Growth, filtration and respiration characteristics of small single-osculum demosponge Halichondria panicea explants.

Filter-feeding demosponges are modular organisms that consist of modules each with one water-exit osculum. Once a mature module has been formed, the weight-specific filtration and respiration rates do not change. Sponge modules only grow to a certain size and for a sponge to increase in size, new modules must be formed. However, the growth characteristics of a small single-osculum module sponge are fundamentally different from those of multi-modular sponges, and a theoretically derived volume-specific filtration rate scales as F/V=V-1/3, indicating a decrease with increasing total module volume (V, cm3). Here, we studied filtration rate (F, l h-1), respiration rate (R, ml O2 h-1), volume-specific (F/V) and weight-specific (F/W) filtration rates, and the ratios F/R and F/W along with growth rates of small single-osculum demosponge Halichondria panicea explants of various sizes exposed to various concentrations of algal cells. The following relationships were found: F/V=7.08V-0.24, F=a1W1.05, and R=a2W0.68 where W is the dry weight (mg). The F/R and F/W ratios were constant and essentially independent of W, and other data indicate exponential growth. It is concluded that the experimental data support the theoretical F/V∝V-1/3.

Measured Air Flow Leakage in Facemask Usage.

The importance of wearing a facemask during a pandemic has been widely discussed, and a number of studies have been undertaken to provide evidence of a reduced infectious virus dose because of wearing facemasks. Here, one aspect that has received little attention is the fraction of breathing flow that is not filtered because it passes as leak flow between the mask and face. Its reduction would be beneficial in reducing the dose response. The results of the present study include the filter material pressure loss parameters, pressure distributions under masks, and the fraction of breathing flow leaked versus steady breathing flow in the range of 5 to 30 L min-1, for two commonly used facemasks mounted on mannequins, in the usual 'casual' way and in a 'tight' way by means of three different fitters placed over the mask to improve the seals. For the 'casual' mount, leaks were high: 83% to 99% for both masks at both exhalation and inhalation flows. For the 'tight' mount with different fitters, the masks showed different lower levels in the range of 18 to 66% of leakage, which, for exhalation, were nearly independent of flow rate, while for inhalation, were decreasing with increasing rates of respiration flows, probably because suction improved the sealing. In practice, masks are worn in a 'casual' mount, which would imply that nearly all contagious viruses found in aerosols small enough to follow air streams would be exhaled to and inhaled from the ambient air.

Hydrodynamics of sponge pumps and evolution of the sponge body plan.

Sponges are suspension feeders that filter vast amounts of water. Pumping is carried out by flagellated chambers that are connected to an inhalant and exhalant canal system. In 'leucon' sponges with relatively high-pressure resistance due to a complex and narrow canal system, pumping and filtering are only possible owing to the presence of a gasket-like structure (forming a canopy above the collar filters). Here, we combine numerical and experimental work and demonstrate how sponges that lack such sealing elements are able to efficiently pump and force the flagella-driven flow through their collar filter, thanks to the formation of a 'hydrodynamic gasket' above the collar. Our findings link the architecture of flagellated chambers to that of the canal system, and lend support to the current view that the sponge aquiferous system evolved from an open-type filtration system, and that the first metazoans were filter feeders.

Hydrodynamics of the leucon sponge pump.

Leuconoid sponges are filter-feeders with a complex system of branching inhalant and exhalant canals leading to and from the close-packed choanocyte chambers. Each of these choanocyte chambers holds many choanocytes that act as pumping units delivering the relatively high pressure rise needed to overcome the system pressure losses in canals and constrictions. Here, we test the hypothesis that, in order to deliver the high pressures observed, each choanocyte operates as a leaky, positive displacement-type pump owing to the interaction between its beating flagellar vane and the collar, open at the base for inflow but sealed above. The leaking backflow is caused by small gaps between the vaned flagellum and the collar. The choanocyte pumps act in parallel, each delivering the same high pressure, because low-pressure and high-pressure zones in the choanocyte chamber are separated by a seal (secondary reticulum). A simple analytical model is derived for the pump characteristic, and by imposing an estimated system characteristic we obtain the back-pressure characteristic that shows good agreement with available experimental data. Computational fluid dynamics is used to verify a simple model for the dependence of leak flow through gaps in a conceptual collar-vane-flagellum system and then applied to models of a choanocyte tailored to the parameters of the freshwater demosponge Spongilla lacustris to study its flows in detail. It is found that both the impermeable glycocalyx mesh covering the upper part of the collar and the secondary reticulum are indispensable features for the choanocyte pump to deliver the observed high pressures. Finally, the mechanical pump power expended by the beating flagellum is compared with the useful (reversible) pumping power received by the water flow to arrive at a typical mechanical pump efficiency of about 70%.

