Substrate Diffusion Limitations Research Articles

Hierarchical metal–organic framework nanoarchitectures for catalysis

Metal–organic frameworks (MOFs) have garnered significant attention in the field of catalysis due to their unique advantages such as diverse coordination geometry, variable metal nodes, and organic linkers, facilitating precise structural and compositional control for achieving programmable catalytic functionalities. Although their inherent microporous structure could provide excellent shape selectivity during catalysis, it typically impedes the mass transfer process, thereby reducing the use of internal active sites and overall catalytic efficiency. Additionally, employing single MOFs as catalysts presents challenges in achieving complex catalytic reactions that require multifunctional active sites. In recent years, considerable research efforts have focused on designing and constructing hierarchical nanostructured MOFs to alleviate substrate diffusion limitations by introducing secondary nanopores, shortening diffusion distances via the construction of low-dimensional nanoarchitectures, and constructing multifunctional catalysts by integrating distinct MOFs with suitable functions. This review provides a comprehensive overview of the design, synthesis methods, and formation mechanisms of MOF-based hierarchical nanostructures in recent years. Subsequently, it further highlights their applications in thermal catalysis, electrocatalysis, and photocatalysis, along with the relationship between their hierarchical nanostructures and catalytic performances. Finally, it provides an outlook on the challenges and potential development directions of hierarchically structured MOF nanocatalysts.

Xylooligosaccharides as emerging prebiotics and their sustainable generation from xylan catalysed by endoxylanase immobilized ordered mesoporous silica

The enzyme endo-1,4-β-xylanase has been a prominent candidate for research in recent years due to its ability to catalyse prebiotic production in an environmentally friendly manner. However, the loss of stability and activity when employed in industrial processes has prompted enzyme immobilization. The present study aims to compare the efficiencies of amorphous (such as fumed silica and silicic acid) and ordered mesoporous silica materials (including SBA-15, IITM-41, and MCM-41) as carriers for endoxylanase immobilization, particularly focussing on the novel silicate IITM-41. Recombinant endoxylanase from Bacillus subtilis KCX006 purified by Ni-NTA columns has been used in this study. XRD, TEM, and BET analyses of these nanostructured materials confirmed the mesoporous structure and the nature of the pores. FT-IR spectroscopy confirmed protein binding through characteristic functional group signatures of silica materials and proteins. Zeta potential values of the nanostructure materials became more negative after enzyme binding, and the confirmation of enzyme immobilization was achieved through fluorescent tagging. Although amorphous silica showed a higher yield of immobilization, it resulted in lower recovered activity due to the formation of protein multilayers. Among the ordered mesoporous silica materials, IITM-41 exhibited the highest recovered activity at 74 %, with a maximum yield of xylooligosaccharides reaching 797.61 mg/g. The optimum pH of the immobilized enzymes remained unchanged, but there was a shift in the optimum temperature from 50 to 60 °C, along with a broader optimal range, attributed to increased enzyme rigidity and substrate diffusion limitations. Kinetic parameters Km and Vmax were higher for immobilized enzymes compared to the free enzyme, particularly with higher Vmax values observed for enzymes immobilized on mesoporous materials compared to those on amorphous silica. Generally, mesoporous materials showed superior performance in terms of xylooligosaccharides production, storage stability, and recycling efficiency. Over 10 cycles of reuse, mesoporous materials exhibited a higher yield of total xylooligosaccharides (ranging from X2 to X6) compared to amorphous silica.

Disintegration of Partial Denitrification Granules at High Nitrate Concentration

It is well acknowledged that high nitrate concentration would negatively affect the biological denitrification activity, especially in the presence of nitrite accumulation. In this study, a novel granular partial denitrification (PD) process was developed, and the impact of nitrate concentration (120–960 mg N/L) on PD granules was investigated by close monitoring of the operational performance, physicochemical characteristics, and microbial community. Results indicated that the effluent nitrite increased with the nitrate concentration, with a maximum value of 492.4 mg N/L obtained. And the maximum denitrification activity in situ was found not to be decreased despite high nitrite accumulation; it was maintained at 0.24 g N/g VSS/h, suggesting the high tolerance of PD granules to the nitrite. The granule size increased with the influent nitrate, and the compact PD granules, however, were observed to be disintegrated at 960 mg N/L of nitrate, which was likely attributed to the substrate diffusion limitation in larger granules, with the cavity formed in its inner part. In addition, microbial analysis revealed that bacterial richness and the diversity of PD granules were decreased, and Gracilibacteria that was supposed to be responsible for maintaining the PD granule structure was largely reduced at a high nitrate concentration, which was likely to be another major reason for the disintegration. This study revealed the potential causes for the disintegration of PD granules, which offer theoretical support and technical guidance to ensure the stability of PD granules in treating high nitrate wastes for efficient nitrite production.

