Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm.

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

"Candidatus Nitrosotenuis aquarius," an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter.

Ammonia is a metabolic waste product excreted by aquatic organisms that causes toxicity when it accumulates. Aquaria and aquaculture systems therefore use biological filters that promote the growth of nitrifiers to convert ammonia to nitrate. Ammonia-oxidizing bacteria (AOB) have been isolated from aquarium biofilters and are available as commercial supplements, but recent evidence suggests that ammonia-oxidizing archaea (AOA) are abundant in aquarium biofilters. In this study, we report the cultivation and closed genome sequence of the novel AOA representative "Candidatus Nitrosotenuis aquarius," which was enriched from a freshwater aquarium biofilter. "Ca Nitrosotenuis aquarius" oxidizes ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells and a generation time of 34.9 h. "Ca Nitrosotenuis aquarius" has an optimal growth temperature of 33°C, tolerates up to 3 mM NH4Cl, and grows optimally at 0.05% salinity. Transmission electron microscopy revealed that "Ca Nitrosotenuis aquarius" cells are rod shaped, with a diameter of ∼0.4 µm and length ranging from 0.6 to 3.6 µm. In addition, these cells possess surface layers (S-layers) and multiple proteinaceous appendages. Phylogenetically, "Ca Nitrosotenuis aquarius" belongs to the group I.1a Thaumarchaeota, clustering with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants. The complete 1.70-Mbp genome contains genes involved in ammonia oxidation, bicarbonate assimilation, flagellum synthesis, chemotaxis, S-layer production, defense, and protein glycosylation. Incubations with differential inhibitors indicate that "Ca Nitrosotenuis aquarius"-like AOA contribute to ammonia oxidation within the aquarium biofilter from which it originated.IMPORTANCE Nitrification is a critical process for preventing ammonia toxicity in engineered biofilter environments. This work describes the cultivation and complete genome sequence of a novel AOA representative enriched from a freshwater aquarium biofilter. In addition, despite the common belief in the aquarium industry that AOB mediate ammonia oxidation, the present study suggests an in situ role for "Ca Nitrosotenuis aquarius"-like AOA in freshwater aquarium biofilters.

Complete genome sequence of the aerobically denitrifying thermophilic bacterium Chelatococcus daeguensis TAD1

ABSTRACT Chelatococcus daeguensis TAD1 is a themophilic bacterium isolated from a biotrickling filter used to treat NOx in Ruiming Power Plant, located in Guangzhou, China, which shows an excellent aerobic denitrification activity at high temperature. The complete genome sequence of this strain was reported in the present study. Genes related to the aerobic denitrification were identified through whole genome analysis. This work will facilitate the mechanism of aerobic denitrification and provide evidence for its potential application in the nitrogen removal.

Complete genome sequence of the aerobically denitrifying thermophilic bacterium Chelatococcus daeguensis TAD1.

Chelatococcus daeguensis TAD1 is a themophilic bacterium isolated from a biotrickling filter used to treat NOx in Ruiming Power Plant, located in Guangzhou, China, which shows an excellent aerobic denitrification activity at high temperature. The complete genome sequence of this strain was reported in the present study. Genes related to the aerobic denitrification were identified through whole genome analysis. This work will facilitate the mechanism of aerobic denitrification and provide evidence for its potential application in the nitrogen removal.

Bacterial Suspensions Deposited on Microbiological Filter Material for Rapid Laser-Induced Breakdown Spectroscopy Identification.

Four species of bacteria, E. coli, S. epidermidis, M. smegmatis, and P. aeruginosa, were harvested from agar nutrient medium growth plates and suspended in water to create liquid specimens for the testing of a new mounting protocol. Aliquots of 30 µL were deposited on standard nitrocellulose filter paper with a mean 0.45 µm pore size to create highly flat and uniform bacterial pads. The introduction of a laser-based lens-to-sample distance measuring device and a pair of matched off-axis parabolic reflectors for light collection improved both spectral reproducibility and the signal-to-noise ratio of optical emission spectra acquired from the bacterial pads by laser-induced breakdown spectroscopy. A discriminant function analysis and a partial least squares-discriminant analysis both showed improved sensitivity and specificity compared to previous mounting techniques. The behavior of the spectra as a function of suspension concentration and filter coverage was investigated, as was the effect on chemometric cell classification of sterilization via autoclaving.

