic characterization of two dominant anammox species was discussed upon calibration and validation of the model. The maximum specific bacterial growth rates (μmax) for Candidatus Brocadia anammoxidans and Ca. Scalindua sp. were estimated at 0.0025 h−1 and 0.0048 h−1 respectively. Decay rate of Ca. Brocadia anammoxidans was estimated at 0.0003 h−1 which is 15% higher than decay rate of species belonging to Ca. Scalindua.
The mainstream anaerobic ammonium oxidation (anammox) has attracted extensive attention recently, particularly due to its potential of transforming current wastewater treatment plants from energy consuming to energy neutral or positive. However, the presence of biodegradable chemical oxygen demanding (COD, 20∼80 mg COD L⁻¹) in the mainstream anammox reactor stimulates the growth of heterotrophic bacteria, which would compete for oxygen with ammonia-oxidizing bacteria (AOB) and for nitrite with anammox bacteria, thus interfering with the autotrophic nitrogen removal process. In the present work, with consideration of granule size distribution, a one-dimensional model describing the mainstream simultaneous partial nitrification, anammox and denitrification (SNAD) in a granule-based reactor was established, calibrated and validated, based on the long-term experimental results. Through applying the verified model, simulation studies were conducted and the results showed that the effluent total nitrogen concentration of < 5 mg N L⁻¹ could be achieved at C/N ratio of 0.2-0.6, DO concentration of 0.2-0.4 mg L⁻¹ and granule radius of 300-600 μm. The combined effects indicated that the SNAD process with TN removal efficiency > 90% was obtained at C/N ratio and DO concentration of 0.2-1.0 and 0.2-0.4 mg O2 L⁻¹ respectively. Finally, the various granule size distribution patterns were simulated, which confirmed that the size distribution needed to be incorporated in the model to accurately describe the granular anammox system, considering a model based on a uniform granule size does not reflect the real situations. These results provide guides to optimize the operation of mainstream granular autotrophic nitrogen removal process.
Achieving maintream anammox is critical for energy-neutral sewage treatment. This study presents a new way to achieve mainstream anammox, which couples anammox with denitratation (nitrate reduction to nitrite) instead of nitritation (ammonium oxidation to nitrite). An anoxic/oxic (A/O) biofilm system treating systhetic domestic wastewater was used to demonstrate this concept for over 400 days. This A/O biofilm system achieved a total nitrogen (TN) removal efficiency of 80±4% from the influent with a low C/N ratio of 2.6 and a TN concentration of 60.5 mg/L. Nitrogen removal via anammox was found to account for 70% of dinitrogen production in the anoxic reactor. Batch tests confirmed that the anoxic biofilm could oxidize ammonium using nitrite as electron acceptor, and that it had a higher nitrate reduction rate than the nitrite reduction rate, thus producing nitrite for the anammox reaction. Metagenomic analysis showed that Candidatus Jettenia caeni and Candidatus Kuenenia stuttgartiensis were the top two dominant species in anoxic biofilm. Genes involved in the metabolism of the anammox process were detected in anoxic biofilm. The abundance of nitrate reductase (73360 hits) was much higher than nitrite reductase (13114 hits) in anoxic biofilm. This system can be easily integrated with the high-rate activated sludge technology, which produces an effluent with a low C/N ratio. While this new design consumes 21% more oxygen in comparison to the currently studied nitritation/anammox process, the nitrite-producing process appears to be more stable.