Hydrodynamic functionality of the lorica in choanoflagellates.

Choanoflagellates are unicellular eukaryotes that are ubiquitous in aquatic habitats. They have a single flagellum that creates a flow toward a collar filter composed of filter strands that extend from the cell. In one common group, the loricate choanoflagellates, the cell is suspended in an elaborate basket-like structure, the lorica, the function of which remains unknown. Here, we use Computational Fluid Dynamics to explore the possible hydrodynamic function of the lorica. We use the choanoflagellate Diaphaoneca grandis as a model organism. It has been hypothesized that the function of the lorica is to prevent refiltration (flow recirculation) and to increase the drag and, hence, increase the feeding rate and reduce the swimming speed. We find no support for these hypotheses. On the contrary, motile prey are encountered at a much lower rate by the loricate organism. The presence of the lorica does not affect the average swimming speed, but it suppresses the lateral motion and rotation of the cell. Without the lorica, the cell jiggles from side to side while swimming. The unsteady flow generated by the beating flagellum causes reversed flow through the collar filter that may wash away captured prey while it is being transported to the cell body for engulfment. The lorica substantially decreases such flow, hence it potentially increases the capture efficiency. This may be the main adaptive value of the lorica.

Swim and fly: escape strategy in neustonic and planktonic copepods.

Copepods can respond to predators by powerful escape jumps that in some surface-dwelling forms may propel the copepod out of the water. We studied the kinematics and energetics of submerged and out-of-water jumps of two neustonic pontellid copepods, Anomalocera patersoni and Pontella mediterranea, and one pelagic calanoid copepod, Calanus helgolandicus (euxinus). We show that jumping out of the water does not happen just by inertia gained during the copepod's acceleration underwater, but also requires the force generated by the thoracic limbs when breaking through the water's surface to overcome surface tension, drag and gravity. The timing of this appears to be necessary for success. At the moment of breaking the water interface, the instantaneous velocity of the two pontellids reached 125 cm s-1, while their maximum underwater speed (115 cm s-1) was close to that of similarly sized C. helgolandicus (106 cm s-1). The average specific power produced by the two pontellids during out-of-water jumps (1700-3300 W kg-1 muscle mass) was close to that during submerged jumps (900-1600 W kg-1 muscle mass) and, in turn, similar to that produced during submerged jumps of C. helgolandicus (1300 W kg-1 muscle mass). The pontellids may shake off water adhering to their body by repeated strokes of the limbs during flight, which leads to a slight acceleration in the air. Our observations suggest that out-of-water jumps of pontellids are not dependent on any exceptional ability to perform this behavior but have the same energetic cost and are based on the same kinematic patterns and contractive capabilities of muscles as those of copepods swimming submerged.

Membrane filtration device for studying compression of fouling layers in membrane bioreactors.

A filtration devise was developed to assess compressibility of fouling layers in membrane bioreactors. The system consists of a flat sheet membrane with air scouring operated at constant transmembrane pressure to assess the influence of pressure on resistance of fouling layers. By fitting a mathematical model, three model parameters were obtained; a back transport parameter describing the kinetics of fouling layer formation, a specific fouling layer resistance, and a compressibility parameter. This stands out from other on-site filterability tests as model parameters to simulate filtration performance are obtained together with a characterization of compressibility. Tests on membrane bioreactor sludge showed high reproducibility. The methodology's ability to assess compressibility was tested by filtrations of sludges from membrane bioreactors and conventional activated sludge wastewater treatment plants from three different sites. These proved that membrane bioreactor sludge showed higher compressibility than conventional activated sludge. In addition, detailed information on the underlying mechanisms of the difference in fouling propensity were obtained, as conventional activated sludge showed slower fouling formation, lower specific resistance and lower compressibility of fouling layers, which is explained by a higher degree of flocculation.

Unified understanding of physico-chemical properties of activated sludge and fouling propensity.

