Nitrifying Biofilm Research Articles

The use of microsensors to study the role of the loading rate and surface velocity on the growth and the composition of nitrifying biofilms

The good composition and activity of biofilms are very important for successful operation and control of fixed-film biological reactors employed in liquid effluents treatment. During the last decade, microsensors have been applied to study microbial ecology. These sensors could provide information regarding the microbial activity concerning nitrification and denitrification that occur inside biofilms. Other techniques of molecular biology, such as fluorescence in situ hybridization (FISH), have also contributed to this matter because their application aids in the identification of the bacterial populations that compose the biofilms. The focus of this paper was to study the loading rate and surface velocity to promote the development of nitrifying biofilms in three distinct flow cells that were employed in the post treatment of a synthetic wastewater simulating the effluent from a UASB (Upflow Anaerobic Sludge Blanket) reactor. Using the FISH technique, it was found that the population of ammonia-oxidizing-bacteria was greater than that of nitrite-oxidizing-bacteria; this was also supported by the lower production of nitrate determined by physicochemical and microsensor analyses. It was verified that the loading rate and surface velocity that promoted the greatest nitrogen removal were 0.25 g N-amon m(-2)biofilm day(-1) and 1 m h(-1), respectively.

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon-Wiener index (H') results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.

Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.

Nitrification potential and population dynamics of nitrifying bacterial biofilms in response to controlled shifts of ammonium concentrations in wastewater trickling filters

Nitrogen removal in wastewater treatment is energy consuming and often carried out in biofilm nitrifying trickling filters (NTFs). We investigated nitrification potential and population dynamics of nitrifying bacteria in pilot-plant NTFs fed with full-scale plant wastewater with high (8–9mg NH4+l-1) or low (<0.5mg NH4+l-1) ammonium concentrations. After ammonium shifts, nitrification potentials stabilized after 10–43days depending on feed regime. An NTF fed with 3days of high, and 4days of low load per week reached a high nitrification potential, whereas a high load for 1day a week gave a low potential. Nitrosomonas oligotropha dominated the AOB and changes in nitrification potentials were not explained by large population shifts to other AOBs. Although nitrification potentials were generally correlated with the relative amounts of AOB and NOB, this was not always the case. Ammonium feed strategies can be used to optimize wastewater treatment performance.

Operational experiences with structured plastic media filters: 10 years on

The performance of 10 cross flow structured plastic media trickling filter plants is discussed. The sites are arranged in a number of configurations with the plastic media filters operating as sole secondary process units, in parallel with other process units or in double filtration configuration. Data for 2003 and 2004 show that the plants are performing well. Data are presented for the plastic media filter stream where it is available. Two operational problems are discussed; overgrazing of nitrifying biofilms by excessive populations of snails and loss of nitrification performance in cold weather, particularly on smaller sites. Overgrazing by snails, identified as Lymnaea peregra has been controlled on one site by the annual treatment of an isolated filter with high ammoniacal nitrogen strength sludge dewatering liquors. On a second site suffering a similar problem the plastic media filter duty was changed from single filtration to being the primary filters in a double filtration system with existing stone media filters used for secondary filters. Loss of nitrification performance has been associated with excessive heat loss on some sites. Filters built as tower structures, with typically 4–5 m media depth allow easy control of airflow via restriction of the engineered ventilation openings at the base of the filter. This has been found to have a direct impact on the degree of cooling. However, airflow control on plastic media retrofitted into wide shallow stone media filter shells with 1.6–2 m media depth is more difficult. These structures offer less opportunity for control of airflow and associated heat losses.