Expanding the activated sludge model no.1 to describe filamentous bulking: The filamentous model

Several wastewater treatment plants (WWTPs) worldwide have documented the occurrence of filamentous bulking in full-scale systems despite the efforts made for filamentous bulking control. The Activated Sludge Models (ASM) can neither describe nor predict filamentous bulking at WWTPs. This research aims to expand the ASM No. 1 to be able to describe filamentous bulking sludge and to model the effects of incorporating an aerobic selector on filamentous bulking. Four theories (hydrolysis of slowly biodegradable organics theory, kinetic selection theory, substrate diffusion limitation theory, and filamentous backbone theory) were combined to expand the ASM1. The results showed that this combination was successful to distinguish between the substrate uptake by filamentous organisms and by floc forming organisms. Moreover, the concentrations of filamentous and floc forming organisms inside the reactor were converted to a “filamentous score” that predicted the outcome of filamentous bulking. Filamentous bulking would occur if the filamentous score was higher than 3, in a range of 1–6. As a case study, the Fuhais WWTP in Jordan was modelled using the expanded-ASM1 “filamentous model” and the filamentous score of 4.2 was in accordance to the visually observed bulking. However, when an aerobic selector with 3 compartments would be added before the aeration tank, the filamentous score decreased to 1.5. The selector changed the hydraulic behaviour from a completely mixed mode to a plug flow mode, which created a substrate gradient in the model, making the floc forming organisms to outcompete the filamentous organisms. Additional experimental results are required to further calibrate and validate the filamentous model.

Soil Respiration Phenology Improves Modeled Phase of Terrestrial net Ecosystem Exchange in Northern Hemisphere

AbstractIn the northern hemisphere, terrestrial ecosystems transition from net sources of CO2 to the atmosphere in winter to net ecosystem carbon sinks during spring. The timing (or phase) of this transition, determined by the balance between ecosystem respiration (RECO) and primary production, is key to estimating the amplitude of the terrestrial carbon sink. We diagnose an apparent phase bias in the RECO and net ecosystem exchange (NEE) seasonal cycles estimated by the terrestrial carbon flux (TCF) model framework and investigate its link to soil respiration mechanisms. Satellite observations of vegetation canopy conditions, surface meteorology, and soil moisture from the NASA SMAP Level 4 Soil Moisture product are used to model a daily carbon budget for a global network of eddy covariance flux towers. Proposed modifications to TCF include: the inhibition of foliar respiration in the light (the Kok effect); a seasonally varying litterfall phenology; an O2 diffusion limitation on heterotrophic respiration (RH); and a vertically resolved soil decomposition model. We find that RECO phase bias can result from bias in RECO magnitude and that mechanisms which reduce northern spring RECO, like substrate and O2 diffusion limitations, can mitigate the phase bias. A vertically resolved soil decomposition model mitigates this bias by temporally segmenting and lagging RH. Applying these model enhancements at continuous soil respiration (COSORE) sites verifies their improvement of RECO and NEE skill compared to in situ observations (up to ΔRMSE = −0.76 g C m−2 d−1). Ultimately, these mechanisms can improve prior estimates of NEE for atmospheric inversion studies.

Long-term effects of mineral precipitation on process performance, granules' morphology and microbial community in anammox granular sludge

When treating wastewaters prone to inert precipitation with granular sludge systems, mineral formation needs to be properly controlled to ensure system’s long-term stability. In this work, an extensive study on mineral precipitation on the surface of anammox granular sludge is presented. A 7-L reactor was inoculated with one-year stored biomass and volumetric load up to 0.48 gN-NO2-/l/d were achieved, with nitrite removal above 95% and total nitrogen removal rate of almost 1 gN/l/d. Severe mineral precipitation was observed on the granules’ surface after three months of hard-water feeding and resulted in a dramatic deterioration of reactor performance and biomass activity. Substrate diffusion limitation in the inner layers, insufficient mixing due to higher granule density and biofilm erosion due to shear stress increase were deemed the main mechanisms that lead to progressive process disruption. Gravimetric selection was applied to discard granules affected by precipitation and allowed for process restoration. Microbial community analyses revealed that mineral composition possibly affected competition between “Ca. Brocadia” and “Ca. Kuenenia”. The knowledge gathered in the present study details the dramatic consequences on process performance lead by severe mineral precipitation and it is presented as a warning for full-scale applications treating wastewaters prone to precipitation.

