Strategies for the reduction of Legionella in biological treatment systems.

A community-wide outbreak of Legionnaire's disease occurred in Warstein, Germany, in August 2013. The epidemic strain, Legionella pneumophila Serogruppe 1, was isolated from an industrial wastewater stream entering the municipal wastewater treatment plant (WWTP) in Wartein, the WWTP itself, the river Wäster and air/water samples from an industrial cooling system 3 km downstream of the WWTP. The present study investigated the effect of physical-chemical disinfection methods on the reduction of the concentration of Legionella in the biological treatment and in the treated effluent entering the river Wäster. Additionally, to gain insight into the factors that promote the growth of Legionella in biological systems, growth experiments were made with different substrates and temperatures. The dosage rates of silver micro-particles, hydrogen peroxide, chlorine dioxide and ozone and pH stress to the activated sludge were not able to decrease the number of culturable Legionella spp. in the effluent. Nevertheless, the UV treatment of secondary treated effluent reduced Legionella spp. on average by 1.6-3.4 log units. Laboratory-scale experiments and full-scale measurements suggested that the aerobic treatment of warm wastewater (30-35 °C) rich in organic nitrogen (protein) is a possible source of Legionella infection.

Nitrogen and me - How little did we, and do we know about "stikstof - azote - nitrogen"?

This retrospective article reflects on the complex and evolving relationship between humans and nitrogen over several decades. Raised on a Flemish farm, the author's early experiences with nitrogen in agriculture - both its benefits and dangers - laid the foundation for a lifelong interest in this element. The article traverses a broad range of topics related to nitrogen, highlighting its critical role in various historical, agricultural, environmental, and industrial contexts. The narrative begins with a historical overview of nitrogen's role in agriculture and warfare. The development of industrial processes like the Haber and Ostwald methods transformed nitrogen into a key ingredient for both fertilizers and explosives. The dual nature of nitrogen - as a life-giver in agriculture and a destructive component in warfare and also in biodiversity - is an important theme. The article delves into the environmental impacts of nitrogen, particularly in the context of modern agriculture and industrialization. Issues like fertilization, water contamination, and the challenges of managing nitrogenous waste highlight the complex interplay between human activities and environmental health. Technological advancements are explored, including the development of bioaugmentation methods and the potential of genetic engineering in optimizing nitrogen fixation. Throughout the narrative, personal anecdotes are weaved with scientific information, offering a unique perspective on the historical and contemporary challenges of managing nitrogen. The discussion extends to the broader implications of nitrogen management in the context of sustainability, climate change, and global food security and its overall regulatory space. All these considerations call for a re-evaluation of our relationship with nitrogen, advocating for innovative solutions and systemic thinking to address the multifaceted challenges posed by this essential, yet often problematic element.

Autotrophic nitrogen removal after ureolytic phosphate precipitation to remove both endogenous and exogenous nitrogen.

Anaerobic digestion yields effluents rich in ammonium and phosphate and poor in biodegradable organic carbon, thereby making them less suitable for conventional biological nitrogen and phosphorus removal. In addition, the demand for fertilizers is increasing, energy prices are rising and global phosphate reserves are declining. This requires both changes in wastewater treatment technologies and implementation of new processes. In this contribution a description is given of the combination of a ureolytic phosphate precipitation (UPP) and an autotrophic nitrogen removal (ANR) process on the anaerobic effluent of a potato processing company. The results obtained show that it is possible to recover phosphate as struvite and to remove the nitrogen with the ANR process. The ANR process was performed in either one or two reactors (partial nitritation + Anammox). The one-reactor configuration operated stably when the dissolved oxygen was kept between 0.1 and 0.35 mg L(-1). The best results for the two-reactor system were obtained when part of the effluent of the UPP was fully nitrified in a nitritation reactor and mixed in a 3:5 volumetric ratio with untreated ammonium-containing effluent. A phosphate and nitrogen removal efficiency of respectively 83 ± 1% and of 86 ± 7% was observed during this experiment.

Sewage pre-concentration for maximum recovery and reuse at decentralized level.