Advanced Wastewater Treatment Engineering-Investigating Membrane Fouling in both Rotational and Static Membrane Bioreactor Systems Using Empirical Modelling.

Advanced wastewater treatment using membranes are popular environmental system processes since they allow reuse and recycling. However, fouling is a key limiting factor and so proprietary systems such as Avanti's RPU-185 Flexidisks membrane bioreactor (MBR) use novel rotating membranes to assist in ameliorating it. In earlier research, this rotating process was studied by creating a simulation model based on first principles and traditional fouling mechanisms. In order to directly compare the potential benefits of this rotational system, this follow-up study was carried out using Avanti's newly developed static (non-rotating) Flexidisks MBR system. The results from operating the static pilot unit were simulated and modelled using the rotational fouling model developed earlier however with rotational switching functions turned off and rotational parameters set to a static mode. The study concluded that a rotating MBR system could increase flux throughput when compared against a similar static system. It is thought that although the slowly rotating spindle induces a weak crossflow shear, it is still able to even out cake build up across the membrane surface, thus reducing the likelihood of localised critical flux being exceeded at the micro level and lessening the potential of rapid trans-membrane pressure increases at the macro level.

Assessment of polycarbonate filter in a molecular analytical system for the microbiological quality monitoring of recycled waters onboard ISS.

On the ISS, as on Earth, water is an essential element for life and its quality control on a regular basis allows to ensure the health of the crew and the integrity of equipment. Currently, microbial water analysis onboard ISS still relies on the traditional culture-based microbiology methods. Molecular methods based on the amplification of nucleic acids for microbiological analysis of water quality show enormous potential and are considered as the best alternative to culture-based methods. For this reason, the Midass, a fully integrated and automated prototype was designed conjointly by ESA and bioMérieux for a rapid monitoring of the microbiological quality of air. The prototype allows air sampling, sample processing and the amplification/detection of nucleic acids. We describe herein the proof of principle of an analytical approach based on molecular biology that could fulfill the ESA's need for a rapid monitoring of the microbiological quality of recycled water onboard ISS. Both concentration and recovery of microorganisms are the main critical steps when the microfiltration technology is used for water analysis. Among filters recommended standards for monitoring the microbiological quality of the water, the polycarbonate filter was fully in line with the requirements of the ISO 7704-1985 standard in terms of efficacy of capture and recovery of bacteria. Moreover, this filter does not retain nucleic acids on the surface and has no inhibitory effect on their downstream processing steps such as purification and amplification/detection. Although the Midass system was designed for the treatment of air samples, the first results on the integration of PC filters were encouraging. Nevertheless, system modifications are needed to better adapt the Midass system for the monitoring of the microbiological water quality.

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community.

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.

Investigations into the fouling mechanism of parvovirus filters during filtration of freeze-thawed mAb drug substance solutions.

Filtration to remove viruses is one of the single most expensive steps in the production of mAb drug products. Therefore, virus filtration steps should be fully optimized, and any decline in flow rates warrants investigation into the causes of such membrane fouling. In the current study, it was found that freezing and thawing of a mAb bulk drug solution caused a substantial decrease in viral filter membrane flow rate. Freezing and thawing also caused formation of aggregates and particles across a broad size range, including particles that could be detected by microflow imaging (≥1 µm in size). However, removal of these particles offered little protection against flow rate decline during viral filtration. Further investigation revealed that trace amounts of aggregates (ca. 10⁻6 of the total mass of protein in solution) approximately 20-40 nm in size were primarily responsible for the observed membrane fouling.