A shortcut nitrogen removal process was investigated for treatment of high ammonium strength wastewater using an algal-bacterial consortium in photo-sequencing batch reactors (PSBRs). In this process, algae provide oxygen for nitritation during the light period, while denitritation takes place during the dark (anoxic) period, reducing overall energy and chemical requirements. Two PSBRs were operated at different solids retention times (SRTs) and fed with a high ammonium concentration wastewater (264 mg NH4⁺-N L⁻¹), with a '12 hour on, 12 hour off' light cycle, and an average surface light intensity of 84 μmol m⁻² s⁻¹. High total inorganic nitrogen removal efficiencies (∼95%) and good biomass settleability (sludge volume index 53-58 mL g⁻¹) were observed in both PSBRs. Higher biomass density was observed at higher SRT, resulting in greater light attenuation and less oxygen production. A mathematical model was developed to describe the algal-bacterial interactions, which was based on Activated Sludge Model No. 3, modified to include algal processes. Model predictions fit the experimental data well. This research also proposes an innovative holistic approach to water and energy recovery. Wastewater can be effectively treated in an anaerobic digester, generating energy from biogas, and later post-treated using an algal-bacterial PSBR, which produces biomass for additional biogas production by co-digestion.
Studies of microorganisms have traditionally focused on single species populations, which have greatly facilitated our understanding of the genetics and physiology that underpin microbial growth, adaptation and biofilm development. However, given that most microorganisms exist as multispecies consortia, the field is increasingly exploring microbial communities using a range of technologies traditionally limited to populations, including meta-omics based approaches and high resolution imaging. The experimental communities currently being explored range from relatively low diversity, e.g. two to four species, to significantly more complex systems, comprised of several hundred species. Results from both defined and undefined communities have revealed a number of emergent properties, including improved stress tolerance, increased biomass production, community level signalling and metabolic cooperation. Based on results published to date, we submit that community-based studies are timely and increasingly reveal new properties associated with multispecies consortia that could not be predicted by studies of the individual component species. Here, we review a range of defined and undefined experimental systems used to study microbial community interactions. This article is protected by copyright. All rights reserved.
In this study, a novel integrated anaerobic ammonium oxidization with partial denitrification process (termed as ANAMMOX-PD) was developed for advanced nitrogen removal from high-strength wastewater, which excess NO3⁻-N produced by ANAMMOX was fed into PD reactor for NO2⁻-N production and then refluxing to ANAMMOX reactor for further removal. Results showed that total nitrogen (TN) removal efficiency as high as 97.8% was achieved and effluent TN-N was below 20 mg/L at influent TN-N of 820 mg/L. Furthermore, the feasibility of simultaneously treating domestic wastewater was demonstrated in ANAMMOX-PD process, and NH4⁺-N removal efficiency of 96.7% was obtained. The nitrogen removal was mainly carried out through ANAMMOX pathway, and high-throughput sequencing revealed that Candidatus_Brocadia was the major ANAMMOX species. The presented process could effectively solve the problem of excess nitrate residual in ANAMMOX effluent, which hold a great potential in application of currently ANAMMOX treating high-strength wastewater (e.g. sludge digestion supernatant).
Anaerobic ammonium oxidation (anammox) is known to autotrophically convert ammonium to dinitrogen gas with nitrite as the electron acceptor, but little is known about their released microbial products and how these are relative to heterotrophic growth in anammox system. In this work, we applied a mathematical model to assess the heterotrophic growth supported by three key microbial products produced by bacteria in anammox biofilm (utilization associated products (UAP), biomass associated products (BAP), and decay released substrate). Both One-dimensional and two-dimensional numerical biofilm models were developed to describe the development of anammox biofilm as a function of the multiple bacteria–substrate interactions. Model simulations show that UAP of anammox is the main organic carbon source for heterotrophs. Heterotrophs are mainly dominant at the surface of the anammox biofilm with small fraction inside the biofilm. 1-D model is sufficient to describe the main substrate concentrations/fluxes within the anammox biofilm, while the 2-D model can give a more detailed biomass distribution. The heterotrophic growth on UAP is mainly present at the outside of anammox biofilm, their growth on BAP (HetB) are present throughout the biofilm, while the growth on decay released substrate (HetD) is mainly located in the inner layers of the biofilm.
From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in nature. ‘Aerobic’ ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol NH+4 min−1 (mg protein)−1 using nitrogen dioxide as electron acceptor. This reaction, which may involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH+4 min−1 (mg protein)−1 was determined. Although this reaction is 25-fold faster than in Nitrosomonas , it allowed growth at a rate of only 0.003 h−1 (doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual P 468 cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations.