Pre-concentration of municipal wastewater by chemically enhanced primary treatment (CEPT) was studied under controlled laboratory conditions. Both iron and aluminium-based coagulants were examined at gradually increasing concentrations (0.23, 0.35, 0.70 and 1.05 mmol/L). The CEPT sludge generated from different coagulation experiments was digested in batch anaerobic reactors, while the supernatant was tested in a dead-end microfiltration setup. The results of the study show that biogas yield was dramatically decreased (from 0.40 to 0.10 m(3)/kg chemical oxygen demand of influent) with increasing coagulant dose. In contrast, supernatant filterability was improved. Based on the laboratory results, a conceptual design was produced for a community of 2000 inhabitant equivalents (IE), using CEPT technology (at low coagulant dose) with anaerobic digestion of the concentrates. According to this, the capital and operational costs were 0.11 and 0.09 €/m(3), respectively. The biogas generated is used for digester heating and the overall process is energy self-sufficient. At a small-scale and in private applications, CEPT technology is preferably operated at higher coagulant dose, followed by membrane filtration for water reuse. Accordingly, sewage purification and reuse is possible without implementing aerobic biological processes.

Diatomaceous earth as a protective vehicle for bacteria applied for self-healing concrete.

Crack repair is crucial since cracks are the main cause for the decreased service life of concrete structures. An original and promising way to repair cracks is to pre-incorporate healing agents inside the concrete matrix to heal cracks the moment they appear. Thus, the concrete obtains self-healing properties. The goal of our research is to apply bacterially precipitated CaCO3 to heal cracks in concrete since the microbial calcium carbonate is more compatible with the concrete matrix and more environmentally friendly relative to the normally used polymeric materials. Diatomaceous earth (DE) was used in this study to protect bacteria from the high-pH environment of concrete. The experimental results showed that DE had a very good protective effect for bacteria. DE immobilized bacteria had much higher ureolytic activity (12-17 g/l urea was decomposed within 3 days) than that of un-immobilized bacteria (less than 1 g/l urea was decomposed within the same time span) in cement slurry. The optimal concentration of DE for immobilization was 60% (w/v, weight of DE/volume of bacterial suspension). Self-healing in cracked specimens was visualized under light microscopy. The images showed that cracks with a width ranging from 0.15 to 0.17 mm in the specimens containing DE immobilized bacteria were completely filled by the precipitation. Scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) were used to characterize the precipitation around the crack wall, which was confirmed to be calcium carbonate. The result from a capillary water absorption test showed that the specimens with DE immobilized bacteria had the lowest water absorption (30% of the reference ones), which indicated that the precipitation inside the cracks increased the water penetration resistance of the cracked specimens.

Evaluation and thermodynamic calculation of ureolytic magnesium ammonium phosphate precipitation from UASB effluent at pilot scale.

The removal of phosphate as magnesium ammonium phosphate (MAP, struvite) has gained a lot of attention. A novel approach using ureolytic MAP crystallization (pH increase by means of bacterial ureases) has been tested on the anaerobic effluent of a potato processing company in a pilot plant and compared with NuReSys(®) technology (pH increase by means of NaOH). The pilot plant showed a high phosphate removal efficiency of 83 ± 7%, resulting in a final effluent concentration of 13 ± 7 mg · L(-1) PO(4)-P. Calculating the evolution of the saturation index (SI) as a function of the remaining concentrations of Mg(2+), PO(4)-P and NH(4)(+) during precipitation in a batch reactor, resulted in a good estimation of the effluent PO(4)-P concentration of the pilot plant, operating under continuous mode. X-ray diffraction (XRD) analyses confirmed the presence of struvite in the small single crystals observed during experiments. The operational cost for the ureolytic MAP crystallization treating high phosphate concentrations (e.g. 100 mg · L(-1) PO(4)-P) was calculated as 3.9 € kg(-1) P(removed). This work shows that the ureolytic MAP crystallization, in combination with an autotrophic nitrogen removal process, is competitive with the NuReSys(®) technology in terms of operational cost and removal efficiency but further research is necessary to obtain larger crystals.

A time-course analysis of four full-scale anaerobic digesters in relation to the dynamics of change of their microbial communities.