Structure of nitrifying biofilms in a high-rate trickling filter designed for potable water pre-treatment

This study examined the composition and structure of nitrifying biofilms sampled from a high-rate nitrifying trickling filter which was designed to pre-treat raw surface water for potable supply. The filter was operated under a range of feed water ammonia and organic carbon concentrations that mimicked the raw water quality of poorly protected catchments. The biofilm structure was examined using a combination of fluorescence in situ hybridisation and scanning electron microscopy. Biopolymers (carbohydrate and protein) were also measured. When the filter was operated under low organic loads, nitrifiers were abundant, representing the majority of microorganisms present. Uniquely, the study identified not only Nitrospira but also the less common Nitrobacter. Small increases in organic carbon promoted the rapid growth of filamentous heterotrophs, as well as the production of large amounts of polysaccharide. Stratification of nitrifiers and heterotrophs, and high polysaccharide were observed at all filter bed depths, which coincided with the impediment of nitrification throughout most of the filter bed. Observations presented here specifically linked biofilm structure with filter functionality, physically validating previous empirical modelling hypotheses regarding competitive interactions between autotrophic and heterotrophic bacteria in biofilms.

Free Chlorine and Monochloramine Application to Nitrifying Biofilm: Comparison of Biofilm Penetration, Activity, and Viability

Biofilm in drinking water systems is undesirable. Free chlorine and monochloramine are commonly used as secondary drinking water disinfectants, but monochloramine is perceived to penetrate biofilm better than free chlorine. However, this hypothesis remains unconfirmed by direct biofilm monochloramine measurement. This study compared free chlorine and monochloramine biofilm penetration into an undefined mixed-culture nitrifying biofilm by use of microelectrodes and assessed the subsequent effect on biofilm activity and viability by use of dissolved oxygen (DO) microelectrodes and confocal laser scanning microscopy (CLSM) with LIVE/DEAD BacLight. For equivalent chlorine concentrations, monochloramine initially penetrated biofilm 170 times faster than free chlorine, and even after subsequent application to a monochloramine penetrated biofilm, free chlorine penetration was limited. DO profiles paralleled monochloramine profiles, providing evidence that either the biofilm was inactivated with monochloramine's penetration or its persistence reduced available substrate (free ammonia). While this research clearly demonstrated monochloramine's greater penetration, this penetration did not necessarily translate to immediate viability loss. Even though free chlorine's penetration was limited compared to that of monochloramine, it more effectively (on a cell membrane integrity basis) inactivated microorganisms near the biofilm surface. Limited free chlorine penetration has implications when converting to free chlorine in full-scale chloraminated systems in response to nitrification episodes.

Nitrification Comparison between Synthetic Wastewater and Secondary Effluent Using Membrane-Aerated Biofilm Reactor (MABR) Process

Nitrification Comparison between Synthetic Wastewater and Secondary Effluent Using Membrane-Aerated Biofilm Reactor (MABR) ProcessTwo pilot-scale membrane aerated biofilm reactors (MABR) were operated for over 530 days to compare the nitrification in both synthetic wastewater and secondary effluent, and to examine the impacts of hydrodynamic shear caused by adjusting effluent recirculation on ammonia removal. The results showed that secondary effluent had enhanced the nitrifying biofilm formation and/or detention. The...Author(s)Zebo LongLori LishmanHongde ZhouPeter SetoSteve N. LissSourceProceedings of the Water Environment FederationSubjectSession 76: Membrane ProcessesDocument typeConference PaperPublisherWater Environment FederationPrint publication date Jan, 2011ISSN1938-6478SICI1938-6478(20110101)2011:11L.4953;1-DOI10.2175/193864711802765723Volume / Issue2011 / 11Content sourceWEFTECFirst / last page(s)4953 - 4966Copyright2011Word count152Subject keywordsAmmonia removalmembrane-aerated biofilm reactormixingtertiary nitrificationoxygen supplywater reclamation

Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge

The composition and the distribution of extracellular polymeric substances (EPS) and pellets of autotrophic nitrifying biofilm and activated sludge were investigated in this work. Fourier-transform Infrared Spectroscopy, fluorescent in situ hybridization and fluorescence staining were used to examine proteins, carbohydrates, humic substances and DNA being present in the biofilms and the sludge samples. To investigate extraction efficiency and its effect on characterization of tightly bounded EPS, four extraction methods (ethylenediamine tetraacetic acid (EDTA), NaOH, cationic exchange resin (CER), ultrasound) were compared. EDTA and ultrasound showed more effective extraction ability than NaOH and CER. NaOH and ultrasound extraction led to high activity of glucose-6-phosphate dehydrogenase from cell lysis, which was confirmed by fluorescence staining analysis. Ultrasound and NaOH extraction yielded 18% and 11% of dead cells in biofilm, respectively, whereas they obtained 11% and 9% of dead cells in activated sludge, respectively. Four layers of extractible products were separated from autotrophic nitrifiering flocs. The extraction results indicated that extraction yield in different layers varied in a wide range: 3–6% of soluble EPS, 5–10% of loosely bound EPS, 34–67% of tightly bound EPS and 30–60% of pellets.

Influence of water hardness on concrete surface deterioration caused by nitrifying biofilms in wastewater treatment plants

In aeration tanks of wastewater treatment plants (WWTP), biodeterioration can lead to a partial dissolution and ensuing erosion of the concrete due to an acid attack caused by the nitrifying biofilm covering the tank surface. In this study, concrete samples were exposed in four different WWTP. The CaO content of the cement used, the water-to-cement ratio, and curing affect the resistance of the concrete to acid attack. However, both experimental data and thermodynamic modeling show that water hardness is the dominating parameter for surface erosion. In the presence of high water hardness, the protons produced by the nitrifiers are buffered and the calcite precipitation occurring in all concrete samples is increased. This results in a decrease of porosity near the surface and higher mass transfer resistance against carbon dioxide and therefore in lower deterioration rates of the concrete.

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane-aerated biofilm reactor (MABR) and the composition of the initial ammonia- and nitrite-oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat-sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO(2)(-)-N/NH(4)(+)-N) were obtained in the Inoculum B- (55.2-56.4%) versus the Inoculum A- (20.2-22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100-150 µm), but the AOB profiles [based on 16S rRNA gene targeted real-time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B- versus A-initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A- and B-initiated reactors were Nitrosospira spp. (48.9-61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8-63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell(-1) h(-1) versus 0.79 fmol cell(-1) h(-1) ). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.

Measuring and modeling the oxygen profile in a nitrifying Moving Bed Biofilm Reactor

In this paper we determine the oxygen profile in a biofilm on suspended carriers in two ways: firstly by microelectrode measurements and secondly by a simple mathematical model. The Moving Bed Biofilm Reactor is well-established for wastewater treatment where bacteria grow as a biofilm on the protective surfaces of suspended carriers. The flat shaped BiofilmChip P was developed to allow good conditions for transport of substrates into the biofilm. The oxygen profile was measured in situ the nitrifying biofilm with a microelectrode and it was simulated with a one-dimensional mathematical model. We extended the model by adding a CSTR equation, to connect the reactor to the biofilm through the boundary conditions. We showed the dependence of the thickness of the mass transfer boundary layer on the bulk flow rate. Finally, we estimated the erosion parameter λ to increase the concordance between the measured and simulated profiles. This lead to a simple empirical relationship between λ and the flow rate. The data gathered by in situ microelectrode measurements can, together with the mathematical model, be used in predictive modeling and give more insight in the design of new carriers, with the ambition of making process operation more energy efficient.

Biologically induced concrete deterioration in a wastewater treatment plant assessed by combining microstructural analysis with thermodynamic modeling

In the nitrification basins of wastewater treatment plants, deterioration of the concrete surface can occur due to acid attack caused by a nitrifying biofilm covering the concrete. To identify the mechanism of deterioration, concrete cubes of different composition were suspended in an aerated nitrification basin of a wastewater treatment plant for two years and analyzed afterwards. The microstructural investigation reveals that not only dissolution of hydrates takes place, but that calcite precipitation close to the surface occurs leading to the formation of a dense layer. The degree of deterioration of the different cubes correlates with the CaO content of the different cements used. Cements which contain a high fraction of CaO form more calcite offering a better protection against the acid attack. The presence of slag, which lowers the amount CaO in the cement, leads to a faster deterioration of the concrete than observed for samples produced with pure OPC.

