How does shear affect aggregation in granular sludge sequencing batch reactors? Relations between shear, hydrophobicity, and extracellular polymeric substances.

The objective was to provide an answer to "how to grow/survive in aggregative physiology" through evaluating the relation between physical stress and observed biomass characteristics. For that, a lab-scale sequencing batch reactor was operated at an anaerobic-aerobic mode and under altered hydraulic selection pressures of settling time (10-1 min) and hydrodynamic shear rates due to mechanical mixing (15.5-12.0 cm/s) and/or aeration (1.76-0.24 cm/s). Main physical stress experienced by the biomass was mechanical mixing, which resulted in extreme shearing conditions at the first operational stage (days 1-86), during which first granules formed but settling properties deteriorated and biomass was almost totally washed out. After relaxing the overall shear stress at the second stage, biomass formation accelerated, settling properties enhanced and granulation proceeded (days 86-136), until disturbance of the process at the last month of operation (days 136-163). Aggregative physiology-related parameters, being cell surface hydrophobicity and extracellular polymeric substances (EPS), followed increasing trends parallel to the progress of granulation, and then decreased upon disturbance of the process. There was an increase in the EPS production also during the first stage under extreme shear, while a substantial amount of biomass was present in the system. A direct correlation was also found between %hydrophobicity and EPS-composition expressed as ExoPN/ExoPS.

Membrane technology revolutionizes water treatment.

Membranes play a crucial role in living cells, plants and animals. They not only serve as barriers between the inside and outside world of cells and organs. More importantly, they are means of selective transport of materials and host for biochemical conversion. Natural membrane systems have demonstrated efficiency and reliability for millions of years and it is remarkable that most of these systems are small, efficient and highly reliable even under rapidly changing ambient conditions. Thus, it appears to be advisable for technology developers to keep a close eye on Mother Nature. By doing so it is most likely that ideas for novel technical solutions are born. Following the concept of natural systems it is hypothesized that the Millennium Development Goals can be best met when counting on small water and wastewater treatment systems. The core of such systems could be membranes in which chemical reactions are integrated allowing recovery and direct utilization of valuable substances.

Biological nitrogen removal with three different SBBR.

In order to compare the performance of biofilms growing on different support media, three reactors were fed with municipal wastewater from the city of Garching, Germany, and operated under the sequencing batch procedure. The support media tested have the commercial names of Kaldnes, polyethylene special support for moving bed reactors with approximate diameter of 12 mm; Liapor, ceramic spheres with diameters between 4 and 6 mm; Linpor, plastic foam cut in cubes of 15 mm. The bench-top reactors were tested for COD, TSS and ammonia nitrogen removal. During 452 days runs with organic loads between 0.5 and 8.0 gCOD/m2 x d were tested. Thin biofilms (Kaldnes and Liapor) perform better for COD and ammonia removal under lower organic loading values (< 2.5 gCOD/m2 x d). For organic loads over 3.0 gCOD/m2 x d, the reactor packed with Linpor (thick biofilm) showed a better COD and ammonia nitrogen removal than the other two. Linpor achieved the highest NOx-N production reaching values between 15 and 20 mg/l. For low organic loading rates Linpor and Liapor present similar average NOx-N concentrations. Kaldnes shows the lowest concentrations throughout the whole experimental period. The difference between ammonia nitrogen removal and NOx-N generation is simultaneous denitrification inside the deep biofilms. The average mean cellular retention times were 5.4 days for Liapor, 10.0 days for Kaldnes and 22.9 days for Linpor. This is the reason why Linpor achieved complete nitrification even with higher organic loads.

Infiltration of a copper roof runoff through artificial barriers.

On-site infiltration of a copper roof runoff may contribute to deterioration of the ground and ground water. To avoid such a negative effect the performance of two different technical systems, equipped with four different barrier materials, regarding copper elimination was examined in a field study. During the period March 2004 to January 2005, 16 rain events were examined. Copper concentrations between 200 and 11,000 microg/L in the roof runoff during a rain event were observed. The cover material of the roof and the drainage system were responsible for the high concentrations of copper in the roof runoff. It was evident that roof aspects facing towards the wind direction were receiving higher rainfall, thus were establishing higher copper runoff rates. The retention facilities have reached a performance of up to 97% regarding copper elimination.