This study describes the microbial community richness, -dynamics, and -organization of four full-scale anaerobic digesters during a time-course study of 45 days. The microbial community was analyzed using a Bacteria- and Archaea-targeting 16S rRNA gene-based Terminal-Restriction Fragment Length Polymorphism approach. Clustering analysis separated meso- and thermophilic reactors for both archaeal and bacterial communities. Regardless of the operating temperature, each installation possessed a distinct community profile. For both microbial domains, about 8 dominant terminal-restriction fragments could be observed, with a minimum of 4 and a maximum of 14. The bacterial community organization (a coefficient which describes the specific degree of evenness) showed a factor 2 more variation in the mesophilic reactors, compared with the thermophilic ones. The archaeal community structure of the mesophilic UASB reactor was found to be more stable. The community composition was highly dynamic for Bacteria and Archaea, with a rate of change between 20-50% per 15 days. This study illustrated that microbial communities in full-scale anaerobic digesters are unique to the installation and that community properties are dynamic. Converging complex microbial processes such as anaerobic digestion which rely on a multitude of microbial teams apparently can be highly dynamic.

Antimicrobial mortar surfaces for the improvement of hygienic conditions.

AIMS: To evaluate the effectiveness of various antimicrobial mortar formulations in inhibiting the growth of a selection of pathogens of environmental and hygienic concern. METHODS AND RESULTS: Mortar prisms containing triclosan-incorporated fibres or different concentrations of silver copper zeolites were incubated with Escherichia coli, Listeria monocytogenes, Salmonella enterica or Staphylococcus aureus at 4 or 20 degrees C for 24 h. From plate counting, a substantial bactericidal effect (>4 log units) could only be observed for the mortar specimens containing more than 3% zeolites on cement weight base, the effect being more pronounced at 20 degrees C compared to 4 degrees C. No inhibitory effect could be observed for mortar specimens containing antimicrobial fibres. Adenosinetriphosphate (ATP) measurements allowed for a rapid indication of the occurrence of antimicrobial activity. CONCLUSIONS: In order to obtain a bactericidal effect on mortar surfaces, concentrations of silver copper zeolites of more then 3% are required. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study in which the effectiveness of various antimicrobial mortar mixtures towards the inhibition of pathogens has been evaluated in a quantitative way. Antimicrobial concrete mixtures can be used for the improvement of the hygienic conditions in a variety of environments.

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi.

AIMS: To study the potential biocontrol activity of bioflocs technology. METHODS AND RESULTS: Glycerol-grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth-inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing-regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. CONCLUSIONS: Bioflocs grown on glycerol as carbon source inhibit quorum sensing-regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.

Fast start-up of a pilot-scale deammonification sequencing batch reactor from an activated sludge inoculum.

Deammonification involves the combined application of aerobic and anoxic ammonium-oxidizing bacteria (AerAOB & AnAOB) and allows to treat wastewaters with a high ammonium concentration in a sustainable and cost-efficient way. So far, it could take more than one year to start up the process, even with the addition of AnAOB enriched inocula. In contrast, we started up a deammonifying reactor for the treatment of sludge digestate in less than four months without any AnAOB enriched inoculum. In a single sequencing batch reactor (SBR) of 3 m(3), nitritation and anammox were performed without nitrite accumulation. Larger biomass aggregates (>1.0 mm) had a typical reddish colour, but FISH also showed that small aggregates (<0.25 mm) contained a considerable amount of AnAOB. The AerAOB were related to Nitrosomonas halophila, N. eutropha and N. halophila, and the AnAOB to "Candidatus Kuenenia & Brocadia", as shown by FISH. Our results show that the deammonification inoculum does not play an important role, and that the AnAOB can quickly develop under the proper aerational conditions. Nitrogen was removed stably at high nitrogen loading rates (740 mg N/L/d) and removal efficiency (90%).

Chemopreventive effects from prebiotic inulin towards microbial 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine bioactivation.

