[Show abstract][Hide abstract]ABSTRACT: The aerobic granular sludge process is a promising technology for the removal of nutrients
and organic contaminants from wastewater. However, a large amount of the sludge is often washed
out during the start-up of granular reactors, which results in reduced process performance and a
protracted start-up phase. In this study, the possibility of a rapid start-up of the nitrification process
through a stepwise decrease of the settling time was investigated, and the bacterial population
dynamics in two lab-scale sequencing batch reactors were studied. The results demonstrated that
the stepwise decrease of the settling time enabled fast granulation and rapid start-up of the process.
Small cores of granules were already observed after 10 days of operation, and the biomass was
dominated by granules after 28 days. The removal of organic matter and ammonium was >95% after
one day and 14 days, respectively. The bacterial community composition changed rapidly during
the first 21 days, resulting in strongly reduced richness and evenness. The diversity increased at a
later stage, and the bacterial community continued changing, albeit at a slower pace. The rate of the
stepwise decrease in settling time strongly affected the abundance of nitrifying organisms, but not
the general composition of the bacterial community. The results of this study support the idea that a
stepwise decrease of the settling time is a successful strategy for the rapid start-up of aerobic granular
[Show abstract][Hide abstract]ABSTRACT: The activated sludge process is commonly used to treat wastewater by aerobic oxidation of organic pollutants into carbon dioxide and water. However, several non-oxidative mechanisms can also contribute to removal of organics. Sorption onto activated sludge can remove a large fraction of the colloidal and particulate wastewater organics. Intracellular storage of e.g. polyhydroxyalkanoates (PHA), triacylglycerides (TAG), or wax esters can convert wastewater organics into precursors for high-value products. Recently, several environmental, economic, and technological drivers have stimulated research on non-oxidative removal of organics for wastewater treatment. In this paper, we review these non-oxidative removal mechanisms as well as the existing and emerging process configurations that make use of them for wastewater treatment. Better utilization of non-oxidative processes in activated sludge could reduce the wasteful aerobic oxidation of organic compounds and lead to more resource-efficient wastewater treatment plants.
No preview · Article · Feb 2016 · Critical Reviews in Environmental Science and Technology
[Show abstract][Hide abstract]ABSTRACT: Storage methods are important to preserve the viability and biochemical characteristics of microbial cultures between experiments or during periods when bioreactors are inactive. Most of the research on storage has focused on isolates; however, there is an increasing interest in methods for mixed cultures, which are of relevance in environmental biotechnology. The purpose of this study was to investigate the effect of different storage methods on electrochemically active enrichment cultures. Acetate-oxidizing bioanodes generating a current density of about 5 A m−2 were enriched in a microbial electrolysis cell. The effect of five weeks of storage was evaluated using electrochemical techniques and microbial community analysis. Storage by refrigeration resulted in quicker re-activation than freezing in 10% glycerol, while the bioelectrochemical activity was entirely lost after storage using dehydration. The results showed that the bioelectrochemical activity of bioanodes stored at low temperature could be retained. However, during the re-activation period the bioanodes only recovered 75% of the current density generated before storage and the bacterial communities were different in composition and more diverse after storage than before.
Full-text · Article · Dec 2015 · Scientific Reports
[Show abstract][Hide abstract]ABSTRACT: Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may result in loss of biomass, which could compromise nitrogen removal by the nitritation-anammox process. However, predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the possible predation in nitritation-anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and confocal scanning laser microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH-CLSM. Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different co-localization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the abundance of AOB and AMX, with implications for nitrogen removal from wastewater.
No preview · Article · Oct 2015 · FEMS Microbiology Ecology
[Show abstract][Hide abstract]ABSTRACT: The study of the strength and stability of aerobic granules is essential for the application of aerobic granular sludge membrane bioreactors (AGMBR). In this study, a coarse pore nylon mesh membrane was used to study the differences in compressibility and breakage of 78 aerobic granules submitted to different water fluxes. Confocal laser scanning microscopy was employed to investigate the distribution of extracellular polymeric substances of cryosectioned granules. The tested granules were able to withstand fluxes much higher than those typically applied in MBRs, with pressures ranging from 0.2 to 4.5 kN m-2 before breakage. Cells, β-polysaccharides and proteins were present in higher abundance in the outer layers, while calcium ions were abundant in the outer- as well as the inner layers of the granule. The results shows that the use of coarse pore meshes operated at very high flux is a feasible technique for biomass separation in AGMBR.