Nitrogen Removal from Wastewater Using a Hybrid Membrane‐Biofilm Process: Pilot‐Scale Studies

The hybrid membrane biofilm process (HMBP) is a new approach to achieving total nitrogen removal from wastewater. Air-filled, hollow-fiber membranes are placed into an activated sludge basin and bulk aeration is suppressed. A nitrifying biofilm develops on the membranes, exporting nitrate and nitrite to the bulk liquid. The nitrate and nitrite are reduced by suspended biomass using influent BOD as the electron donor. Previous research demonstrated the HMBP concept at the bench scale and explored process fundamentals. This research explored the HMBP at the pilot scale, with a 120-L reaction tank, real wastewater, and a potentially scalable configuration. Nitrification rates averaged 0.5 g N m(-2)/d(-1), which were lower than found at the bench scale, and lower than predicted by a mathematical model, but still allowed effluent total nitrogen concentrations below 6 mg N/L with an average influent total nitrogen concentration of 25 mg N/L and a hydraulic retention time of 12 hours. More than 75% of the produced nitrate and nitrite was reduced with an average influent sCOD of only 68 mg/L and an average C:N ratio of 3.1. Mass balances on carbon and nitrogen suggest that nitrogen removal via nitrite occurred. This research confirms that the HMBP process is effective for BOD and nitrogen removal from wastewater, and suggests that the grid configuration is viable for scale-up.

Monitoring structure and activity of nitrifying bacterial biofilm in an automatic biodetector of water toxicity

Automatic biodetector of water toxicity is a biosensor based on monitoring of catalytic activity of the nitrifying bacteria. To create a standardized biosensing system, development of the biofilm must be characterized to determine the prerequisites for its biological (biocatalytic) stability. In this paper, growth of biofilm comprising ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the open cellular polyurethane material polyurethane sponge bioreactor has been investigated. Dynamics of the biofilm formation was estimated using AOB and NOB metabolic activity and the volume occupied by these two types of bacteria in the biofilm. Spectrophotometry liquid ion chromatography and image cytometry were used, respectively, for these measurements. A mathematical model of the dynamics of biofilm formation was established. These data indicate that open cellular polyurethane material is a good basis for the immobilization of nitrifying bacteria. Moreover, growth of the biofilm leads to its stable structural form, whose biocatalytic activity (12.29 for AOB and 6.84 μmol min −1 for NOB) is constant in the long term.

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi-species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite-oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia-oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients.

Dissolved organic matter and nitrogen removal by advanced aerated submerged bio-film reactor

Aerated submerged bio-film (ASBF) pilot plant has been developed to optimize an inexpensive method of enhanced wastewater treatment. This study describes pilot scale experiments for efficient removal of dissolved organic and nitrogen compounds by using ASBF. This research explores the possibility of enhancing the performance of shallow wastewater treatment lagoons through the addition of specially designed structures. These structures are designed to encourage the growth of a nitrifying bacterial bio-film on a submerged surface. These structures also force the direct contact of rising air bubbles against the submerged bio-film. This direct gas-phase contact should increase the oxygen transfer rate into the bio-film, as well as increase the micro-climate mixing of water, nutrients, and waste products into and out of the bio-film. This research investigated the efficiency of dissolved organic matter and ammonia nitrogen removals at cold temperatures. As it was anticipated, nitrification activity was highest during periods when the flow rate was lower, but it seemed to decline during times when the flow rate was highest. And ammonia nitrogen removal rates were more sensitive than dissolved organic matter removal rates when flow rates exceeded 2.2L/min.