Combined effects of EPS and HRT enhanced biofouling on a submerged and hybrid PAC-MF membrane bioreactor.

The goal of this study was to quantify and demonstrate the dynamic effects of hydraulic retention time (HRT), organic carbon and various components of extracellular polymeric substances (EPS) produced by microorganisms on the performance of submersed hollow-fiber microfiltration (MF) membrane in a hybrid powdered activated carbon (PAC)-MF membrane bioreactor (MBR). The reactors were operated continuously for 45 days to treat surface (river) water before and after pretreatment using a biofiltration unit. The real-time levels of organic carbon and the major components of EPS including five different carbohydrates (D(+) glucose and D(+) mannose, D(+) galactose, N-acetyl-D-galactosamine and D-galactose, oligosaccharides and L(-) fucose), proteins, and polysaccharides were quantified in the influent water, foulants, and in the bulk phases of different reactors. The presence of PAC extended the filtration cycle and enhanced the organic carbon adsorption and removal more than two fold. Biological filtration improved the filtrate quality and decreased membrane fouling. However, HRT influenced the length of the filtration cycle and had less effect on organic carbon and EPS component removal and/or biodegradation. The abundance of carbohydrates in the foulants on MF surfaces was more than 40 times higher than in the bulk phase, which demonstrates that the accumulation of carbohydrates on membrane surfaces contributed to the increase in transmembrane pressure significantly and PAC was not a potential adsorbent of carbohydrates. The abundance of N-acetyl-d-galactosamine and d-galactose was the highest in the foulants on membranes receiving biofilter-treated river water. Most of the biological fouling compounds were produced inside the reactors due to biodegradation. PAC inside the reactor enhanced the biodegradation of polysaccharides up to 97% and that of proteins by more than 95%. This real-time extensive and novel study demonstrates that the PAC-MF hybrid MBR is a sustainable technology for treating river water.

Evaluation of the bioremoval of Cr(VI) and TOC in biofilters under continuous operation using response surface methodology.

In the present study, the bioremoval of Cr(VI) and the removal of total organic carbon (TOC) were achieved with a system composed by an anaerobic filter and a submerged biofilter with intermittent aeration using a mixed culture of microorganisms originating from contaminated sludge. In the aforementioned biofilters, the concentrations of chromium, carbon, and nitrogen were optimized according to response surface methodology. The initial concentration of Cr(VI) was 137.35 mg l(-1), and a bioremoval of 85.23% was attained. The optimal conditions for the removal of TOC were 4 to 8 g l(-1) of sodium acetate, >0.8 g l(-1) of ammonium chloride and 60 to 100 mg l(-1) of Cr(VI). The results revealed that ammonium chloride had the strongest effect on the TOC removal, and 120 mg l(-1) of Cr(VI) could be removed after 156 h of operation. Moreover, 100% of the Cr(VI) and the total chromium content of the aerobic reactor output were removed, and TOC removals of 80 and 87% were attained after operating the anaerobic and aerobic reactors for 130 and 142 h, respectively. The concentrations of cells in both reactors remained nearly constant over time. The residence time distribution was obtained to evaluate the flow through the bioreactors.

Composition and variability of biofouling organisms in seawater reverse osmosis desalination plants.

Seawater reverse osmosis (SWRO) membrane biofouling remains a common challenge in the desalination industry, but the marine bacterial community that causes membrane fouling is poorly understood. Microbial communities at different stages of treatment processes (intake, cartridge filtration, and SWRO) of a desalination pilot plant were examined by both culture-based and culture-independent approaches. Bacterial isolates were identified to match the genera Shewanella, Alteromonas, Vibrio, and Cellulophaga based on 16S rRNA gene sequencing analysis. The 16S rRNA gene clone library of the SWRO membrane biofilm showed that a filamentous bacterium, Leucothrix mucor, which belongs to the gammaproteobacteria, accounted for nearly 30% of the clone library, while the rest of the microorganisms (61.2% of the total clones) were related to the alphaproteobacteria. 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that bacteria colonizing the SWRO membrane represented a subportion of microbes in the source seawater; however, they were quite different from those colonizing the cartridge filter. The examination of five SWRO membranes from desalination plants located in different parts of the world showed that although the bacterial communities from the membranes were not identical to each other, some dominant bacteria were commonly observed. In contrast, bacterial communities in source seawater were significantly different based on location and season. Microbial profiles from 14 cartridge filters collected from different plants also revealed spatial trends.