Three-dimensional ordered magnetic macroporous metal-organic frameworks for enzyme immobilization

Metal-organic frameworks (MOFs) have been emerged as a promising support for immobilizing enzymes owing to the tunable porosity, high surface area, and structural diversity. However, most of these possess nanometer size and small pores, which are difficult to recover them from the reaction medium and present low immobilization efficiency and protein loading capacity, and high substrate diffusion limitations. Herein, a novel magnetic amino-functionalized zeolitic imidazolate framework-8 (ZIF-8) with 3D highly ordered macroporous structure was synthesized using the assembled polystyrene (PS) nanosphere monoliths as a template. Subsequently, catalase (CAT) molecules were immobilized on the surface of macroporous magnetic ZIF-8 and inside the macropores by precipitation, covalent binding and cross-linking. The resultant immobilized CAT showed high immobilization efficiency (58%) and protein loading capacity (29%), leading to 500% higher activity than the immobilized CAT on ZIF-8 (CAT/ZIF-8). Meanwhile, the immobilized CAT could be easily recovered with a magnet without obvious activity loss. The traditional CAT/ZIF-8 lost its activity after 6 cycles, whereas, the immobilized CAT retained 90% activity of its initial activity after reusing for 8 cycles, indicating excellent reusability. In conclusion, this study provides a facile and efficient approach to immobilize enzymes on/in MOFs with enhanced activity and excellent recyclability.

Composites of Crosslinked Aggregates of Eversa® Transform and Magnetic Nanoparticles. Performance in the Ethanolysis of Soybean Oil

Eversa® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the available tools to improve its performance. Several variables in the CLEA preparation were optimized to improve the recovered activity, such as precipitant nature and crosslinker concentration. Moreover, some feeders were co-precipitated to improve the crosslinking step, such as bovine serum albumin, soy protein, or polyethyleneimine. Starch (later enzymatically degraded) was utilized as a porogenic agent to decrease the substrate diffusion limitations. Silica magnetic nanoparticles were also utilized to simplify the CLEA handling, but it was found that a large percentage of the Eversa activity could be immobilized on these nanoparticles before aggregation. The best CLEA protocol gave a 98.9% immobilization yield and 30.1% recovered activity, exhibited a porous structure, and an excellent performance in the transesterification of soybean oil with ethanol: 89.8 wt% of fatty acid ethyl esters (FAEEs) yield after 12 h of reaction, while the free enzyme required a 48 h reaction to give the same yield. A caustic polishing step of the product yielded a biodiesel containing 98.9 wt% of FAEEs and a free fatty acids content lower than 0.25%, thus the final product met the international standards for biodiesel. The immobilized biocatalyst could be reused for at least five 12 h-batches maintaining 89.6% of the first-batch yield, showing the efficient catalyst recovery by applying an external magnetic field.

Carbohydrate-binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity.

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a "non-catalytic" lectin-like domain or carbohydrate-binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intradomain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers.

Coimmobilization of different lipases: Simple layer by layer enzyme spatial ordering

This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution.

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%).

Growth and photosynthetic activity of Chlamydomonas reinhardtii entrapped in lens-shaped silica hydrogels

Entrapment of microalgae in silica hydrogels enables the application as biocatalysts in continuous production of secreted products. Despite a mitigation of substrate and product diffusion limitations by lens-shaped particles, there are no reports on light supply and limitation. This study investigated the impact of hydrogel structure, particle size and biomass loading on the behaviour of the microalga Chlamydomonas reinhardtii entrapped in lens-shaped silica particles.Entrapment in tetraethyl orthosilicate and tetra(n-propylamino)silane based hydrogels reduced the growth rate by 30% and 23%, respectively. In contrast, cells entrapped in sodium silicate based hydrogels displayed a growth rate similar to free cells and cells entrapped in calcium alginate (1.13 d−1), indicating a suitable biocompatibility. Reduction of lens height by 26% maintained the growth rate in silica hydrogel. A fourfold increase in biomass loading reduced the growth rate by 20% and elevated the yield coefficient by 211%, indicating the impact of biomass loading on light and nutrient supply on photosynthetic growth. Finally, hydrogen production was observed by entrapped cells.The results of this work will pave the way for robust biocatalytic processes where photosynthetically active cells are protected against harmful mechanical and biological influences.