Modelling COD and N removal in the water and in the benthic biofilm for the River Wupper in Germany.

The River Wupper, a tributary of the River Rhine, is at several locations influenced by anthropogenous nitrogen input, hydraulic structures, and influents from other tributaries. These influences have an impact both on the water quality and on the hydrodynamic conditions. The model approaches used for this article are based on work of Rauch et al. and the River Water Quality Model No. 1; they allow the simulation of the nitrogen conversion in the River Wupper. They are compatible with the activated sludge models and can thus be used also for integrated approaches. The calibration and validation of the model was realized using actual data of the River Wupper over a length of 60 km with one dam, 10 weirs, three wastewater treatment plants and 11 tributaries. The model considers the nitrogen conversion and COD removal and has a strong focus on biofilm processes in the benthic zone. Additional information is given about the sedimentation processes, the physical oxygen input processes, biofilm detachment processes, molecular diffusion, the influence of the laminar border layer and the changing of COD fractions and biofilm densities.

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests.

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Mesophilic-thermophilic-mesophilic anaerobic digestion of liquid dairy cattle manure.

The potential of a mesophilic-thermophilic-mesophilic anaerobic digestion system was investigated with respect to improvement of both digestion and sanitation efficiencies during treatment of liquid cattle manure. The pilot plant produced a high methane yield from liquid dairy cattle manure of 0.24 m3 (kg VSfed)(-1) Considering the low system loading rate of 1.4-1.5 kg VS (m3 d)(-1), digestion efficiency compared to conventional processes did not appear improved. The minimum guaranteed retention time in the tubular thermophilic reactor was increased compared to a continuously stirred tank reactor. Levels of intestinal enterococci in raw liquid manure as determined with cultivation methods were reduced by 2.5 -3 log units to a level of around 10(2) cfu/mL. This sanitizing effect was achieved both during mesophilic-thermophilic-mesophilic and thermophilic-mesophilic treatment, provided the thermophilic digester was operated at 53-55 degrees C. A change in feeding interval from 1 h to 4 h did not significantly alter methane yield and sanitation efficiency. It was proposed that a two-stage, thermophilic-mesophilic anaerobic digestion system would be able to achieve the same sanitizing effect and equal or better digestion efficiency at lower costs.

Biodegradation of nitrilotriacetic acid (NTA) and ferric-NTA complex by aerobic microbial granules.

Development of mixed-culture microbial granules under aerobic conditions in a sequencing batch reactor (SBR), capable of completely degrading a recalcitrant metal chelating agent is reported. In laboratory-scale reactor studies, the microbial granules degraded 2mM of free nitrilotriacetic acid (NTA) and Fe(III)-NTA completely in 14 and 40 h, respectively. Free NTA was degraded at a specific rate of 0.7 mM (gMLSS)(-1)h(-1), while Fe(III)-NTA was degraded at a specific rate of 0.37 mM (gMLSS)(-1)h(-1). Achievement of significant degradation rates of NTA and ferric-NTA in double-distilled water suggests that the microbial metabolism is not constrained by lack of essential elements. Efficient degradation of recalcitrant synthetic chelating agents by aerobic microbial granules suggests their potential application in a variety of situations where heavy metals or radionuclides are to be co-disposed with metal chelating agents.

Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

Sustainable water management in rural and peri-urban areas: what technology do we need to meet the UN millennium development goals?

Installation of advanced urban water management systems is one of the most important first steps in the attempt to overcome poverty on earth, outbreak of diseases, crime and even terrorism. Because world wide application of traditional water supply, sewerage and wastewater treatment technology requires financial resources which are basically not available within a reasonable short time frame novel solutions must be found, developed and implemented. The combination of high-tech on-site treatment of the various waste streams generated in households, enterprises and industrial sites, and reuse of the valuable materials obtained from the treatment plants, including the purified water, is one of the options which is investigated by various groups of researchers and technology developers, nowadays. This concept may help meeting the UN Millennium Development Goals, provided people are ready to accept this new way of dealing with household wastes. Education is necessary to build up the foundation which modern water technology can be based upon. In parallel, tailored modifications are to be considered to satisfy the specific demands of local communities. In this context, female participation appears to be extremely important in the decision making process.