Biological nitrogen removal from sewage via anammox is a promising and feasible technology to make sewage treatment energy-neutral or energy-positive. Good retention of anammox bacteria is the premise of achieving sewage treatment via anammox. Therefore the anammox metabolism and its factors were critically reviewed so as to form biofilm/granules for retaining anammox bacteria. A stable supply of nitrite for anammox bacteria is a real bottleneck for applying anammox in sewage treatment. Nitritation and partial-denitrification are two promising methods of offering nitrite. As such, the strategies for achieving nitritation in sewage treatment were summarized by reviewing the factors affecting nitrite oxidation bacteria growth. Meanwhile, the methods of achieving partial-denitrification have been developed through understanding the microorganisms related with nitrite accumulation and their factors. Furthermore, two cases of applying anammox in the mainstream sewage treatment plants were documented.
A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.
The frequency and distribution of putatively nitrite-oxidizing, Nitrospira-like bacteria in nitrifying biofilms from two reactors receiving wastewater with different ammonia and salt concentrations were observed by fluorescent in situ hybridization. For this purpose, new 16S rRNA-directed oligonucleotide probes targeting the bacterial phylum Nitrospira and the three main lineages within this phylum were developed and evaluated. The diversity of Nitrospira-like bacteria in the reactors was additionally investigated by retrieval and comparative analysis of full 16S rRNA sequences from the biofilms. We found that, despite of the differences in the influent composition, Nitrospira-like bacteria form dominant populations in both reactors. In addition, first insights into the physiology of these still unculturable bacteria were obtained by the incubation of active biofilm samples with radioactively labeled substrates followed by the combined application of fluorescent in situ hybridization and microautoradiography. The results are discussed in consideration of the frequently observed dominance of Nitrospira-like bacteria in nitrifying bioreactors. Consequently, high priority should be assigned to future studies on the ecology and physiology of these organisms in order to increase our fundamental understanding of nitrogen cycling and to enable knowledge-driven future improvements of nitrifying wastewater treatment plants.
Full-scale application of partial nitritation and anammox in a single floc-based sequencing batch reactor (SBR) has been achieved for high-rate nitrogen (N) removal, but mechanisms resulting in reliable operation are not well understood. In this work, a mathematical model was calibrated and validated to evaluate operating conditions that lead to out-competition of nitrite oxidizers (NOB) from the SBRs and allow to maintain high anammox activity during long-term operation. The validity of the model was tested using experimental data from two independent previously reported floc-based full-scale SBRs for N-removal via partial nitritation and anammox, with different aeration strategies at aeration phase (continuous vs. intermittent aeration). The model described the SBR cycle profiles and long-term dynamic data from the two SBR plants sufficiently and provided insights into the dynamics of microbial population fractions and N-removal performance. Ammonium oxidation and anammox reaction could occur simultaneously at DO range of 0.15-0.3 mg O2 L(-1) at aeration phase under continuous aeration condition, allowing simplified process control compared to intermittent aeration. The oxygen supply beyond prompt depletion by ammonium oxidizers (AOB) would lead to the growth of NOB competing with anammox for nitrite. NOB could also be washed out of the system and high anammox fractions could be maintained by controlling sludge age higher than 40 days and DO at around 0.2 mg O2 L(-1). Furthermore, the results suggest that N-removal in SBR occurs via both alternating nitritation/anammox and simultaneous nitritation/anammox, supporting an alternative strategy to improve N-removal in this promising treatment process, i.e., different anaerobic phases can be implemented in the SBR-cycle configuration.