Sensitive optical detection of clinically relevant biomarkers in affordable microfluidic devices: Overcoming substrate diffusion limitations

Abstract One of the biggest challenges in miniaturization of optical immunoassays is the short light path distance of microchannels/microcapillaries. Protein biomarkers are often presented in circulating blood in the picomolar-femtomolar range, requiring exceptional levels of sensitivity that cannot be met with traditional chromogenic substrates and without sophisticated, bulky detection systems. This study discloses an effective strategy for increasing the sensitivity and shorten the total test time for sandwich ELISAs in microfluidic devices optically interrogated, based on enhancing enzymatic amplification. We found that activity of Horseradish Peroxidase (HRP) in mesofluidic systems is highly limited by diffusion, therefore increasing the concentration of enzymatic substrate in these systems does not translate into an enhancement in enzymatic conversation. The opposite happens in microfluidic systems due to short diffusion distances, however increased concentration of the second enzymatic substrate, hydrogen peroxide (H2O2), leads to enzyme inhibition as herein reported. Consequently, we found that the molar ratio of o-phenylenediamine (OPD) to hydrogen peroxide from commercially substrate formulations is not suitable for miniaturized systems. Sandwich ELISA quantitation of a cancer biomarker PSA and human cytokine IL-1β in fluoropolymer microfluidic strips revealed over one order of magnitude increase in sensitivity and 10-fold decrease in incubation time by simply changing the molar ratio of OPD:H2O2 from 1:3 to 1:1 and increasing OPD concentration from 1 to 4 mg/ml. This enhancement in enzymatic amplification offers finally the sensitivity required for optical interrogation of novel portable and affordable microfluidic devices with inexpensive and ubiquitous smartphones and flatbed scanners.

Properties of lysozyme/sodium alginate complexes for the development of antimicrobial films

Complexation study of lysozyme (0.714g/L) by sodium alginate at pH7 showed that aggregates formation was a two-phase process. The first phase (from 0 to 0.1g/L sodium alginate) corresponded to the combination of individual complexes to form aggregates which caused an increase of turbidity and average size and a rapid sedimentation. Charge neutralization estimated by ζ-potential measurements occurred at 0.1g/L sodium alginate concentration. The second phase (from 0.1 to 4g/L of sodium alginate) was characterized by the formation of aggregates having a less dense structure with higher average size despite the drop in turbidity and the high dispersion in the medium. Lysozyme enzymatic activity decreased upon complexation with sodium alginate but was fully recovered after calcium chloride addition. In order to check whether lysozyme reversible inactivation was only due to substrate diffusion limitation or to conformational changes upon complexation, fluorescence and UV–Vis absorption measurements were performed. Moreover, lysozyme/sodium alginate complexes were used to manufacture an edible antimicrobial film to target lysozyme sensitive microorganisms.

Modeling Growth of Filaments and Floc Formers in Activated Sludge Flocs: Integrating the Effects of Kinetics and Diffusion

Filamentous bulking is the most common solid-separation problem in wastewater treatment systems. The phenomenon is attributed to competition between filamentous bacteria and floc-forming bacteria, a tradeoff between kinetic selection (KST) and substrate diffusion limitation (SDL). Previously, we explained our contradictory experimental results by both effects and developed a conceptual framework with three regions (bulking, intermediate, and non-bulking). However, within this purely qualitative framework, the range of substrate concentrations for each region was not determined. To test the framework and quantify the critical concentrations for the three regions, we here integrate KST and SDL into a model that simulates the growth rates of filaments and floc formers inside activated sludge flocs. Bulking and non-bulking sludges were produced in laboratory-scale reactors, and the kinetic model parameters were measured by respirometry. We then extended the model to verify our previous experimental results from sequencing batch reactors (SBRs). The effects of oxygen limitation and decay rates of floc formers and filaments on the modeled growth rates were also evaluated. In basic simulations, the model successfully determined the critical values of substrate concentrations in the framework. The results of the extended model were consistent with our experimental data, indicating that large flocs induce bulking whereas small flocs do not. Simulation results were more sensitive to the kinetic parameters than to the diffusion parameters, and the parameters more sensitively affected the growth of floc formers than filament growth.

Immobilized Enzymes: Strategies for Overcoming the Substrate Diffusion- Limitation Problem

Immobilized enzymes offer many advantages over the free counterparts. These advantages include, but are not limited to, easy separation, reuse, induced stabilities and potential of continuous operation. However, the immobilization process at the same time causes some drawbacks including loss of catalytic activity of the immobilized enzymes. Many interpretations have been given to explain the reasons behind such drawback. Some of them are related to the enzymes themselves, such as deactivation of enzymes and miss-orientation. Others refer to diffusion limitation of substrates and/or to inhibitory effects of the products. Substrate diffusion limitation presents the most serious one. In this review some strategies for overcoming this drawback are reported. Among these strategies are the ones related to enzyme immobilization on: a) soluble-insoluble matrices, b) thermally reversible hydrogels, c) pressure-sensitive gels, d) on catalytic hydrophobic membranes operating under non-isothermal conditions, and finally, on carriers' surface, are presented and discussed. Keywords: Activity decay, diffusion limitation, easy separation, enzymes immobilization, inhibition, non-isothermal bioreactors, pressure-sensitive gels, products, reuse ability, soluble-insoluble matrices, stabilities, substrates, surface immobilization, thermally reversible hydrogels.

