Louise Vanysacker

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene. Microbial community fingerprints were assessed by automated ribosomal intergenic spacer analysis. The resulting community profiles were analyzed with canonical correspondence analysis. Our results clearly demonstrate that direct DNA extraction methods can significantly influence the DNA quantity, purity, and observed community patterns of microbiota in activated sludge. Fast and Mobio generated high amounts of good quality DNA compared to Bead and Qiagen. Mobio also resulted in the detection of the highest number of species while Fast scored the best in discriminating between the community patterns of different activated sludge types. With respect to the characterization of community profiles, our analyses demonstrated a strong sludge type dependent variability among methods. Taking into account our results, we recommend Fast as the most suitable DNA extraction method for activated sludge samples used for bacterial community studies.

Biofouling ecology as a means to better understand membrane biofouling

Despite more than a decade of worldwide research on membrane fouling in membrane bioreactors, many questions remain to be answered. Biofouling, which is referred to as the unwanted deposition and growth of biofilms, remains the main problem. Due to its complexity, most of the existing anti-biofouling strategies are not completely successful. To unravel this complexity and finally to developed well-adapted control strategies, a microbial-based description of the biofouling development is needed. Therefore, in this review, the biofouling formation will be described as a typical biofilm formation in five steps including the formation of a conditioning film, the bacterial attachment, the production of extracellular polymeric substances, the biofilm maturation, and the bacterial detachment. Moreover, important processes such as hydrodynamics and bacterial communication or quorum sensing will be taken into account. It is finally discussed whether biofouling formation is an active or inactive biofilm process together with suggestion for further research.

Spherical nucleic acid enhanced FO-SPR DNA melting for detection of mutations in Legionella pneumophila.

A home-built fiber optic surface plasmon resonance platform (FO-SPR) was applied to directly screen PCR amplified DNA for mutations. The FO-SPR sensor was used for real-time monitoring of DNA duplex melting during high resolution temperature cycling. The signal of the DNA melting was enhanced by means of gold nanoparticle labels. This FO-SPR genetic assay allowed for detection of single-point mutations (SNP) in less than 20 min. The concept was demonstrated for the analysis of 9 different serogroups of the bacterium Legionella pneumophila, a common human pathogen responsible for atypical pneumonia. FO-SPR allowed us to detect genetic mutations inhibiting PCR, which could lead to amplification bias when molecular diagnostics are applied for L. pneumophila detection. All serogroups were found to display unique melting temperatures, indicating that mutations have accumulated in the target sequence. In a next step, clinical samples of L. pneumophila were analyzed using the FO-SPR sensor. This technology was proven to be reliable for the detection of mutations for those samples that previously displayed ambiguous qPCR quantification results. When these results were benchmarked, FO-SPR results were found to be consistent with Sanger sequencing but not with fluorescence based DNA melting. The presented results convincingly advocate the advantages of FO-SPR as a high resolution and fast genetic screening tool that can compete with the current standard techniques for SNP detection.

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration.

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93×10(9) to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R(2)=0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

Evaluation of power ultrasound for disinfection of both Legionella pneumophila and its environmental host Acanthamoeba castellanii

The objectives of this study were to (1) examine the effect of power ultrasound on the viability of both Legionella pneumophila and Acanthamoeba castellanii trophozoites and cysts, (2) investigate if intracellular Legionella replication in trophozoites positively affects bacterial resistance to ultrasound and (3) study if Legionella renders viable but non-culturable (VBNC) due to ultrasound treatments. Using laboratory scale experiments, microorganisms were exposed for various time periods to power ultrasound at a frequency of 36 kHz and an ultrasound power setting of 50 and 100%. Due to a fast destruction, trophozoite hosts were not able to protect intracellular Legionella from eradication by ultrasound, in contrast to cysts. No significant effects of ultrasound on cyst viability could be detected and power settings of 100% for 30 min only made intracellular Legionella concentrations decrease with 1.3 log units. Due to intracellular replication of Legionella in trophozoites, ultrasound no longer affected bacterial viability. Concerning the VBNC state, ultrasound treatments using a power setting of 50% partly induced Legionella (+/-7%) to transform into VBNC bacteria, in contrast to power settings of 100%. Promising results obtained in this study indicate the possible application of power ultrasound in the control of both Legionella and Acanthamoeba concentrations in anthropogenic water systems.

Microbial Adhesion and Biofilm Formation on Microfiltration Membranes: A Detailed Characterization Using Model Organisms with Increasing Complexity

Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development.

Core-shell nanoparticles as enhanced probes for imaging applications

The development of highly specific markers for fluorescent microscopy has become a very popular research topic. Organic fluorophores have several drawbacks, such as photobleaching and autofluorescence. Therefore increasing interest in inorganic nanoparticles has been observed because of their unseen photostability, chemical robustness and straightforward synthesis. The surface of iron oxide nanoparticles was coated with trialkoxy silanes, which introduced functional groups for possible subsequent coupling reactions. An additional gold layer was added to the surface of the particle to show the enhanced contrast improvement. The nanoparticles were imaged by an optical microscope, in dark field mode, on a glass substrate and inside microorganisms. This proved that the reported method could have great potential as a labelling technique, since it combines the non-photobleaching, photostable nanoparticles with a straightforward and rapid imaging technique.

Towards a low complexity carbon removal model for the optimal design of compact decentralised wastewater treatment systems

On-site decentralised wastewater treatment systems can provide a financially attractive alternative to a sewer connection in locations far from existing sewer networks. Operational problems and shortcomings in the design of these systems still occur frequently. The aim of this paper is to provide a low complexity (i.e. easy to calibrate) but still accurate mathematical model that can be used to optimise the operational design of compact individual wastewater treatment systems. An integrated hydraulic and biological carbon removal model of a biofilm-based compact decentralised treatment system is developed. The procedure for drafting the model is generic and can be used for similar types of wastewater treatment systems since (i) the hydraulic model is based on an N-tanks-in-series model inferred from tracer test experiments and (ii) (biofilm) respirometry experiments are exploited to determine the biodegradation kinetics of the biomass. Based on the preliminary validation results of the integrated model, the carbon removal in the system can be predicted quite accurately. While some adjustments could further improve the modelling strategy, the here presented results can already assist the manufacturers of compact treatment systems in efficiently (re)designing their systems.