Anammox related technologies are currently widely applied for nitrogen removal from sewage sludge digester rejection water. Nevertheless, many aspects of the anammox process like the kinetic characteristics and the reaction stoichiometry are still subject of debate. Parameter values reported in literature are often hampered by mass transfer limitation or by the presence of a significant side population. In this study a membrane bioreactor (MBR) based method for growing a highly enriched anammox microbial community is described. The almost pure free-cells suspension of highly active anammox bacteria was used for detailed kinetic and stoichiometric analysis of the anammox process. The anammox culture enriched during this study had a biomass specific maximum growth rate of 0.21 d(-)(1) which is higher than ever reported before in literature. Using an experimental methodology based on imposing dynamic process conditions combined with process modeling and parameter estimation, the intrinsic nitrite half saturation constant was identified to be as low as 35 μg-N L(-)(1). This was confirmed to be an accurate estimation in the pH range of 6.8-7.5.
Anaerobic Ammonia Oxidising (Anammox) biomass was enriched from sludge collected at a municipal wastewater treatment plant, employing a Sequential Batch Reactor (SBR). After 60 days Anammox activity started to be detected, by consumption of stoichiometric amounts of NO2− and NH4+ in the system. Fluorescence In Situ Hybridisation analysis confirmed the increase of Anammox bacteria concentration with time. A final concentration of enriched biomass of 3–3.5 gVSS dm−3 was obtained, showing a Specific Anammox Activity of 0.18 gNH4+-N gVSS−1 d−1 The reactor was able to treat nitrogen loading rates of up to 1.4 kgN m−3 d−1, achieving a removal efficiency of 82 %. On the other hand, the start-up and operation of the Anammox SBR reactor were consequentially modelled with the Activated Sludge Model nr 1, extended for Anammox. The simulations predicted quite well the experimental data in relation to the concentrations of nitrogenous compounds and can be used to estimate the evolution of Anammox and heterotrophic biomass in the reactor. These simulations reveal that heterotrophs still remain in the system after the start-up of the reactor and can protect the Anammox microorganisms from a negative effect of the oxygen. Copyright © 2004 Society of Chemical Industry
Soluble microbial products (SMP) formation kinetics were investigated by employing a laboratory-scale biofilm reactor and naturally-grown oligotrophs. The experimental results indicated that the majority of effluent soluble organic carbon(SOC) was SMP, while only a small fraction of the effluent SOC was the residual original substrate. Utilization-associated products (UAP), which were produced directly from substrate metabolism, were more important than biomass-associated products (BAP), which were produced by basic metabolism. The SMP contained mainly high-molecular-weight organic compounds, although the organic carbon source to a biofilm reactor was a low-molecular-weight compound. The steady-state concentrations of the effluent SMP and SOC were directly proportional to the influent substrate concentrations in this study. An extended steady-state biofilm model was developed by incorporating into the steady-state biofilm model an SMP formation model based on two types of SMP (i.e. UAP and BAP). The model described successfully the experimental substrate utilization, SMP formation, and the removal of total soluble organic matter (SOC).
A mathematical model was developed to describe the anaerobic ammonium oxidation (ANAMMOX) process in a granular upflow anaerobic sludge blanket (UASB) reactor. ANAMMOX granules were cultivated in the UASB reactor by seeding aerobic granules. The granule-based reactor had a great N-loading resistant capacity. The model simulation results on the 1-year reactor performance matched the experimental data well. The yield coefficient for the growth and the decay rate coefficient of the ANAMMOX granules were estimated to be 0.164 g COD g(-1) N and 0.00016 h(-1), respectively. With this model, the effects of process parameters on the reactor performance were evaluated. Results showed that the optimum granule diameter for the maximum N-removal should be between 1.0 and 1.3 mm and that the optimum N loading rate should be 0.8 kg N m(-3) d(-1). In addition, the substrate micro-profiles in the ANAMMOX granules were measured with a microelectrode to explore the diffusion dynamics within the granules, and the measured profiles matched the predicted results well.
Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gram-negative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.