Simulation of batch-operated experimental wetland mesocosms in AQUASIM biofilm reactor compartment

In this study, a mathematical biofilm reactor model based on the structure of the Constructed Wetland Model No.1 (CWM1) coupled to AQUASIM's biofilm reactor compartment has been used to reproduce the sequence of transformation and degradation of organic matter, nitrogen and sulphur observed in a set of constructed wetland mesocosms and to elucidate the development over time of microbial species as well as the biofilm thickness of a multispecies bacterial biofilm in a subsurface constructed wetland. Experimental data from 16 wetland mesocosms operated under greenhouse conditions, planted with three different plant species (Typha latifolia, Carex rostrata, Schoenoplectus acutus) and an unplanted control were used in the calibration of this mechanistic model. Within the mesocosms, a thin (predominantly anaerobic) biofilm was simulated with an initial thickness of 49 μm (average) and in which no concentration gradients developed. The biofilm density and area, and the distribution of the microbial species within the biofilm were evaluated to be the most sensitive biofilm properties; while the substrate diffusion limitations were not significantly sensitive to influence the bulk volume concentrations. The simulated biofilm density ranging between 105,000 and 153,000 gCOD/m3 in the mesocosms was observed to vary with temperature, the presence as well as the species of macrophyte. The biofilm modeling was found to be a better tool than the suspended bacterial modeling approach to show the influence of the rhizosphere configuration on the performance of the constructed wetlands.

Carbon and hydrogen stable isotope fractionation associated with the anaerobic degradation of propane and butane by marine sulfate‐reducing bacteria

The anaerobic degradation of propane and butane is typically initiated by activation via addition to fumarate. Here we investigated the mechanism of activation under sulfate-reducing conditions by one pure culture (strain BuS5) and three enrichment cultures employing stable isotope analysis. Stable isotope fractionation was compared for cultures incubated with or without substrate diffusion limitation. Bulk enrichment factors were significantly higher in mixed vs. static incubations. Two dimensional factors, given by the correlation of stable isotope fractionation of both carbon and hydrogen at their reactive positions (Lambda reactive position, Λrp), were compared to analyse the activation mechanisms. A characteristic reactive position isotope fractionation pattern was observed, distinct from aerobic degradation. Λrp values ranged from 10.5 to 11.8 for propane and from 7.8 to 9.4 for butane. Incubations of strain BuS5 with deuterium-labelled n-alkanes indicated that butane was activated solely at the subterminal C atom. In contrast, propane was activated mainly at the subterminal C atom but also significantly at the terminal C atoms. A conservative estimate suggests that about 70% of the propane activation events occurred at the subterminal C atom and about 30% at the terminal C atoms.

Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor

We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6 mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m −3 d −1. The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, “ Candidatus Brocadia–like species”, affiliated with “ Candidatus Brocadia anammoxidans” with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4′,6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups, Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 μm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-N L −1) than did the homogenized biomass (200 mg-N L −1) probably due to substrate diffusion limitation.

Effect of loading rate and oxygen supply on nitrification in a non-porous membrane biofilm reactor

A nitrifying membrane biofilm reactor (MBfR) was operated over 170 days, to assess the effect of ammonia loading rate under O 2-excess conditions, and the effect of dissolved oxygen under O 2-limiting conditions on nitrification efficiency. The MBfR was fed pure oxygen by diffusion through a non-porous membrane. Five different loading rates, ranging from 1.92 to 5.53 g N/m 2 d, were tested, yielding specific nitrification rates (SNR) ranging from 1.54 to 2.60 g N/m 2 d. SNR increased linearly with specific loading rate, up to the load of 3.5 g N/m 2 d, which indicated that mass transfer was linearly related to the bulk ammonia concentration. Beyond that load, substrate diffusion limitation inhibited further increase of SNR. When operating the system under limited oxygen supply conditions, 100% oxygen utilization was achievable. Maintenance of higher oxygen supply allowed a slightly higher SNR due to the growth of nitrifiers at the outer side of the biofilm (away from the membrane surface). Nitrification batch tests confirmed that the fraction of nitrifiers in the solids detached from the surface of the biofilm (and washed out with the effluent), was twice as high during oxygen-excess conditions when compared to oxygen-limiting conditions.