Analysis of microbial contamination in nanoparticle formulations.

This chapter describes a procedure for quantitative determination of microbial contamination of a nanoparticle formulation. The protocol includes tests for yeast, mold, and bacteria using Millipore sampler devices. This approach is primarily intended to avoid contamination of cell cultures and transmitting potential microbial contaminants to animals in preclinical studies of efficacy, biodistribution, and toxicity. Other methods common to microbiology will likely work equally well.

Aspects of tests and assessment of filtering materials used for respiratory protection against bioaerosols. Part I: type of active substance, contact time, microorganism species.

This paper presents the results of a study on antimicrobial activity of polymer filter nonwovens produced by needle-punching or melt-blowing with an addition of disinfecting agents. The first part of the paper discusses how the biocidal activity of nonwovens is a function of the active agent added to the nonwovens, the duration of the contact of microorganisms with nonwovens and the type of microorganisms. The types of fibres and disinfecting agents had a considerable effect on the biocidal activity of nonwovens. The biocidal effect of nonwovens increased with the duration of their contact with microorganisms. Fibre activity differed considerably depending on the species of the microorganism. The microorganisms most sensitive to biocidal activity of the active filter nonwoven were S. aureus, M. flavus and E. coli. There were no biocidal effects on spore-forming bacterium B. subtilis.

Aspects of tests and assessment of filtering materials used for respiratory protection against bioaerosols. Part II: sweat in the environment, microorganisms in the form of a bioaerosol.

The second part of the article presents the results of a study of antimicrobial activity of filter nonwovens with an addition of biocides, as a function of the presence of sweat in the environment and the method of microbe deposition on a nonwoven in the form of a liquid and a bioaerosol. At the same time, the filtration efficiency of nonwovens against microorganisms in the form of a bioaerosol was tested with the dynamic method. The results showed that the addition of sweat on the surface of a nonwoven resulted in an insignificant decrease of biological activity that still remained high. Moreover, an active nonwoven showed biostatic and biocidal activity only when microbes were deposited on the surface in the form of a solution. The nonwoven did not show any biological activity after deposition of microorganisms with the dynamical method in the form of a bioaerosol.

Characterization of the biofouling and cleaning efficiency of nanofiltration membranes.

The nanofiltration (NF) drinking water production unit of the Mery-sur-Oise plant (Val d'Oise, France) consists of eight identical filtration trains composed of three stages positioned in steps for a production capacity of 140,000 m(3) day(-1). To gain a better understanding of the irreversible fouling of the NF membranes, spiral wound modules in operation for 8 years from each of the three stages of the plant were autopsied before and after chemical cleaning and analysis by Attenuated Total Reflection Fourier Transform Infrared spectroscopy, Inductive Coupled Plasma-Atomic Emission Spectrometry, contact angles, adenosine triphosphate (ATP) content measurements, and rheometry. The fouled membranes from the three stages had similar contact angles of approximately 60 degrees . Relative infrared signals typical of biofilms were classified in descending order from stage 1 to stage 3. The foulant matter of stages 1 and 2 contained similar but weaker ATP concentrations than stage 3. During rheometry experiments, rotation and oscillation analyses demonstrated that the biofilm of stage 3 was less viscous and less elastic than the biofilms of stages 1 and 2. After cleaning, all the parameters analyzed demonstrated a quantitative decrease in the fouling matter at the NF membrane surface, but a biofilm with intact viscoelastic properties (unchanged G' and G'' values) remained at the membrane surface for the three stages. The persistence of biofilm material with intact mechanical properties at the NF membrane surface after chemical cleaning may result in permanent permeability decreases.