UN water action decade: a unique challenge and chance for water engineers.

In the year 2000, the UN General Assembly resolved the Millennium Development Goals (MDGs), and all the Member States of the UN pledged to take any measures to get the MDGs achieved. Concerning water supply and sanitation, a rough calculation clearly tells that it is impossible to meet the MDGs by just copying and pasting the traditional technology as it has been developed in the industrialized countries. Novel concepts and technologies have to be developed, tested and made available which allow fast implementation for a reasonable price, and which provide high treatment efficiency, reliability and robustness. Based on the latest estimates on population growth and migration rates it appears that top priority should be given to investments in the water infrastructure of municipalities. Introduction of water re-use systems should be favoured to avoid over-exploitation of local water resources. Suggested are step changes of the infrastructural system components with emphasis on technology innovation. Many small steps are often more effective than a single big one. In the attempt to quickly improve local situations specific attention should be paid to modern physico-chemical methods based on membrane technology. Decentralized wastewater treatment and re-use methods should be installed simultaneously to the construction of new houses. Further development of methods for separate collection and treatment of particularly composed waste streams should be encouraged. Wastewater should be rated as a source of valuable materials rather than a nuisance. Technology development should proceed hand-in-hand with the development of the societal, economical and political framework. Concerted actions are necessary to make novel technology function. Otherwise, meeting of the MDGs will remain an illusion.

Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions.

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge.

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.

Treatment of dairy effluents in an aerobic granular sludge sequencing batch reactor.

Aerobic granular sludge can successfully be cultivated in a sequencing batch reactor (SBR) treating dairy wastewater. Attention has to be paid to the fact that suspended solids are always present in the effluent of aerobic granular sludge reactors, making a post-treatment step necessary. Sufficient post-treatment can be achieved through a sedimentation process with a hydraulic retention time of 15-30 min. After complete granulation and the separation of biomass from the effluent, removal efficiencies of 90% CODtotal, 80% Ntotal and 67% Ptotal can be achieved at a volumetric exchange ratio of 50% and a cycle duration of 8 h. Effluent values stabilize at around 125 mg l-1 CODdissolved. The maximum applicable loading rate is nevertheless limited, as the stability of aerobic granules very much depends on the presence of distinct feast and famine conditions and the degradation of real wastewaters shows slower kinetics compared with synthetic wastewaters. As loading rate and volumetric exchange ratio are coupled in an SBR system, the potential of granular sludge for improving process efficiency is also limited.

Compatibility of the green fluorescent protein and a general nucleic acid stain for quantitative description of a Pseudomonas putida biofilm.

Better understanding of biofilm development is essential for making optimal use of beneficial biofilms as well as for devising effective control strategies for detrimental biofilms. Analysis of biofilm structure and quantification of biofilm parameters using optical (including confocal) microscopy and digital image analysis techniques are becoming routine in many laboratories. The purpose of this study was to evaluate a dual labeling technique based on fluorescence signals from the green fluorescent protein (GFP) and those resulting from staining with the general nucleic acid stain SYTO 60 for the quantitative description of a model biofilm. For this purpose, a Pseudomonas putida KT2442 derivative was genetically tagged with the green fluorescent protein gene. Biofilm formation by this strain was investigated using flow cells and confocal laser scanning microscopy (CLSM). Percentage surface coverage as well as microcolony size quantified using GFP and SYTO 60 signals showed significant correlation (R=0.99). The results indicated that intrinsic labelling of this model biofilm using constitutively expressed proteins such as GFP can be used for real-time biofilm observation and generation of reliable quantitative data, comparable to those obtained using conventional methods such as nucleic acid staining. Non-destructive time series observation of GFP-expressing biofilms in flow-cells can thus be confidently used for four-dimensional (x, y, z, t) analysis and quantification of biofilm development. The results also point to the possibility of using GFP and SYTO 60 to study dual species biofilms, as quantitative data generated using both fluorophore signals are comparable.

The effect of intermittent feeding on aerobic granule structure.