A range of parameters affecting floc characteristics, sludge composition and filtration properties was investigated by analyzing 29 sludge samples from municipal and industrial conventional activated sludge systems and municipal membrane bioreactors (MBR). Samples were characterized by physico-chemical parameters, composition of ions and EPS, degree of flocculation, settling properties, dewatering properties, and filtration properties. By analyzing the interplay between various metrics instead of single parameters, a unified understanding of the influence of sludge composition and characteristics was developed. From this, a conceptual model was proposed to describe the interplay between sludge composition, characteristics, and filtration properties. The article shows three major results contributing to describe the interplay between sludge characteristics and fouling propensity: First, the degree of flocculation could be quantified by the ratio between floc size and residual turbidity and was a key parameter to assess fouling propensity. Second, extracted EPS to polyvalent cations ratio was used as an indicator of the flocculation. A high ratio combined with a high concentration of EPS resulted in large, loosely bound, and weak flocs that were easily deformed, hence giving compressible fouling layers. Finally, high amounts of carbohydrates in both total and extracted EPS resulted in more pronounced fouling, which may be explained by carbohydrates forming poorer flocs than humic substances and proteins. Accordingly, samples with high humic content showed lower specific resistance to filtration due to better floc structure. The amount of carbohydrates in EPS correlated positively to the influent COD/N ratio, which may explain why systems with high influent COD/N ratio demonstrated higher fouling propensity.

Dynamics of the Fouling Layer Microbial Community in a Membrane Bioreactor.

Membrane fouling presents the greatest challenge to the application of membrane bioreactor (MBR) technology. Formation of biofilms on the membrane surface is the suggested cause, yet little is known of the composition or dynamics of the microbial community responsible. To gain an insight into this important question, we applied 16S rRNA gene amplicon sequencing with a curated taxonomy and fluorescent in situ hybridization to monitor the community of a pilot-scale MBR carrying out enhanced biological nitrogen and phosphorus removal with municipal wastewater. In order to track the dynamics of the fouling process, we concurrently investigated the communities of the biofilm, MBR bulk sludge, and the conventional activated sludge system used to seed the MBR system over several weeks from start-up. As the biofilm matured the initially abundant betaproteobacterial genera Limnohabitans, Hydrogenophaga and Malikia were succeeded by filamentous Chloroflexi and Gordonia as the abundant species. This study indicates that, although putative pioneer species appear, the biofilm became increasingly similar to the bulk community with time. This suggests that the microbial population in bulk water will largely determine the community structure of the mature biofilm.

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids.

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.

Filtration properties of activated sludge in municipal MBR wastewater treatment plants are related to microbial community structure.

In the conventional activated sludge process, a number of important parameters determining the efficiency of settling and dewatering are often linked to specific groups of bacteria in the sludge--namely floc size, residual turbidity, shear sensitivity and composition of extracellular polymeric substances (EPS). In membrane bioreactors (MBRs) the nature of solids separation at the membrane has much in common with sludge dewaterability but less is known about the effect of specific microbial groups on the sludge characteristics that affect this process. In this study, six full-scale MBR plants were investigated to identify correlations between sludge filterability, sludge characteristics, and microbial community structure. The microbial community structure was described by quantitative fluorescence in situ hybridization and sludge filterability by a low-pressure filtration method. A strong correlation between the degree of flocculation (ratio between floc size and residual turbidity) and sludge filterability at low pressure was found. A good balance between EPS and cations in the sludge correlated with good flocculation, relatively large sludge flocs, and low amounts of small particles and single cells in the bulk phase (measured as residual turbidity), all leading to a good filterability. Floc properties could also be linked to the microbial community structure. Bacterial species forming strong microcolonies such as Nitrospira and Accumulibacter were present in plants with good flocculation and filtration properties, while few strong microcolonies and many filamentous bacteria in the plants correlated with poor flocculation and filtration problems. In conclusion this study extends the hitherto accepted perception that plant operation affects floc properties which affects fouling. Additionally, plant operation also affects species composition, which affects floc properties and in the end fouling propensity.

Validation of the flow-through chamber (FTC) and steady-state (SS) methods for clearance rate measurements in bivalves.