AIMS: Using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME), we investigated the chemopreventive potential of prebiotic chicory inulin towards the in vitro bioactivation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human intestinal microbiota. METHODS AND RESULTS: HPLC data revealed that inulin significantly decreased the formation of the genotoxic PhIP-M1 metabolite, with the highest inhibitory activity in the colon ascendens (87% decrease). Interestingly, this chemopreventive effect correlated with alterations of bacterial community composition and metabolism in the different colon compartments. Conventional culture-based techniques and PCR-DGGE analysis on the SHIME colon suspension revealed significant bifidogenic effects during inulin treatment, whereas the overall microbial community kept relatively unchanged. Additionally, the production of short-chain fatty acids increased with 12%, 3% and 7%, while ammonia concentrations decreased with 3%, 4% and 3% in the ascending, transverse and descending colon compartments, respectively. CONCLUSIONS: These results indicate that the prebiotic effects from inulin may also purport protective effects towards microbial PhIP bioactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: As the colonic microbiota may contribute significantly to the carcinogenic potential of PhIP, the search for dietary constituents that decrease the formation of this harmful metabolite, may help in preventing its risk towards human health.

High reproducibility of ammonia-oxidizing bacterial communities in parallel sequential batch reactors.

AIMS: To investigate whether the ammonia-oxidizing bacterial (AOB) communities of replicate nitrifying bioreactors (i) co-evolve or diverge over time and (ii) are stable or dynamic during periods of complete nitrification. METHODS AND RESULTS: Three sequential batch reactors (SBR) were inoculated with sludge from a municipal wastewater treatment plant, fed with ammonium-enriched tap water and operated in parallel for 134 days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) demonstrated co-evolvement of the AOB communities over time. During start-up, temporary decreases in nitrification were noticed, and the AOB community rate of change values (Delta(t(week))) were medium to high (12-22%). During the adjacent period of complete nitrification, low AOB community dynamics were observed (Delta(t(week)) < 5%). Further pragmatic processing of the DGGE profiles revealed a high range-weighted richness and a medium functional organization of the AOB communities. CONCLUSIONS: After a start-up period, high functional stability and low dynamics of the AOB communities were observed. Deterministic rather than stochastic driving forces led to AOB community co-evolvement in the replicate SBR. SIGNIFICANCE AND IMPACT OF THE STUDY: Replicates in identical set-ups are reproducible, and pragmatic processing of DGGE patterns is a straightforward tool to score and compare the functionality of the bacterial communities.

Bioaugmentation to degrade the organic de-icers acetate and monopropylene glycol at low temperatures.

Two de-icers, potassium acetate and monopropylene glycol (MPG), used widespread as a runway and wing de-icer respectively, can exert high BOD in the surrounding waters. A bioaugmentation approach to degrade these de-icer compounds in the drainage water prior to discharge has been tested. A microbial consortium originating from soil was enriched at low temperatures (4 degrees C) in order to adapt to wintertime conditions. With 0.05 g CDW/L of biocatalyst, maximum specific removal rates up to 1.46 and 3.33 g acetate/g CDW d at 4 degrees C were achieved with and without biostimulation respectively. An acetate:MPG mixture of 1:3 at a total COD concentration of 0.80 and 1.20 g/L was degraded in 12 days by 83 and 70% respectively. Bioaugmentation in the field over a period of 25 days showed a removal of 88% MPG compared to 46% in the control. These results demonstrate that bioaugmentation of airport runoff water can be successfully applied to prevent organic de-icer compounds from entering the receiving surface waters.

Upflow anaerobic clarification tank (UACT) to upgrade existing anaerobic effluents.

The Upflow Anaerobic Clarification Tank (UACT) is of interest as a post-treatment step for high-rate anaerobic wastewater treatment systems. The UACT contributes to extra capture of methane from the raw wastewater. It also significantly facilitates the reuse of the treated water. The efficiency of the UACT process is demonstrated in this study using laboratory-scale reactors. The water produced by the UACT had a low total COD, in most cases below 145 mg/L and a soluble COD below 60 mg/L at surface loading rates between 0.1-0.4 m(3)/(m(2) h) and hydraulic retention times between 5-6 h. The sludge bed of the UACT was characterised by 6-10 times higher substrate affinity (for ethanol, acetate and propionate) compared to the parent UASB and similar specific activity. The results suggest that a UASB followed by the UACT can, upon subsequent application of a rapid filtration, qualify as a total COD removal, no longer necessitating aerobic treatment.

