| 1. |
- Almstrand, Robert
(författare)
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Biofilms in Nitrogen Removal - Bacterial Population Dynamics and Spatial Distribution
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Efficient nitrogen removal at wastewater treatment plants (WWTPs) is necessary to avoid eutrophication of recipient waters. The most commonly used approach consists of aerobic nitrification and subsequent anaerobic denitrification resulting in the release of dinitrogen gas into the atmosphere. Nitrification is a two-step process performed by ammoniaoxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) often grown in biofilms at WWTPs. An alternative approach is anaerobic ammonium oxidation (anammox) where anammox bacteria convert ammonium and nitrite directly into dinitrogen gas. These groups of bacteria grow very slowly and are sensitive to perturbations, which may result in decreased efficiency or even breakdown of the process. Therefore, the ecology and activity of these bacteria and the structure of the biofilms in which they grow require detailed investigation to improve the understanding of nitrification and to facilitate the design of efficient nitrogen-removal strategies. To facilitate such studies of relevance for wastewater treatment, a nitrifying pilotplant was built where environmental conditions and especially ammonium concentrations could be controlled. In an experiment on model nitrifying trickling filters (NTFs), it was shown that biofilms subjected to intermittent feeding regimes of alternating high and low ammonium concentration in the water, could maintain a higher nitrification potential than biofilms constantly fed with low ammonium water. Such ammonium feed strategies can be used to optimize wastewater treatment performance. Different AOB populations within the N. oligotropha lineage were shown to respond differently to changes in environmental conditions, indicating microdiversity within this lineage which may be of importance for wastewater treatment. This diversity was further investigated through the development of new image analysis methods for analyzing bacterial spatial distribution in biofilms. The diversity within the N. oligotropha lineage was also reflected in the positioning of two such populations in the biofilm, where the vertical distribution patterns and relative positions compared to the NOB Nitrospira were different. In combination with a cryosectioning approach for retrieval of intact biofilm from small biofilm carrier compartments, the new image analysis methods showed a threedimensonal stratification of AOB-anammox biofilms. This may be of importance for mathematical modeling of such biofilms and the design of new biofilm carriers.
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| 2. |
- Almstrand, Robert, et al.
(författare)
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Biofilms in Nitrogen Removal: Population Dynamics and Spatial Distribution of Nitrifying- and Anammox Bacteria
- 2014
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Ingår i: Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications. - 9781908230485
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Efficient nitrogen removal at wastewater treatment plants (WWTPs) is necessary to avoid eutrophication of recipient waters. The most commonly used approach consists of aerobic nitrification and subsequent anaerobic denitrification resulting in the release of dinitrogen gas into the atmosphere. Nitrification is a two-step process performed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) often grown in biofilms at WWTPs. Alternatively, anaerobic ammonium oxidation (anammox) where anammox bacteria convert ammonium and nitrite directly into dinitrogen gas may be utilized. These groups of recalcitrant bacteria grow very slowly and are sensitive to perturbations, which may result in decreased efficiency or even breakdown of the respective process. Thus, their ecology, activity and the structure of the biofilms in which they grow require detailed investigation to improve our understanding of the nitrification and anammox processes. This in turn will facilitate the design of more efficient nitrogen-removal strategies. To assess the population dynamics and spatial distribution of nitrifying and anammox bacteria, culture-independent methods are essential. Therefore, the application of methods such as quantitative PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP) and Fluorescence In Situ Hybridization (FISH) in combination with advanced microscopy techniques and digital image analysis for the study of nitrifying and anammox biofilms will be reviewed in this chapter.
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| 3. |
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| 4. |
- Almstrand, Robert, et al.
(författare)
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Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater
- 2013
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Verlag (Germany). - 0175-7598 .- 1432-0614. ; 97:5, s. 2183-2191
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Tidskriftsartikel (refereegranskat)abstract
- Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.
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| 5. |
- Almstrand, Robert, et al.
(författare)
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New methods for analysis of spatial distribution and co-aggregation of microbial populations in complex biofilms.
- 2013
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 79:19, s. 5978-5987
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Tidskriftsartikel (refereegranskat)abstract
- In biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.
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| 6. |
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| 7. |
- Almstrand, Robert
(författare)
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Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes
- 2017
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Ingår i: Bio-protocol. - 2331-8325. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solidphase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.