Self-immobilized biofilms, or aerobic granules without the addition of carrier material, have only been reported in one suspended growth system, the Sequencing Batch Reactor (SBR) with a very short fill time (dump fill). The SBR utilizes intermittent feeding which creates a period of high load followed by starvation (often referred to as feast-famine). In this experiment, three identical SBRs were operated with different feeding conditions to determine the role of feast-famine on granule formation. All three SBRs were operated with a total volumetric load of 2.4 kg/m3 x d. The 90 minute Fill phase was altered for each reactor, providing an increasing time of Aerated Fill. A dump fill condition was applied for one reactor, while the other two reactors were aerated for different times during Fill, resulting in a smaller COD load at the beginning of each React phase. Aerobic granules formed in all reactors, but the structural properties and content of filamentous organisms were clearly dependent on a high feast condition. Only the reactor with dump fill formed compact, stable granules. It is concluded that intermittent feeding associated with the SBR affects the selection and growth of filamentous organisms and has a critical role in granule structure and composition.

Aerobic granular sludge in an SBR-system treating wastewater rich in particulate matter.

Aerobic granular sludge was successfully cultivated in a lab-scale SBR-system treating malting wastewater with a high content of particulate organic matter (0.9 gTSS/L). At an organic loading rate (CODtotal) of 3.4 kg/(m3 x d) an average removal efficiency of 50% in CODtotal and 80% in CODdissolved was achieved. Fractionation of the COD by means of particle size showed that particles with a diameter less than 25-50 microm could be removed at 80% efficiency, whereas particles bigger than 50 microm were only removed at 40% efficiency. Tracer experiments revealed a dense sessile protozoa population covering the granules. The protozoa appeared to be responsible for primary particle uptake from the wastewater.

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples.

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.

Reject water: treating of process water in large wastewater treatment plants in Germany--a case study.

The increase of the hydraulic loading of a municipal wastewater treatment plant caused by the sludge dewatering process is of minor importance. However, the rejected nitrogen load accounts for up to 25% of the nitrogen load in the raw sewage. As a result, the process water significantly influences the stage volume of the biological treatment steps. Rejection management can prevent critical nitrogen concentrations in the effluent of the plant. In some cases, the treating capacity must be increased. Separate treatment of the process water can be an alternative to the enlargement of the main wastewater treatment stages. This article describes the compositions of the process waters from different sludge treatment steps, the influence on the main wastewater treatment steps, possible strategies to handle the rejection problem, and processes for separate treatment of the sludge liquor. Some results of a German case study on large wastewater treatment plants illustrate the situation concerning separate process water treatment in Germany. These results suggest that side stream treatment can be a practical alternative to a classic increase in the length of the main stream stages. Moreover, they prove that many different processes are useful for the separate treatment of sludge liquor.

Applying sustainable water management concepts in rural and urban areas: some thoughts about reasons, means and needs.

Serving the world population with adequate drinking water and sanitation is an important prerequisite, not only to hygienic safety, but to prosperity and political stability as well, and will foster the adaptive capacity of the societies in the developing countries and beyond. To avoid hygienic and political disasters impacting the world economy, investment in water supply and sanitation must urgently be made. Whether the classical system of urban water supply and sanitation is appropriate to satisfy the needs of the developing world, however, and whether this system meets the general criteria of sustainability is questionable. The costs and the time needed for installation of sewers and wastewater treatment plants are tremendous. In water shortage areas, the amount of tap water required to transport pollutants to the treatment plant is hardly affordable. Recovery and re-introduction of valuable substances, including water, into the urban cycle of materials is impossible because of mixing and dilution effects inherent in the system. Decentralized water and wastewater management should be seriously taken into account as an alternative. Source separation of specific fractions of domestic and industrial wastewater, separate treatment of these fractions and recovery of water and raw materials including fertilizer and energy are the main characteristics of modern high-tech on-site treatment/reuse systems. Mass production of the key components of the system could reduce the costs of the treatment units to a reasonable level. On-site units could be installed independently of the development stage of the urban sewer system. In conjunction with building new housing complexes a stepwise improvement of the hygienic situation in urban and peri-urban areas could be achieved, therefore. Remote control of the satellite systems using modern telecommunication methods would allow reliable operation, and comfort for the users. Intensive research is required, however, to develop this system and bring it to a standard allowing efficient application worldwide.