Decreasing sulfide generation in sewage by dosing formaldehyde and its derivatives under anaerobic conditions.

Hydrogen sulfide emission in sewers is associated with toxicity, corrosion, odour nuisance and high costs. In this study, a new method to inhibit sulfide generation by means of formaldehyde and its derivatives has been evaluated under anaerobic conditions. The possible impact of formaldehyde on an activated sludge system and an appraisal of the economic aspects are presented as well. A dosage of 19 mg L(-1) formaldehyde resulted in a decrease of the sulfide production of 90%. Dosing of 32 mg L(-1) paraformaldehyde and addition of 111 mg L(-1) ureaformaldehyde were not sufficient to inhibit the sulfide generation in sewage to the same extent. The impacts of 19 mg L(-1) formaldehyde on activated sludge, in terms of COD removal, nitrification rate and oxygen uptake rates, were negligible. This suggests that formaldehyde dosage is a feasible technique to abate the sulfide problem in sewers.

Ureolytic phosphate precipitation from anaerobic effluents.

In this work, the elimination of phosphate from industrial anaerobic effluents was evaluated at lab-scale. For that purpose, the ureolytic method previously developed for the precipitation of Ca(2 + ) from wastewater as calcite was adapted for the precipitation of phosphate as struvite. In the first part of the study, computer simulations using MAPLE and PHREEQC were performed to model phosphate precipitation from wastewater as struvite. The results obtained showed that relative high concentrations of ammonium and magnesium are needed to precipitate phosphate as struvite. The total molar concentrations ratio of Mg(2 + ):PO(4) (3-)-P:NH(4) (+) required to decrease PO(4) (3-)-P concentrations from 20 to 6 mg PO(4) (3-)-P/l at pH 8.4-8.5 was estimated on 4.6:1:8. In the second part of the study, lab-scale experiments with either synthetic wastewater or the anaerobic effluent from a vegetable processing industry were carried out in batch and continuous mode. Overall, the continuous operation at a hydraulic retention time (HRT) of 2.4 h and an added molar concentration [Mg(2 + )]:[PO(4) (3-)-P]:[NH(4) (+)] ratio of 1.6:1:2.3 resulted in a constant pH value in the reactor (around 8.5) and an efficient phosphate removal (>90%) to residual levels of 1-2 mg PO(4) (3-)-P/l. Different operational conditions, such as the initial phosphate concentration, HRT and the use of CaCl(2) or MgO instead of MgCl(2), were analysed and the performance of the reactor was satisfactory under a broad range of them. Yet, overall, optimal results (higher phosphate removal) were obtained with MgCl(2).

Granular biomass capable of partial nitritation and anammox.

Water Science and Technology 58(5) 1113-1120. Publisher's note. We regret that an outdated version of this article was used in production; the correct final version, which incorporates several amendments and different authorship, is printed below.

Legionella occurrence in municipal and industrial wastewater treatment plants and risks of reclaimed wastewater reuse: Review.

Wastewater treatment plants (WWTPs) have been identified as confirmed but until today underestimated sources of Legionella, playing an important role in local and community cases and outbreaks of Legionnaires' disease. In general, aerobic biological systems provide an optimum environment for the growth of Legionella due to high organic nitrogen and oxygen concentrations, ideal temperatures and the presence of protozoa. However, few studies have investigated the occurrence of Legionella in WWTPs, and many questions in regards to the interacting factors that promote the proliferation and persistence of Legionella in these treatment systems are still unanswered. This critical review summarizes the current knowledge about Legionella in municipal and industrial WWTPs, the conditions that might support their growth, as well as control strategies that have been applied. Furthermore, an overview of current quantification methods, guidelines and health risks associated with Legionella in reclaimed wastewater is also discussed in depth. A better understanding of the conditions promoting the occurrence of Legionella in WWTPs will contribute to the development of improved wastewater treatment technologies and/or innovative mitigation approaches to minimize future Legionella outbreaks.

Probiotic bacteria as biological control agents in aquaculture.

There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates.

AIMS: To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. METHODS AND RESULTS: Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. CONCLUSIONS: There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.