To obtain precise and reliable laboratory clearance rate (filtration rate) measurements with the 'flow-through chamber method' (FTC) the design must ensure that only inflow water reaches the bivalve's inhalant aperture and that exit flow is fully mixed. As earlier recommended these prerequisites can be checked by a plot of clearance rate (CR) versus increasing through-flow (Fl) to reach a plateau, which is the true CR, but we also recommend to plot percent particles cleared versus reciprocal through-flow where the plateau becomes the straight line CR/Fl, and we emphasize that the percent of particles cleared is in itself neither a criterion for valid CR measurement, nor an indicator of appropriate 'chamber geometry' as hitherto adapted in many studies. For the 'steady-state method' (SS), the design must ensure that inflow water becomes fully mixed with the bivalve's excurrent flow to establish a uniform chamber concentration prevailing at its incurrent flow and at the chamber outlet. These prerequisites can be checked by a plot of CR versus increasing Fl, which should give the true CR at all through-flows. Theoretically, the experimental uncertainty of CR for a given accuracy of concentration measurements depends on the percent reduction in particle concentration (100×P) from inlet to outlet of the ideal 'chamber geomety'. For FTC, it decreases with increasing values of P while for SS it first decreases but then increases again, suggesting the use of an intermediate value of P. In practice, the optimal value of P may depend on the given 'chamber geometry'. The fundamental differences between the FTC and the SS methods and practical guidelines for their use are pointed out, and new data on CR for the blue mussel, Mytilus edulis, illustrate a design and use of the SS method which may be employed in e.g. long-term growth experiments at constant algal concentrations.

Microbial communities involved in enhanced biological phosphorus removal from wastewater--a model system in environmental biotechnology.

Enhanced biological phosphorus removal (EBPR) is one of the most advanced and complicated wastewater treatment processes applied today, and it is becoming increasingly popular worldwide as a sustainable way to remove and potentially reuse P. It is carried out by complex microbial communities consisting primarily of uncultured microorganisms. The EBPR process is a well-studied system with clearly defined boundaries which makes it very suitable as a model ecosystem in microbial ecology. Of particular importance are the transformations of C, N, and P, the solid-liquid separation properties and the functional and structural stability. A range of modern molecular methods has been used to study these communities in great detail including single cell microbiology, various -omics methods, flux analyses, and modeling making this one of the best studied microbial ecosystems so far. Recently, an EBPR core microbiome has been described and we present in this article some highlights and show how this complex microbial community can be used as model ecosystem in environmental biotechnology.

Functional amyloid in Pseudomonas.

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

Widespread abundance of functional bacterial amyloid in mycolata and other gram-positive bacteria.

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phyla Actinobacteria and Firmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the genera Streptomyces, Bacillus, and Nocardia were all coated with amyloids. FuBA was purified from Gordonia amarae (from the cell envelope) and Geodermatophilus obscurus, and they had the morphology, tinctorial properties, and beta-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope of G. amarae was visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.

Quantification of lipids and protein in thin biofilms by fluorescence staining.

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised.

Adhesion characteristics of nitrifying bacteria in activated sludge.

Nitrifiers are known to form relatively dense and strong microcolonies in activated sludge flocs, but little is known about their adhesion characteristics and how these are relative to other floc components. The size distribution of ammonia and nitrite-oxidizing bacteria (Nitrosomonas oligotropha and Nitrospira spp.) in activated sludge from a nutrient removal plant showed that the majority of N. oligotropha cells formed microcolonies with a diameter of 13-22.5 microm, and most Nitrospira spp. cells formed microcolonies with a diameter of 9-22.5 microm. By applying high shear forces (2200 s(-1)), the largest microcolonies of N. oligotropha fragmented to a level well below the Kolmogorov microscale (approx. 15-25 microm). Only very little erosion of single cells took place. Nitrospira spp. microcolonies were generally slightly stronger than N. oligotropha. In order to characterize the adhesion/binding mechanisms for the individual microcolonies, a number of different physico-chemical treatments were combined with shear, and even though this did not lead to any explicit characterization of the species-specific adhesion mechanisms, entanglement of extracellular polymers was proposed as a plausible important adhesion mechanism. When compared to other floc components, the deflocculated fractions of N. oligotropha and Nitrospira spp. were much lower than those of cells in general (total cell count, DAPI) or the organic matter. Deflocculation of N. oligotropha ranged from 3% to 11% of the total N. oligotropha biovolume, Nitrospira spp. from 1% to 4% of the total Nitrospira spp. biovolume, whereas the number of deflocculated cells was 9-54% of the total cell count, and the deflocculated organic matter constituted 8-43% of the total amount of organic matter. These results clearly showed that activated sludge contained a large pool of loosely attached cells and extracellular polymeric substances, and that the nitrifiers and some other microcolony formers were very strong and remained almost intact even under extreme physical and chemical conditions.

Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants.

Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.

Amyloid-like adhesins produced by floc-forming and filamentous bacteria in activated sludge.

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.