We present a critical review of the relationships among three microbial products: extracellular polymeric substances (EPS), soluble microbial products (SMP), and inert biomass. Up to now, two different "schools" of researchers have treated these products separately. The "EPS school" has considered active biomass and EPS, while the "SMP school" has considered active biomass, SMP, and inert biomass. Here, we provide a critical review of each of the microbial products. Then, we develop a unified theory that couples them and reconciles apparent contradictions. In our unified theory, cells use electrons from the electron-donor substrate to build active biomass, and they also produce bound EPS and utilization-associated products (UAP) at the same time and in proportion to substrate utilization. Bound EPS are hydrolyzed to biomass-associated products (BAP), while active biomass undergoes endogenous decay to form residual dead cells. Finally, UAP and BAP, being biodegradable, are utilized by active biomass as recycled electron-donors substrates. Our unified theory shows that the apparently distinct products from the SMP and EPS schools overlap each other. Soluble EPS is actually SMP, or the sum of UAP and BAP. Furthermore, active biomass, as defined by the SMP school, includes bound EPS, while inert biomass includes bound EPS and the residual dead cells.
Combinations of microscopy and molecular techniques to detect, identify and characterize microorganisms in environmental and medical samples are widely used in microbial ecology and biofilm research. The scope of these methods, which include fluorescence in situ hybridization (FISH) with rRNA-targeted probes, is extended by digital image analysis routines that extract from micrographs important quantitative data. Here we introduce daime (digital image analysis in microbial ecology), a new computer program integrating 2-D and 3-D image analysis and visualization functionality, which has previously not been available in a single open-source software package. For example, daime automatically finds 2-D and 3-D objects in images and confocal image stacks, and offers special functions for quantifying microbial populations and evaluating new FISH probes. A novel feature is the quantification of spatial localization patterns of microorganisms in complex samples like biofilms. In combination with '3D-FISH', which preserves the 3-D structure of samples, this stereological technique was applied in a proof of principle experiment on activated sludge and provided quantitative evidence that functionally linked ammonia and nitrite oxidizers cluster together in their habitat. This image analysis method complements recent molecular techniques for analysing structure-function relationships in microbial communities and will help to characterize symbiotic interactions among microorganisms.
For the successful application of anaerobic ammonium oxidation (anammox) in wastewater practice it is important to know how to seed new anammox reactors with biomass from existing reactors. In this study, a new high salinity anammox reactor was inoculated with biomass from a freshwater system. The changes in activity and population shifts were monitored. It was shown that freshwater anammox bacteria could adapt to salt concentrations as high as 30 gl(-1) provided the salt concentration was gradually increased. Higher salt concentrations reversibly inhibited anammox bacteria. The nitrogen removal efficiency and maximum anammox activity of the salt adapted sludge was very similar to the reference freshwater sludge. Fluorescence in situ hybridization analysis revealed that the freshwater anammox species Candidatus "Kuenenia stuttgartiensis" was the dominant in both salt adapted sludge and freshwater sludge. These results show that gradual adaptation may be the key to successful seeding of anammox bioreactors.
Scalindua brodae', sp. nov., Candidatus 'Scalindua wagneri', sp. nov., two new species of anaerobic ammonium oxidizing bacteria
- M Schmid
- K Walsh
- R Webb
- W I C Rijpstra
- K Van De Passchoonen
- M J Verbruggen
- T Hill
- B Moffett
- J Fuerst
- S Schouten
- J S S Damsté
- J Harris
- P Shaw
- M Jetten
- M Strous
- Candidatus
Schmid, M., Walsh, K., Webb, R., Rijpstra, W. I. C., van de PasSchoonen, K., Verbruggen, M. J., Hill, T., Moffett, B., Fuerst,
J., Schouten, S., Damsté, J. S. S., Harris, J., Shaw, P., Jetten,
M. & Strous, M. Candidatus 'Scalindua brodae', sp. nov.,
Candidatus 'Scalindua wagneri', sp. nov., two new species of
anaerobic ammonium oxidizing bacteria. Syst. Appl.
Microbiol. 26, 529-538. doi:10.1078/072320203770865837.
A novel protocol for model calibration in biological wastewater treatment
- Sánchez