Biofouling and pollutant removal during long-term operation of an anaerobic membrane bioreactor treating municipal wastewater.

Two different sludge retention times (SRTs) were tested in order to assess the impact on membrane fouling and effluent quality in an anaerobic membrane bioreactor (AnMBR). Two up-flow anaerobic sludge bed (UASB) reactors (1 l volume) coupled to external tubular ultrafiltration membranes (filtration area = 81 cm(2)) were operated at a hydraulic retention time of 3 h and two different SRTs (100 and 60 days). The transmembrane pressure (TMP), flux (J) and relevant parameters to assess water quality were measured. Effluents from UASB reactors were filtered for 500 h without intermediate cleaning. The permeate met Mexican standards for wastewater reclamation in both tested conditions. Abrupt and periodical changes in the TMP and J were noticed during the experimental period. A fouling layer collapse and compression hypothesis was set forth in order to explain these changes. An autopsy performed on biofouled membranes indicated that deposited mass was mainly composed of volatile solids (85%) and the rest related to mineral matter, with the presence of inorganic salts containing Ca, Mg, Fe, P and Si. Biomass in the fouling layer was estimated at 0.27% based on the DNA/biomass ratio for the bacterial biofilm. No clear difference in membrane fouling was detected under the two SRTs applied to the systems. However, when operated over 500 h, repetitive sudden TMP and flux changes occurred later in system A (SRT of 100 days) than in system B (SRT of 60 days) suggesting a stronger fouling layer structure in the former.

Feasibility of atmospheric methane removal using methanotrophic biotrickling filters.

Methane is a potent greenhouse gas with a global warming potential ~23 times that of carbon dioxide. Here, we describe the modeling of a biotrickling filtration system composed of methane-consuming bacteria, i.e., methanotrophs, to assess the utility of these systems in removing methane from the atmosphere. Model results indicate that assuming the global average atmospheric concentration of methane, 1.7 ppmv, methane removal is ineffective using these methanotrophic biofilters as the methane concentration is too low to enable cell survival. If the concentration is increased to 500-6,000 ppmv, however, similar to that found above landfills and in concentrated animal feeding operations (factory farms), 4.98-35.7 tons of methane can be removed per biofilter per year assuming biotrickling filters of typical size (3.66 m in diameter and 11.5 m in height). Using reported ranges of capital, operational, and maintenance costs, the cost of the equivalent ton of CO(2) removal using these systems is $90-$910 ($2,070-$20,900 per ton of methane), depending on the influent concentration of methane and if heating is required. The use of methanotrophic biofilters for controlling methane emissions is technically feasible and, provided that either the costs of biofilter construction and operation are reduced or the value of CO(2) credits is increased, can also be economically attractive.

A novel method and simple apparatus for the detection of thermophilic Campylobacter spp. in chicken meat products.

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.

Comparative study of using different materials as bacterial carriers to treat hydrogen sulfide.

The use of support media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and protect the microorganisms from inhibitory compounds. In this study, molecular sieve, granular porous carbon, and ferric oxide desulfurizer, immobilized with autotrophic bacteria capable of oxidizing ferrous iron to ferric iron, were developed to treat hydrogen sulfide (H(2)S). Their corresponding bioreactors were referred to as BMS, BPC, and BFO, respectively. H(2)S loading, gas retention time, hydrogen ion, and aluminous, ferric, and ferrous iron concentrations of recycling effluents were evaluated. Thermogravimetric analysis, Brauner-Emmett-Teller method, and scanning electron microscopy were used to characterize packing materials. Results showed that the elimination capacity was in the order of BFO > BPC > BMS. This study suggested that the material characteristics progressively influenced the deodorization capacities of bioreactors. H(2)S was oxidized into elemental sulfur and oxidized sulfur species, according to differences of carriers. Furthermore, this study revealed the potential application of simultaneously treating of H(2)S under extremely acidic conditions.