[Show abstract][Hide abstract]ABSTRACT: Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.
Full-text · Article · Dec 2014 · Environmental Science and Technology
[Show abstract][Hide abstract]ABSTRACT: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed.
The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data.
The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.
Full-text · Article · Dec 2014 · BMC Bioinformatics
[Show abstract][Hide abstract]ABSTRACT: Background
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples.ResultsVariations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a data set from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters.ConclusionsA novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.
Full-text · Article · Nov 2014 · BMC Bioinformatics
[Show abstract][Hide abstract]ABSTRACT: Biofilms are complex communities. Their growth and the presence of different bacterial populations can lead to physicochemical gradients in the biofilm. Those gradients could form microhabitats, allowing different bacterial populations to live in different parts of the biofilm.
One-stage partial nitritation-anammox biofilms, growing on carriers in Moving Bed Biofilm Reactors (MBBR) for nitrogen removal in wastewater treatment, is an example of a system with a strong spatial stratification. The layers facing the bulk water have access to oxygen and ammonium allowing the establishment of ammonia oxidizing bacteria (AOB). AOB in turn produce nitrite, and together with other bacteria, they create the anoxic condition that allows the establishment of anammox bacteria in deeper biofilm layers. MBBR biofilms are often several millimeters thick. These provide opportunities to studying large-scale process in biofilms.
We have studied biofilms from one-stage partial nitritation-anammox processes in MBBR. The biofilms are fixed and embedded in OCT, which allows lengthwise or crosswise cryosections of whole “wall-to wall” biofilms to be studied. Fluorescence in Situ Hybridization (FISH) and confocal laser scanning microscope (CLSM) together with novel imaging analyses tools are used to study population composition and biofilm positions of anammox and AOB, as well as eukaryotic predators.
After analyzing 16SrRNA gene clone library sequences we reported that Brocadia sp. 40 strongly dominated the anammox community in an MBBR biofilm. However, when studying “wall-to wall” biofilm cryosections with CLSM-FISH we observed that one of the minor members of the anammox community was present in the biofilm layers close to the bulk water, near the AOB. Thus, spatial distinct distribution between anammox populations may be due to their eco-physiological differences, just as we have shown earlier for AOB populations.
Biofilm structure and positioning of bacteria can be formed by gradients of electron donors and acceptors, but also by other forcing factors such as predation. We are using the same approach of combining cryosections and CLSM-FISH to study relationship between protozoa and bacteria in biofilms. We have shown that eukaryotic predators of different types are present at different depths in the biofilm. Preliminary result suggests the some predators penetrate into deeper biofilm parts and show evidence of grazing of anammox bacteria. Thus, neither the formation of microcolonies nor living deep within the biofilm can fully protect bacteria from predation. Little is known about bacterial predation in anammox biofilms. We discuss the importance of predation for the function of one stage MBBR nitrogen removal in wastewater.
Studies of bacterial activity in biofilms, using molecular methods, together with CLSM-FISH analyses of cryosections are underway and will be discussed in relation to electron donor and acceptor gradients and predation pressure.
The use of cryosections, combined with CLSM-FISH allows us to study large scale spatial patterns in biofilms. Together with techniques for cell activity it is possible to finally start seeing the big picture in biofilms, such as structures formed by limiting resources and predation.
[Show abstract][Hide abstract]ABSTRACT: The complete nucleotide sequence of plasmids pMCBF1 and pMCBF6 was determined and analyzed. pMCBF1 and pMCBF6 form a novel clade within the IncP-1 plasmid family designated IncP-1 ς. The plasmids were exogenously isolated earlier from a marine biofilm. pMCBF1 (62 689 base pairs; bp) and pMCBF6 (66 729 bp) have identical backbones, but differ in their mercury resistance transposons. pMCBF1 carries Tn5053 and pMCBF6 carries Tn5058. Both are flanked by 5 bp direct repeats, typical of replicative transposition. Both insertions are in the vicinity of a resolvase gene in the backbone, supporting the idea that both transposons are "res-site hunters" that preferably insert close to and use external resolvase functions. The similarity of the backbones indicates recent insertion of the two transposons and the ongoing dynamics of plasmid evolution in marine biofilms. Both plasmids also carry the insertion sequence ISPst1, albeit without flanking repeats. ISPs1is located in an unusual site within the control region of the plasmid. In contrast to most known IncP-1 plasmids the pMCBF1/pMCBF6 backbone has no insert between the replication initiation gene (trfA) and the vegetative replication origin (oriV). One pMCBF1/pMCBF6 block of about 2.5 kilo bases (kb) has no similarity with known sequences in the databases. Furthermore, insertion of three genes with similarity to the multidrug efflux pump operon mexEF and a gene from the NodT family of the tripartite multi-drug resistance-nodulation-division (RND) system in Pseudomonas aeruginosa was found. They do not seem to confer antibiotic resistance to the hosts of pMCBF1/pMCBF6, but the presence of RND on promiscuous plasmids may have serious implications for the spread of antibiotic multi-resistance.