Biodegradation of 1,2-dichloroethane (1,2-DCA) by cometabolism in a nitrifying biofilm reactor

Biodegradation of 1,2-dichloroethane (1,2-DCA) by cometabolism was investigated in a continuous-flow nitrifying biofilm reactor over a time period of 218 days. The removal efficiency of 1,2-DCA ranged between 70 and 90%. Using the generation of chloride (Cl −) as an indicator of 1,2-DCA mineralization, it was shown that the cometabolic degradation of 1,2-DCA was initiated through oxidative dechlorination. However, Cl − production rates were observed to be lower than the stoichiometric ones which indicated the partial mineralization of 1,2-DCA and the possibility of by-product formation due to incomplete dechlorination. At high 1,2-DCA removal rates, Cl − release seemed to reach a saturation due to 1,2-DCA-dependent inactivation of NH 4–N oxidation. The cometabolic 1,2-DCA degradation capacity of nitrifiers was quite comparable to metabolic 1,2-DCA degradation capacities of pure cultures. A strong linear relationship was found between 1,2-DCA transformation yields and NH 4–N and 1,2-DCA loadings. The effect of 1,2-DCA loading on nitrifier population was monitored using molecular microbiological tools. Long-term input of 1,2-DCA to the biofilm reactor resulted in no significant changes in the quantities of Nitrosomonas, Nitrobacter and Nitrospira species and no shift in the diversities of ammonia oxidizing species. Those findings provide an insight into both the operation and the community structure in natural and managed nitrifying biofilm systems where cometabolic 1,2-DCA takes place.

Enhancing the formation and shear resistance of nitrifying biofilms on membranes by surface modification

Polypropylene (PP) membranes and polyethylene (PE) surfaces were modified to enhance formation and shear resistance of nitrifying biofilms for wastewater treatment applications. A combination of plasma polymerization and wet chemistry was employed to ultimately introduce poly(ethyleneglycol) (PEG) chains with two different functional groups (–PEG–NH 2 and –PEG–CH 3). Biofilm growth experiments using a mixed nitrifying bacterial culture revealed that the specific combination of PEG chains with amino groups resulted in most biofilm formation on both PP and PE samples. Detachment experiments showed similar trends: biofilms on –PEG–NH 2 modified surfaces were much stronger compared to the other modifications and the unmodified reference surfaces. Electrostatic interactions between the protonated amino group and negatively charged bacteria as well as PEG chain density which can affect the surface structure might be possible explanations of the superiority of the –PEG–NH 2 modification. The success of the–PEG–NH 2 modification was independent of the original surface and might, therefore, be used in wastewater treatment bioreactors to improve reactor performance by making biofilm formation more stable and predictable.

In situ characterization of nitrifying biofilm: Minimizing biomass loss and preserving perspective

Methods for characterizing nitrifying bacteria within biofilms are of key importance to understand and optimize the nitrification kinetics of attached growth treatment facilities. In this work, we propose an analytical protocol based upon environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CSLM) in combination with fluorescent in situ hybridization (FISH) to characterize the structure of nitrifying biofilm as it remains attached to the original reactor substratum. This protocol minimizes the loss of mass and distortion of in situ perspective commonly associated with traditionally applied microscopic techniques and thereby enables a more accurate estimation of the nitrifying biomass within biofilm attached to the substratum. The use of ESEM eliminates the destructive preparatory procedures associated with traditional scanning electron microscopy and thus the loss of mass and shrinking of the samples. ESEM is used in this study to evaluate the percent coverage of the substratum with biofilm and the biofilm thickness. CLSM–FISH is used to determine cell counts in the biofilm and to characterize the undisturbed substratum/biofilm interface. By hybridizing and analyzing the nitrifying biofilm using CLSM as it remains attached to the substratum, the loss of material and distortion of in situ perspective associated with the biofilm detachment process is minimized. Moreover, by conducting the CLSM analysis directly on the nitrifying biofilm as it remains attached to the substratum it is shown that cell counts at the substratum/biofilm interface differ significantly from that located above the interface.