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| 8. |
- Almstrand, Robert
(författare)
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Spatial impacts of inorganic ligand availability and localized microbial community structure on mitigation of zinc laden mine water in sulfate-reducing bioreactors
- 2017
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Ingår i: Water Research. - : Elsevier BV. - 0043-1354 .- 1879-2448. ; 115, s. 50-59
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Tidskriftsartikel (refereegranskat)abstract
- Sulfate-reducing bioreactors (SRBRs) represent a passive, sustainable, and long-term option for mitigating mining influenced water (MIW) during release. Here we investigate spatial zinc precipitation profiles as influenced by substrate differentiation, inorganic ligand availability (inorganic carbon and sulfide), and microbial community structure in pilot-scale SRBR columns fed with sulfate and zinc-rich MIW. Through a combination of aqueous sampling, geochemical digests, electron microscopy and energy-dispersive x-ray spectroscopy, we were able to delineate zones of enhanced zinc removal, identify precipitates of varying stability, and discern the temporal and spatial evolution of zinc, sulfur, and calcium associations. These geochemical insights revealed spatially variable immobilization regimes between SRBR columns that could be further contrasted as a function of labile (alfalfa-dominated) versus recalcitrant (woodchip-dominated) solid-phase substrate content. Both column subsets exhibited initial zinc removal as carbonates; however precipitation in association with labile substrates was more pronounced and dominated by metal-sulfide formation in the upper portions of the down flow columns with micrographs visually suggestive of sphalerite (ZnS). In contrast, a more diffuse and lower mass of zinc precipitation in the presence of gypsum-like precipitates occurred within the more recalcitrant column systems. While removal and sulfide-associated precipitation were spatially variable, whole bacterial community structure (ANOSIM) and diversity estimates were comparatively homogeneous. However, two phyla exhibited a potentially selective relationship with a significant positive correlation between the ratio of Firmicutes to Bacteroidetes and sulfide-bound zinc. Collectively these biogeochemical insights indicate that depths of maximal zinc sulfide precipitation are temporally dynamic, influenced by substrate composition and broaden our understanding of bio-immobilized zinc species, microbial interactions and potential operational and monitoring tools in these types of passive bioreactors. (C) 2017 Elsevier Ltd. All rights reserved.
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| 9. |
- Almstrand, Robert
(författare)
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Succession of founding microbiota in an anaerobic baffled bioreactor treating low-temperature raw domestic wastewater
- 2022
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Ingår i: Environmental Science: Water Research & Technology. - : Royal Society of Chemistry (RSC). - 2053-1400 .- 2053-1419. ; 8, s. 792-806
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Tidskriftsartikel (refereegranskat)abstract
- Continuously operated pilot- and full-scale anaerobic baffled reactors (ABRs) treating low-temperature raw domestic wastewater are currently few in number but offer significant advantages. As ABRs treating domestic wastewater become more prevalent, engineers and operators face the practical challenge of effectively transferring and seeding anaerobic sludge from existing "donor" ABRs to newly constructed ABRs. Unlike activated sludge, which predominantly consists of relatively fast-growing aerobic heterotrophic bacteria, anaerobic microbiota are slower-growing and the community structure may be impacted by process disturbances during bioreactor start-up. Examining the spatiotemporal development of anaerobic microbiota after transfer can enhance understanding of start-up dynamics in engineered anaerobic bioreactor systems. To understand the impacts of sludge transfer and seeding from an existing ABR operated for 3.5 years treating raw, low-temperature domestic wastewater to a new, similarly configured ABR treating a different raw domestic wastewater stream, influent wastewater and sludge microbiota samples were withdrawn biweekly for 275 days and used to characterize changes to the microbial community structure over time in both ABRs. Results suggest that the donor ABR communities maintained relatively consistent structure over time, but the microbial communities in the receiving bioreactor experienced two apparent successional trajectories post-inoculation. The first trajectory, which lasted for similar to 120 days, showed increasing divergence between communities in the two ABRs. This trajectory was marked by lower wastewater temperatures (12-14 degrees C, with extreme lows of 8 degrees C) and numerous disturbances to the sludge blankets. A second successional trajectory, observed when wastewater temperatures increased (>16 degrees C) and disturbances were eliminated, was marked by significant increases in the relative abundance of Euryarchaeota, especially Methanosaeta ("Methanothrix"), and increasing convergence of microbial communities in complementary donor and receiving bioreactor compartments. Further, the relative abundance of founding microbial community members significantly decreased during the first successional trajectory but significantly increased, or rebounded, during the second successional trajectory. The results of this study indicate that an anaerobic sludge inoculum can be effectively transferred from a long-running ABR treating raw, low-temperature domestic wastewater to a new ABR, and that similar performance can be achieved despite differing environmental conditions and disturbances to the sludge blanket microbial communities during start-up of the new ABR.
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| 10. |
- Almstrand, Robert, et al.
(författare)
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Three-dimensional stratification of bacterial biofilm populations in a moving bed biofilm reactor for nitritation-anammox.
- 2014
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 15:2, s. 2191-206
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Tidskriftsartikel (refereegranskat)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.
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