[Show abstract][Hide abstract]ABSTRACT: A simple and efficient principle for nanopatterning with wide applicability in the sub‐50 nanometer regime is chemisorption of nanoparticles; at homogeneous substrates, particles carrying surface charge may spontaneously self‐organize due to the electrostatic repulsion between adjacent particles. Guided by this principle, a method is presented to design, self‐assemble, and chemically functionalize gradient nanopatterns where the size of molecular domains can be tuned to match the level corresponding to single protein binding events. To modulate the binding of negatively charged gold nanoparticles both locally (100 μm) onto a single modified gold substrate, ion diffusion is used to achieve spatial control of the particles’ mutual electrostatic interactions. By subsequent tailoring of different molecules to surface‐immobilized particles and the void areas surrounding them, nanopatterns are obtained with variable chemical domains along the gradient surface. Fimbriated Escherichia coli bacteria are bound to gradient nanopatterns with similar molecular composition and macroscopic contact angle, but different sizes of nanoscopic presentation of adhesive (hydrophobic) and repellent poly(ethylene) glycol (PEG) domains. It is shown that small hydrophobic domains, similar in size to the diameter of the bacterial fimbriae, supported firmly attached bacteria resembling catch‐bond binding, whereas a high number of loosely adhered bacteria are observed on larger hydrophobic domains. Chemical gradients with the resolution needed to address complex biological binding events at the single protein level are prepared using surface‐deposited gold nanoparticles as a versatile template for orthogonal chemical modifications. The effect of hydrophobic domain arrangement on the sub‐50 nm scale is shown to influence binding of fimbriae carrying E. coli bacteria.
No preview · Article · Feb 2014 · Particle and Particle Systems Characterization
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Feb 2014 · International Journal of Molecular Sciences
[Show abstract][Hide abstract]ABSTRACT: Scatter plot of total biomass areal density fraction vs. depth into the carrier. Each data point corresponds to the percentage coverage of the Eub338 probe mix, as determined by FISH, of the community in an assembled “wall-to-wall” micrograph from the indicated depth in the mature biofilm.
[Show abstract][Hide abstract]ABSTRACT: Scatter plot of area versus depth (z) for the assembled confocal micrographs (open circles) and Microchip compartment area as determined by light microscopy (closed circles). N.B. due to the production method the carrier area decreases with depth down to the mid-point of the carrier. That is, the total carrier compartment, from one carrier surface to the other, has a slight “waist” at the center. The area of the confocal micrographs was measured in the biofilm reference (Eub338) channel. Error bars = 95% confidence interval.
[Show abstract][Hide abstract]ABSTRACT: It is a challenge to apply anaerobic ammonium oxidation (anammox) for nitrogen removal from wastewater at low temperatures. Maintenance of anammox- and aerobic ammonia oxidizing bacteria (AOB) and suppression of nitrite oxidizing bacteria (NOB) are key issues. In this work, a nitritation-anammox moving bed biofilm pilot reactor was operated at 19-10°C for 300d. Nitrogen removal was decreasing, but stable, at 19-13°C. At 10°C removal became unstable. Quantitative PCR, fluorescence in situ hybridization and gene sequencing showed that no major microbial community changes were observed with decreased temperature. Anammox bacteria dominated the biofilm (0.9-1.2×10(14) 16S rRNA copies m(-2)). Most anammox bacteria were similar to Brocadia sp. 40, but another smaller Brocadia population was present near the biofilm-water interface, where also the AOB community (Nitrosomonas) was concentrated in thin layers (1.8-5.3×10(12) amoA copies m(-2)). NOB (Nitrobacter, Nitrospira) were always present at low concentrations (<1.3×10(11) 16S rRNA copies m(-2)).
No preview · Article · Dec 2013 · Bioresource Technology
[Show abstract][Hide abstract]ABSTRACT: Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample.