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The characteristics of biomass and microbial community dynamics, in relation to autotrophic nitrification, were studied in two 20 L stirred tank reactors (STR) with oxic/hypoxic/oxic zones. The bioreactors were fed with synthetic wastewater with stepwise increasing ammonia concentrations (50-200 N mg/L) without organic substrate in the first phase (autotrophic phase) for 35 days (R1) and 15 days (R2), followed by a heterotrophic phase (with supplementation of organic substrate). The settling properties of the biomass, represented by pin-point flocs, gradually improved in both reactors during the autotrophic phase. The pin-point flocs of R1 exhibited granule-like settling properties. The SVI 30 in RI gradually improved to 29 mL/g MLSS, and the corresponding SVI 30 / SVI 10 gradually improved to 0.88 during the autotrophic phase. The settling properties of the biomass deteriorated in both bioreactors during the heterotrophic phase. The protein to polysaccharide ratio (PN:PS ratio) gradually increased in the extracted EPS (in both, loosely bound (LB) and tightly bound (TB) EPS) during the autotrophic phase, in both bioreactors. The TB:LB EPS ratio was higher when the pin-point flocs of R1 showed granule-like settling properties, followed by a decline in TB:LB EPS ratio during the heterotrophic phase. A combination of molecular approaches (droplet digital-PCR (dd-PCR) and 16S rRNA gene sequencing) revealed that Nitrospira were the predominant nitrifying bacteria in the pin-point flocs that show granular sludge-like settling properties during autotrophic phase in R1. Comammox Nitrospira was the dominant ammonia oxidizer in seed biomass and at low ammonia concentrations in both bioreactors. The relative abundance of canonical ammonia-oxidizing bacteria increased with an increase in influent-ammonia concentrations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aqeel, Asefa and Liss.)

This study investigated the impact of the solid sludge content concentrations (SC) on hydrothermal pretreatment (HTP) before fermentation and anaerobic digestion. Five different SC of 3.5%, 7%, 10%, 12%, and 16% were investigated in two different scenarios. The first scenario entailed using only the pretreated samples as substrates, whereas in scenario two, the substrates included pretreated samples combined with the supernatant. Results revealed that the highest overall pCOD solubilization (considering HTP and fermentation) of 64% was achieved for the sample with 12% SC combined with supernatant. The maximum volatile fatty acids production of 2.8 g COD/L occurred with 10% SC without supernatant. The maximum methane yield of 291 mL CH 4 /g VSS added was attained at 7% SC without supernatant. Furthermore, the results indicated that increasing the SC beyond 7% in scenario 1 and 10% in scenario two led to a decrease in methane yield. Additionally, optimizing for all desired endpoints may be difficult, and there are limits on the increase in SC concerning methane production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Anaerobic digestion and fermentation processes in wastewater sludge treatment are limited by several factors, including the slow breakdown of complex organic matter and solubilization of solids. In this study, thermochemical pretreatment of thickened waste activated sludge using high temperature (>170 °C) was investigated to understand the impact of the pretreatment on the volatile fatty acids (VFA) production and its fractions during the fermentation process. Furthermore, the influence the thermochemical pretreatment on sludge disintegration and methane recovery was investigated. A range of acidic and alkaline conditions over the pH range of 4.5-10 was examined. Sludge (pH adjusted) was exposed to hydrothermal pretreatment (HTP) at a temperature of 170 °C for 30 min. Pretreated samples were then subjected to batch fermentation and methane potential tests which revealed that acidic and alkaline conditions resulted in increased sludge solubilization during HTP. Acidic conditions were associated with a higher VFA production yield of up to 185 mg chemical oxygen demand/g total chemical oxygen demand. Alkaline conditions led to a higher methane production yield where the maximum yield (276 mL CH 4 /g total chemical oxygen demand added ) was found to occur at pH 10. Therefore, alkaline sludge used for fermentation has shown technical and economic feasibility for sludge carbon recovery.

Fate of biofilm sloughing was assessed in a laboratory-scale (LS) integrated fixed-film sequencing batch reactor (IF-SBR) treating synthetic wastewater and in a full-scale (FS) integrated fixed-film activated sludge (IFAS) system treating municipal wastewater. It was observed that the properties of biofilms and flocs, including sludge volume index (SVI), mixed liquor suspended solids (MLSS), effluent suspended solids (ESS), relative hydrophobicity, and composition of extracellular polymeric substance (EPS) were associated with biofilm sloughing and formation of large granular flocs in the LS IF-SBR. In the FS IFAS system, the changes were studied at the molecular level. For example, the extracted EPS content results (the protein to polysaccharide ratio decreased in the flocs and increased in the biofilms, with biofilm sloughing) were complemented with the confocal laser scanning microscopy (CLSM) coupled with molecular specific staining. CLSM analyses revealed that micro-colonies rich in polysaccharides readily sloughed from the carriers. Live-dead staining revealed areas of the biofilm where the viability of biomass was a contributing factor associated with areas of the biofilm susceptible to sloughing. 16S rRNA gene sequencing (Illumina) of FS IFAS samples revealed greater diversity (α-diversity) in biofilms compared to flocs. Biofilm sloughing resulted in a decrease in diversity in biofilms and a corresponding increase in the flocs during sloughing. Microbial population dynamics revealed that bacteria known for denitrification (for example, Comamonadaceae) detached from the biofilms during sloughing, readily associated with the suspended biomass, and were retained in the bioreactors., Competing Interests: The authors have declared that no competing interests exist.

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO 2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO 2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO 2 emissions should be considered distinct from the biogenic CO 2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO 2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO 2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO 2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO 2 emission rates. A linear relationship between ammonia removal and CO 2 emissions was demonstrated and the quantified CO 2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO 2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO 2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO 2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The estimated global population growth of 81 million people per year, combined with increased rates of urbanization and associated industrial processes, result in volumes of high strength ammonia wastewater that cannot be treated in a cost-effective or sustainable manner using the floc-based conventional activated sludge approach of nitrification and denitrification. Biofilm and aerobic granular sludge technologies have shown promise to significantly improve the performance of biological nitrogen removal systems treating high strength wastewater. This is partly due to enhanced biomass retention and their ability to sustain diverse microbial populations with juxtaposing growth requirements. Recent research has also demonstrated the value of hybrid systems with heterogeneous bioaggregates to mitigate biofilm and granule instability during long-term operation. In the context of high strength ammonia wastewater treatment, conventional nitrification-denitrification is hampered by high energy costs and greenhouse gas emissions. Anammox-based processes such as partial nitritation-anammox and partial denitrification-anammox represent more cost-effective and sustainable methods of removing reactive nitrogen from wastewater. There is also growing interest in the use of photosynthetic bacteria for ammonia recovery from high strength waste streams, such that nitrogen can be captured and concentrated in its reactive form and recycled into high value products. The purpose of this review is to explore recent advancements and emerging approaches related to high strength ammonia wastewater treatment., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

This study focused on investigating the effect of combined chemical and hydrothermal pretreatment (HTP) on the anaerobic digestibility of thickened waste activated sludge (TWAS). Three different combined pretreatment conditions of HTP + free nitrous acid (FNA), HTP + Acid, and HTP + Alkaline were applied to TWAS. To control and compare the effect of combined pretreatments and a single pretreatment, Acid, Alkaline, FNA and HTP pretreatments were applied done prior to AD. The results of this study revealed that combined pretreatments have higher potential to improve methane production yield and rate but not in the solubilization of COD. The highest methane yield of 275 mL CH 4 /g TCOD added was achieved for the combined pretreatment with FNA and HTP. HTP + FNA pretreatment was found to produce higher methane yields compared to the combination of other typical acid and alkaline reagents with hydrothermal pretreatment. Methane yields of 594, 527, and 544 L CH 4 /g VSS added, were achieved for HTP + FNA, HTP + ALK, and HTP + ACID pretreatments, respectively. The preliminary economic analysis showed that out of the combined pretreatment, only combining HTP with FNA is economically feasible., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As the effects of climate change become increasingly evident, the need for effective CO2 management is clear. Microalgae are well-suited for CO2 sequestration, given their ability to rapidly uptake and fix CO2. They also readily assimilate inorganic nutrients and produce a biomass with inherent commercial value, leading to a paradigm in which CO2-sequestration, enhanced wastewater treatment, and biomass generation could be effectively combined. Natural non-axenic phototrophic cultures comprising both autotrophic and heterotrophic fractions are particularly attractive in this endeavour, given their increased robustness and innate O2-CO2 exchange. In this study, the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy in a non-axenic phototrophic biofilm was examined. When the biofilm was cultivated under autotrophic conditions (i.e. no organic carbon), it grew autotrophically and exhibited CO2 uptake. After amending its growth medium with organic carbon (0.25 g/L glucose and 0.28 g/L sodium acetate), the biofilm rapidly toggled from net-autotrophic to net-heterotrophic growth, reaching a CO2 production rate of 60 μmol/h after 31 hours. When the organic carbon sources were provided at a lower concentration (0.125 g/L glucose and 0.14 g/L sodium acetate), the biofilm exhibited distinct, longitudinally discrete regions of heterotrophic and autotrophic metabolism in the proximal and distal halves of the biofilm respectively, within 4 hours of carbon amendment. Interestingly, this upstream and downstream partitioning of heterotrophic and autotrophic metabolism appeared to be reversible, as the position of these regions began to flip once the direction of medium flow (and hence nutrient availability) was reversed. The insight generated here can inform new and important research questions and contribute to efforts aimed at scaling and industrializing algal growth systems, where the ability to understand, predict, and optimize biofilm growth and activity is critical., Competing Interests: The authors have declared that no competing interests exist.

The aim of the study was enrichment of nitrifying bacteria and to investigate the potential of autotrophic fixed-film and hybrid bioreactors to treat high strength ammonia wastewater (up to 1,000 mg N/L). Two types of fixed-film systems [moving bed biofilm reactor (MBBR) and BioCord TM ] in two different configurations [sequencing batch reactor (SBR) and a continuous stirred tank reactor (CSTR)] were operated for 306 days. The laboratory-scale bioreactors were seeded with activated sludge from a municipal wastewater treatment plant and fed synthetic wastewater with no organics. Strategies for acclimation included biomass reseeding (during bioreactor start-up), and gradual increase in the influent ammonia concentration [from 130 to 1,000 mg N/L (10% every 5 days)]. Stable ammonia removal was observed up to 750 mg N/L from 45 to 145 days in the MBBR SBR (94-100%) and CSTR (72-100%), and BioCord TM SBR (96-100%) and CSTR (92-100%). Ammonia removal declined to 87% ± 6, in all bioreactors treating 1,000 mg N/L (on day 185). Following long-term operation at 1,000 mg N/L (on day 306), ammonia removal was 93-94% in both the MBBR SBR and BioCord TM CSTR; whereas, ammonia removal was relatively lower in MBBR CSTR (20-35%) and BioCord TM SBR (45-54%). Acclimation to increasing concentrations of ammonia led to the enrichment of nitrifying ( Nitrosomonas , Nitrospira , and Nitrobacter ) and denitrifying ( Comamonas , OLB8 , and Rhodanobacter ) bacteria [16S rRNA gene sequencing (Illumina)] in all bioreactors. In the hybrid bioreactor, the nitrifying and denitrifying bacteria were relatively more abundant in flocs and biofilms, respectively. The presence of dead cells (in biofilms) suggests that in the absence of an organic substrate, endogenous decay is a likely contributor of nutrients for denitrifying bacteria. The nitrite accumulation and abundance of denitrifying bacteria indicate partial denitrification in fixed-film bioreactors operated under limited carbon conditions. Further studies are required to assess the contribution of organic material produced in autotrophic biofilms (by endogenous decay and soluble microbial products) to the overall treatment process. Furthermore, the possibility of sustaining autotrophic nitrogen in high strength waste-streams in the presence of organic substrates warrants further investigation., (Copyright © 2020 Aqeel and Liss.)

Climate change brought about by anthropogenic CO 2 emissions has created a critical need for effective CO 2 management solutions. Microalgae are well suited to contribute to efforts aimed at addressing this challenge, given their ability to rapidly sequester CO 2 coupled with the commercial value of their biomass. Recently, microalgal biofilms have garnered significant attention over the more conventional suspended algal growth systems, since they allow for easier and cheaper biomass harvesting, among other key benefits. However, the path to cost-effectiveness and scaling up is hindered by a need for new tools and methodologies which can help evaluate, and in turn optimize, algal biofilm growth. Presented here is a novel system which facilitates the real-time in situ monitoring of algal biofilm CO 2 sequestration. Utilizing a CO 2 -permeable membrane and a tube-within-a-tube design, the CO 2 sequestration monitoring system (CSMS) was able to reliably detect slight changes in algal biofilm CO 2 uptake brought about by light-dark cycling, light intensity shifts, and varying amounts of phototrophic biomass. This work presents an approach to advance our understanding of carbon flux in algal biofilms, and a base for potentially useful innovations to optimize, and eventually realize, algae biofilm-based CO 2 sequestration.

Background: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted., Results: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers., Conclusions: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

Biofilms are multifaceted and robust microbiological systems that enable microorganisms to withstand a multitude of environmental stresses and expand their habitat range. We have shown previously that nutritional status alters antibiotic susceptibility in a mixed-species biofilm. To further elucidate the effects of nutrient addition on inter-species dynamics and whole-biofilm susceptibility to high-dose streptomycin exposures, a CO 2 Evolution Measurement System was used to monitor the metabolic activity of early steady state pure-culture and mixed-species biofilms containing Pseudomonas aeruginosa and Stenotrophomonas maltophilia , with and without added carbon. Carbon supplementation was needed for biofilm recovery from high-dose streptomycin exposures when P. aeruginosa was either the dominant community member in a mixed-species biofilm (containing predominantly P. aeruginosa and S. maltophilia ) or as a pure culture. By contrast, S. maltophilia biofilms could recover from high-dose streptomycin exposures without the need for carbon addition during antibiotic exposure. Metagenomic analysis revealed that even when inocula were dominated by Pseudomonas , the relative abundance of Stenotrophomonas increased upon biofilm development to ultimately become the dominant species post-streptomycin exposure. The combined metabolic and metagenomic results demonstrated the relevance of inter-species influence on survival and that nutritional status has a strong influence on the survival of P. aeruginosa dominated biofilms., (Copyright © 2019 Jackson, Kroukamp, Yeung, Ronan, Liss and Wolfaardt.)

Wastewater treatment plants (WWTPs) are points of control for the environmental dissemination of antimicrobial resistant bacteria. Vancomycin-resistant enterococci (VRE) were used as indicators of antimicrobial resistance (AMR) in two WWTPs (biologically aerated filter (BAF) and conventional activated sludge (CAS)) in the same municipality. The removal and abundance of enterococci and VRE as well as the species and antimicrobial resistance profiles of VRE were assessed. Enterococci and VRE from the primary and final effluents were enumerated. Results were assessed from an ecological context. VRE was not selected for by either WWTP but the BAF system outperformed the CAS system for the removal of enterococci/VRE. Enterococcus faecalis ( n = 151), E. faecium ( n = 94) and E. casseliflavus / E. gallinarum ( n = 59) were the dominant VRE species isolated. A decrease in levofloxacin resistance in enterococci was observed in the BAF WWTP. An increase in nitrofurantoin resistant ( p < 0.001) and a decrease in quinupristin/dalfopristin ( p = 0.003) and streptomycin ( p = 0.022) resistant enterococci were observed in the CAS WWTP, corresponding to a shift of VRE from E. faecalis to E. faecium . Wastewater treatment processes can be managed to limit the dissemination of antimicrobial resistance determinants into the surrounding environment.

Enterococci species in wastewater including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus and Enterococcus gallinarum isolates (n = 308) with low or high level vancomycin resistance were determined and compared using a phenotypic method (RapID™ STR system), 16S rRNA sequencing, and multi-locus (atpA, groESL, and pheS) sequence analysis (MLSA). Error rates for the RapID™ STR system were E. faecalis (15.9%), E. faecium (21.5%), and E. casseliflavus/E. gallinarum (56.9%) when referenced to the consensus of all methods tested. Comparison of single nucleotide polymorphism (SNP) distances and phylogenetic trees suggested that the groESL locus delineated species more effectively than other loci. The groESL locus was the most reliable loci for the correct identification of Enterococcus spp., including E. casseliflavus and E. gallinarum, with high congruence compared to the consensus (Adjusted Rand Index = 0.954; Adjusted Wallace Co-efficient = 0.941). All of the methods were compared to whole genome sequencing, which acted as a gold standard, for the isolates from this study and those downloaded from NCBI., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The effect of COD:N ratio on sludge properties and their role in membrane fouling were examined using a well-controlled aerobic membrane bioreactor receiving a synthetic high strength wastewater containing glucose. Membrane performance was improved with an increase in the COD/N ratio (100:5-100:1.8) (i.e. reduced N dosage). Surface analysis of sludge by X-ray photoelectron spectroscopy (XPS) indicates significant differences in surface concentrations of elements C, O and N that were observed under different COD/N ratios, implying changes in the composition of extracellular polymeric substances (EPS). Fourier transform-infrared spectroscopy (FTIR) revealed a unique characteristic peak (CO bonds) at 1735 cm(-1) under nitrogen limitation conditions. Total EPS decreased with an increase in COD/N ratio, corresponding to a decrease in the proteins (PN) to carbohydrates (CH) ratio in EPS. There were no significant differences in the total soluble microbial products (SMPs) but the ratio of PN/CH in SMPs decreased with an increase in COD/N ratios. The results suggest that EPS and SMP composition and the presence of a small quantity of filamentous microorganisms played an important role in controlling membrane fouling., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aerobic granules offer enhanced biological nutrient removal and are compact and dense structures resulting in efficient settling properties. Granule instability, however, is still a challenge as understanding of the drivers of instability is poorly understood. In this study, transient instability of aerobic granules, associated with filamentous outgrowth, was observed in laboratory-scale sequencing batch reactors (SBRs). The transient phase was followed by the formation of stable granules. Loosely bound, dispersed, and pinpoint seed flocs gradually turned into granular flocs within 60 days of SBR operation. In stage 1, the granular flocs were compact in structure and typically 0.2 mm in diameter, with excellent settling properties. Filaments appeared and dominated by stage 2, resulting in poor settleability. By stage 3, the SBRs were selected for larger granules and better settling structures, which included filaments that became enmeshed within the granule, eventually forming structures 2-5 mm in diameter. Corresponding changes in sludge volume index were observed that reflected changes in settleability. The protein-to-polysaccharide ratio in the extracted extracellular polymeric substance (EPS) from stage 1 and stage 3 granules was higher (2.8 and 5.7, respectively), as compared to stage 2 filamentous bulking (1.5). Confocal laser scanning microscopic (CLSM) imaging of the biomass samples, coupled with molecule-specific fluorescent staining, confirmed that protein was predominant in stage 1 and stage 3 granules. During stage 2 bulking, there was a decrease in live cells; dead cells predominated. Denaturing gradient gel electrophoresis (DGGE) fingerprint results indicated a shift in bacterial community composition during granulation, which was confirmed by 16S rRNA gene sequencing. In particular, Janthinobacterium (known denitrifier and producer of antimicrobial pigment) and Auxenochlorella protothecoides (mixotrophic green algae) were predominant during stage 2 bulking. The chitinolytic activity of Chitinophaga is likely antagonistic towards Auxenochlorella and may have contributed to stage 3 stable granule formation. Rhodanobacter, known to support complete denitrification, were predominant in stage 1 and stage 3 granules. The relative abundance of Rhodanobacter coincided with high protein concentrations in EPS, suggesting a role in microbial aggregation and granule formation.

A full-scale (FS) activated sludge system treating wastewater from a meat rendering plant with a long history of sludge management problems (pin-point flocs; >80% of floc <50 μm diameter; poor settling) was the focus of a study that entailed characterization of floc properties. This was coupled with parallel well-controlled lab-scale (LS) sequencing batch reactors (SBRs) treating the same wastewater and operated continuously over 1.5 years. Distinct differences in the proportion of proteins and polysaccharides associated with extracellular polymeric substances (EPS) were observed when comparing the properties of flocs from the FS and the LB systems. Further differences in the proportion of tightly bound (TB) and loosely bound (LB) fractions of EPS were also observed for flocs derived from conditions where differences in settling and dewatering properties of flocs occurred (i.e. FS and LS systems). FS flocs contained higher levels of EPS along with a higher proportion of LB than TB EPS, and possessing characteristics associated with non-filamentous bulking (SVI >150 mL/g). Floc formed in the LS system, following inoculation from sludge taken from the FS system, was markedly larger in size (>70% of floc >300 μm diameter), spherical in shape, compact and firm, and appeared to be granular in form. Flocs formed in the LS system, when an anoxic phase was introduced into the react stage of the SBR cycle, were found to be more hydrophobic and contained more TB and less loosely bound (LB) EPS when compared to the FS floc. TB-EPS contained a greater amount of protein, whereas the polysaccharide content of LB-EPS was larger. Protein was predominantly localized in the core region of granular flocs where cells were compactly packed. When assessing the operating conditions of the FS and LS systems parameters that appear to impact the floc properties and the transition to a granular form include dissolved oxygen (DO) concentration and food to microorganism (F/M) ratio., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dynamic interaction of bacteria within bed sediment and suspended sediment (i.e., floc) in a wave-dominated beach environment was assessed using a laboratory wave flume. The influence of shear stress (wave energy) on bacterial concentrations and on the partitioning and transport of unattached and floc-associated bacteria was investigated. The study showed that increasing wave energy (0.60 and 5.35 N/s) resulted in a 0.5 to 1.5 log increase in unattached cells of the test bacterium Pseudomonas sp. strain CTO7::gfp-2 in the water column. There was a positive correlation between the bacterial concentrations in water and the total suspended solids, with the latter increasing from values of near 0 to up to 200 mg/L over the same wave energy increase. The median equivalent spherical diameter of flocs in suspension also increased by an order of magnitude in all experimental trials. Under both low (0.60 N/s) and high (5.35 N/s) energy regime, bacteria were shown to preferentially associate with flocs upon cessation of wave activity. The results suggest that collecting water samples during periods of low wave action for the purpose of monitoring the microbiological quality of water may underestimate bacterial concentrations partly because of an inability to account for the effect of shear stress on the erosion and mobilization of bacteria from bed sediment to the water column. This highlights the need to develop a more comprehensive beach analysis strategy that not only addresses presently uncharacterized shores and sediments but also recognizes the importance of eroded flocs as a vector for the transport of bacteria in aquatic environments.

Huwa-San peroxide (hydrogen peroxide; HSP) is a NSF Standard 60 (maximum 8 mg/L(-1)) new generation peroxide stabilized with ionic silver suitable for continuous disinfection of potable water. Experiments were undertaken to examine the mechanism of HSP against planktonic and biofilm cultures of indicator bacterial strains. Contact/kill time (CT) relationships that achieve effective control were explored to determine the potential utility in primary disinfection. Inhibitory assays were conducted using both nutrient rich media and a medium based on synthetic wastewater. Assays were compared for exposures to three disinfectants (HSP, laboratory grade hydrogen peroxide (HP) and sodium hypochlorite) at concentrations of 20 ppm (therefore at 2.5 and 5 times the NSF limit for HP and sodium hypochlorite, respectively) and at pH 7.0 and 8.5 in dechlorinated tap water. HSP was found to be more or equally effective as hypochlorite or HP. Results from CT assays comparing HSP and HP at different bacterial concentrations with neutralization of residual peroxide with catalase suggested that at a high bacterial concentration HSP, but not HP, was protected from catalase degradation possibly through sequestration by bacterial cells. Consistent with this hypothesis, at a low bacterial cell density residual HSP was more effectively neutralized as less HSP was associated with bacteria and therefore accessible to catalase. Silver in HSP may facilitate this association through electrostatic interactions at the cell surface. This was supported by experiments where the addition of mono (K(+)) and divalent (Ca(+2)) cations (0.005-0.05M) reduced the killing efficacy of HSP but not HP. Experiments designed to distinguish any inhibitory effect of silver from that of peroxide in HSP were carried out by monitoring the metabolic activity of established P. aeruginosa PAO1 biofilms. Concentrations of 70-500 ppm HSP had a pronounced effect on metabolic activity while the equivalent concentrations of ionic silver (50- 375 ppb) had a negligible effect, demonstrating that the microbiocidal activity of HSP was due to peroxide rather than silver. Overall, it was found that the antimicrobial activity of HSP is enhanced over that of hydrogen peroxide; the presence of the ionic silver enhances interactions of HSP with the bacterial cell surface rather than acting directly as a biocide at the tested concentrations.

Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.

Background: Microbial cellulose conversion by Clostridium thermocellum 27405 occurs predominantly through the activity of substrate-adherent bacteria organized in thin, primarily single cell-layered biofilms. The importance of cellulosic surface exposure to microbial hydrolysis has received little attention despite its implied impact on conversion kinetics., Results: We showed the spatial heterogeneity of fiber distribution in pure cellulosic sheets, which made direct measurements of biofilm colonization and surface penetration impossible. Therefore, we utilized on-line measurements of carbon dioxide (CO2) production in continuous-flow reactors, in conjunction with confocal imaging, to observe patterns of biofilm invasion and to indirectly estimate microbial accessibility to the substrate's surface and the resulting limitations on conversion kinetics. A strong positive correlation was found between cellulose consumption and CO2 production (R2 = 0.996) and between surface area and maximum biofilm activity (R2 = 0.981). We observed an initial biofilm development rate (0.46 h-1, 0.34 h-1 and 0.33 h-1) on Whatman sheets (#1, #598 and #3, respectively) that stabilized when the accessible surface was maximally colonized. The results suggest that cellulose conversion kinetics is initially subject to a microbial limitation period where the substrate is in excess, followed by a substrate limitation period where cellular mass, in the form of biofilms, is not limiting. Accessible surface area acts as an important determinant of the respective lengths of these two distinct periods. At end-point fermentation, all sheets were digested predominantly under substrate accessibility limitations (e.g., up to 81% of total CO2 production for Whatman #1). Integration of CO2 production rates over time showed Whatman #3 underwent the fastest conversion efficiency under microbial limitation, suggestive of best biofilm penetration, while Whatman #1 exhibited the least recalcitrance and the faster degradation during the substrate limitation period., Conclusion: The results showed that the specific biofilm development rate of cellulolytic bacteria such as C. thermocellum has a notable effect on overall reactor kinetics during the period of microbial limitation, when ca. 20% of cellulose conversion occurs. The study further demonstrated the utility of on-line CO2 measurements as a method to assess biofilm development and substrate digestibility pertaining to microbial solubilization of cellulose, which is relevant when considering feedstock pre-treatment options.

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h(-1)) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.

Integrated fixed-film activated sludge systems (IFFAS) may achieve year-round nitrification or gain additional treatment capacity due to the presence of both flocs and biofilms, and the potential for multiple redox states and long solids retention time. Flocs and biofilms are distinctive microbial structures and characterization of the physicochemical and structural properties of these may provide insight into their respective roles in wastewater treatment and contaminant removal in IFFAS. Flocs and biofilms were examined from five different pilot media systems being evaluated for potential full scale implementation at a large municipal wastewater treatment plant. Flocs and biofilms within the same system possessed different surface characteristics; flocs were found to have a higher negative surface charge (-0.35 to -0.65 meq./g VSS) and are more hydrophobic (60%-75%) than biofilms (-0.05 to -0.07 meq/g VSS; 19-34%). The EPS content of flocs was significantly higher (range of 2.1-4.5 folds) than that of biofilms. In floc-derived extracellular polymeric substances (EPS), protein (PN) was clearly dominant; whereas in biofilm-derived EPS, PN and polysaccharide (PS) were present in approximately equal proportions. Biofilm EPS had a higher proportion of DNA when compared to flocs. Biofilm growth was preferential on the protected internal surfaces of the media. Colonization of the external surfaces of the media was evident by the presence of small microcolonies. The structural heterogeneity of the biofilms examined was supported by observed differences in biomass content, thickness and roughness of biofilm surface. The biofilm on the interior surface of media was found to be patchy with clusters of cells connected by an irregular arrangement of interconnecting EPS projections. Biofilm thickness ranged between 139 μm and 253 μm. The pattern of oxygen penetration is expected to be complex. Nitrifiers and denitrifiers were predominantly associated with the biofilms, and the latter were found to be dispersed throughout the film and arranged in micro-clusters, suggesting partial oxygen penetration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The bioeconomy, and in particular, biorefining and bioenergy production, have received considerable attention in recent years as a shift to renewable bioresources to produce similar energy and chemicals derived from fossil energy sources, represents a more sustainable path. Membrane technologies have been shown to play a key role in process intensification and products recovery and purification in biorefining and bioenergy production processes. Among the various separation technologies used, membrane technologies provide excellent fractionation and separation capabilities, low chemical consumption, and reduced energy requirements. This article presents a state-of-the-art review on membrane technologies related to various processes of biorefining and bioenergy production, including: (i) separation and purification of individual molecules from biomass, (ii) removal of fermentation inhibitors, (iii) enzyme recovery from hydrolysis processes, (iv) membrane bioreactors for bioenergy and chemical production, such as bioethanol, biogas and acetic acid, (v) bioethanol dehydration, (vi) bio-oil and biodiesel production, and (vii) algae harvesting. The advantages and limitations of membrane technologies for these applications are discussed and new membrane-based integrated processes are proposed. Finally, challenges and opportunities of membrane technologies for biorefining and bioenergy production in the coming years are addressed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hydrodynamic forces generated by an orifice plate under low pressure were examined as a means of disrupting flocs, in order to improve disinfection of treated wastewater effluents. Changes in cavitation conditions were found to have little impact on the extent of particle breakage in this experimental setup. The rate of strain (flow rate divided by the hole radius cubed), however, was found to be the best predictor of floc breakage. Floc breakage was not affected by changes in floc concentration, but was very sensitive to differences between flocs collected from different sources. Larger flocs (90 to 106 microm) were broken apart to a greater extent than smaller ones (53 to 63 microm). Hydrodynamic treatment decreased the viability of bacteria associated with large flocs, and also increased the ultraviolet dose response by up to one log unit (i.e., a factor of ten). Subjecting final effluent wastewaters to hydrodynamic treatment, therefore, provides a treatment strategy for conditions in which the presence of flocs limits the level of disinfection that can be achieved.

The analysis of persistent organic pollutants is a real challenge due to the large number of compounds with varying chemical and physical properties. Gas chromatography with electron capture detection or mass spectrometry has been the method of choice for the past 50 years. Comprehensive two-dimensional gas chromatography (GCxGC) coupled with micro-electron capture detector (μECD) is a new method that can analyze polychlorinated biphenyls (PCBs), organochlorine pesticides (OCs) and chlorobenzenes (CBz) in a single analytical run with enhanced selectivity and sensitivity over single column methods and can also be used to screen for other halogenated organics in environmental samples. An accredited routine method using commercially available LECO GCxGC-μECD and a column combination DB-1 × Rtx-PCB has been developed to analyse PCBs/OCs/CBz in soils, sediments and sludges. The method provides quantification of Aroclors and Aroclor mixtures to within 15% of target values and sub-nanogrammes per gramme detection limits.

Runoff from agricultural watersheds can carry a number of agricultural pollutants and pathogens; often associated with the sediment fraction. Deposition of this sediment can impact water quality and the ecology of the river, and the re-suspension of such sediment can become sources of contamination for reaches downstream. In this paper a modelling framework to predict sediment and associated microbial erosion, transport and deposition is proposed for the South Nation River, Ontario, Canada. The modelling framework is based on empirical relationships (deposition and re-suspension fluxes), derived from laboratory experiments in a rotating circular flume using sediment collected from the river bed. The bed shear stress governing the deposition and re-suspension processes in the stream was predicted using a one dimensional mobile boundary flow model called MOBED. Counts of live bacteria associated with the suspended and bed sediments were used in conjunction with measured suspended sediment concentration at an upstream section to allow for the estimation of sediment associated microbial erosion, transport and deposition within the modelled river reach. Results suggest that the South Nation River is dominated by deposition periods with erosion only occurring at flows above approximately 250 m(3) s(-1) (above this threshold, all sediment (suspended and eroded) with associated bacteria are transported through the modelled reach). As microbes are often associated with sediments, and can survive for extended periods of time, the river bed is shown to be a possible source of pathogenic organisms for erosion and transport downstream during large storm events. It is clear that, shear levels, bacteria concentrations and suspended sediment are interrelated requiring that these parameters be studied together in order to understand aquatic microbial dynamics. It is important that any management strategies and operational assessments for the protection of human and aquatic health incorporate the sediment compartments (suspended and bed sediment) and the energy dynamics within the system in order to better predict the concentration of indicator organism., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Understanding the transport behavior, survival, and persistence of pathogens such as Escherichia coli O157:H7 in the subsurface is essential to protection of public health. In this study, the transport of E. coli O157:H7 in a two-dimensional bench-scale sand aquifer system, hereafter referred to as the sandbox, was investigated, with a focus on the impact of biofilm development on E. coli retention and survival. Biofilm growth was initiated through flushing with unsterilized groundwater and addition of glucose, nitrate, and phosphate. Retention of E. coli from an injection test in clean sand, prior to promotion of biofilm growth, was approximately 9%. Subsequent to biofilm growth, 47% of injected E. coli cells were retained under similar flow conditions. After 10 d of no flow, sterile water was flushed through the biofouled sandbox and substantial concentrations (up to 1.5 × 10(5) cells/mL) of E. coli were measured in the effluent indicating that E. coli had survived the starvation period. Confocal laser scanning microscopy revealed that E. coli were located not only on the surface but also within the biofilm. Imposition of starvation conditions resulted in biofilm sloughing and possible mobilization of biofilm-associated E. coli., (Copyright © 2010 The Author(s). Journal compilation © 2010 National Ground Water Association.)

The growth kinetics of Hyphomicrobium spp. and Thiobacillus spp. on dimethyl sulfide (DMS) and methanol (in the case of Hyphomicrobium spp.) in an enrichment culture created from a biofilter cotreating DMS and methanol were studied. Specific growth rates of 0.099 h(-1) and 0.11 h(-1) were determined for Hyphomicrobium spp. and Thiobacillus spp., respectively, growing on DMS at pH 7. These specific growth rates are double the highest maximum specific growth rate for bacterial growth on DMS reported to date in the literature. When the pH of the medium was decreased from pH 7 to pH 5, the specific growth rate of Hyphomicrobium spp. decreased by 85%, with a near 100-fold decline in the yield of Hyphomicrobium 16S rRNA gene copies in the mixed culture. Through the same pH shift, the specific growth rate and 16S rRNA gene yield of Thiobacillus spp. remained similar. When methanol was used as a substrate, the specific growth rate of Hyphomicrobium spp. declined much less over the same pH range (up to 30%) while the yield of 16S rRNA gene copies declined by only 50%. Switching from an NH(4)(+)-N-based source to a NO(3)(-)-N-based source resulted in the same trends for the specific growth rate of these microorganisms with respect to pH. This suggests that pH has far more impact on the growth kinetics of these microorganisms than the nitrogen source. The results of these mixed-culture batch experiments indicate that the increased DMS removal rates observed in previous studies of biofilters cotreating DMS and methanol are due to the proliferation of DMS-degrading Hyphomicrobium spp. on methanol at pH levels not conducive to high growth rates on DMS alone.

Submerged anaerobic membrane bioreactor (SAnMBR) technology was studied for kraft evaporator condensate treatment at 37 +/- 1 degrees C over a period of 9 months. Under tested organic loading rates of 1-24 kg COD/m3/day, a chemical oxygen demand (COD) removal efficiency of 93-99% was achieved with a methane production rate of 0.35 +/- 0.05 L methane/g COD removed and a methane content of 80-90% in produced biogas. Bubbling of recycled biogas was effective for in-situ membrane cleaning, depending on the biogas sparging rate used. The membrane critical flux increased and the membrane fouling rate decreased with an increase in the biogas sparging rate. The scanning electron microscopy images showed membrane pore clogging was not significant and sludge cake formation on the membrane surface was the dominant mechanism of membrane fouling. The results suggest that the SAnMBR is a promising technology for energy recovery from kraft evaporator condensate.

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO2 production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O2 flux, with CO2 production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO2 production rate of cells from this region increased within 0.25 h from 9.45 +/- 5.40 fmol of CO2 x cell(-1) x h(-1) to 22.6 +/- 7.58 fmol of CO2 x cell(-1) x h(-1) when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.

The performance and microbiology of two inorganic biofilters treating dimethyl sulphide (DMS) in the presence and absence of methanol was investigated. Addition of methanol was shown to result in an increase in DMS removal for methanol loadings below 90 g MeOH per cubic metre per hour with the optimal methanol loading around 10-15 g MeOH per cubic metre per hour for a DMS loading of 3.4 g DMS per cubic metre per hour, a fivefold increase in the DMS removal rate compared to the biofilter treating DMS alone. Microbial community analysis revealed that the addition of methanol led to a significant increase of up to an order of magnitude in the abundance of Hyphomicrobium spp. in the biofilter co-treating DMS and methanol compared to the biofilter treating DMS alone, whilst there was no significant difference in the abundance of Thiobacillus spp. between the two biofilters. Given the behaviour of the biofilter co-treating DMS and methanol, the magnitude of the increase in Hyphomicrobium spp. in the biofilter co-treating DMS and methanol and the ability of Hyphomicrobium spp. to use both methanol and DMS as growth substrates, it was concluded that Hyphomicrobium spp. were the microorganisms responsible for the bulk of the DMS degradation in the biofilter co-treating DMS and methanol.

For proper treatment, recycling, or disposal of the pulp and paper mill secondary sludge qualitative and quantitative determination of its characteristics are necessary. Chemical extraction, quantitative characterization, and spectroscopic experiments have been performed to determine the molecular composition and chemical functionality of a pulp and paper mill secondary sludge. In order to extract the low-molecular-weight substances, soxhlet extraction with polar and non-polar solvents was performed where most of the target substances (17±1.3%.) were extracted after 2 hours. Over time, this extraction followed a first-order kinetics. Fiber analyses have shown 12±3% lignin, 28±3% cellulose, and 12±4% hemicelluloses content. The ash content was about 17±0.5%. In this work, 7 and 16% intra- and extracellular polymeric substances, respectively, were extracted from the secondary sludge. EPS and mixture of intra- and extracellular biopolymers have shown similar chemical functionalities. These analyses confirmed that the paper secondary sludge consisted mainly of wood fiber, i.e. lignocellulosic substances, along with proteins and polysaccharides originated from microorganisms.

Two submerged anaerobic membrane bioreactors (SAnMBRs) (thermophilic vs. mesophilic) were operated for a period of 3.5 months with kraft evaporator condensate at a feed chemical oxygen demand of 10,000 mg/L. The results show that the filtration behavior of the two systems was significantly different. The filtration resistance in the thermophilic SAnMBR was about 5-10 times higher than that of the mesophilic system when operated under similar hydrodynamic conditions. Comparison of sludge properties and cake layer structure from the two systems was made to elucidate major factors governing the different filtration characteristics. There were more soluble microbial products (SMP) and biopolymer clusters (BPC) produced and a larger portion of fine flocs (<15 microm) in the thermophilic SAnMBR. Analysis of bound extracellular polymeric substances (EPS) showed that the thermophilic sludge had a higher protein/polysaccharide ratio in EPS, as compared to that in the mesophilic sludge. A series of analyses, including Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray spectroscopy (EDX), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and particle size analyzer showed that the cake layer formed in the thermophilic SAnMBR contained higher levels of both organic and inorganic foulants, smaller particle sizes, and especially, a denser and more compact sludge cake structure. These results indicate that floc size, SMP, BPC, bound EPS as well as cake layer structure are the major factors governing membrane fouling in SAnMBR systems.

The transport and fate of indicator E. coli and Salmonella are shown to be highly influenced by their relationship with flocculated suspended and bed sediment particles. Flocs were found to dominate the suspended sediment load and have the effect of increasing the downward flux of the sediment to the river bed. Bacteria counts were consistently higher within sediment compartments (suspended and bed) than for the water alone, with the bed sediment found to represent a possible reservoir of pathogens for subsequent remobilization and transport to potentially high risk areas. The mechanism of microbial attachment and entrapment within the sediment was strongly linked to the EPS fibrils secreted by the biological consortium of the aquatic system. It is suggested that the sediment/pathogen relationship should be of concern to public health officials because of its potential effects on pathogen source fate and effect with implications on public health risk assessment. Current standard sampling strategies, however, are based on an assumption that bacteria are entirely planktonic and do not account for the potentially significant concentration of bacteria from the sediment compartments. The lack of understanding around pathogen/sediment associations may lead to an inaccurate estimate of public health risk, and, as such, possible modification of sampling strategies to reflect this association may be warranted.

Membrane-aerated biofilms with oxygen and nutrients diffusing from the opposite sides possess distinct properties, including the ability to couple aerobic and anaerobic processes. The objective of this study was to examine the effects of oxygen partial pressure and chemical oxygen demand (COD) loading on biofilm properties. Two laboratory-scale membrane-aerated bioreactors were operated for a total of 283 days, with one reactor operated at 42, 60, and 89 kPa (0.41, 0.59, and 0.88 atm) oxygen, and the other reactor at 25 kPa (0.25 atm) oxygen (air control). The biofilm detached at the oxygen partial pressures of 60 and 89 kPa (0.59 and 0.88 atm) at a COD loading of 11.3 kg COD/1000 m2/d, but was sustained at the oxygen partial pressures of 25 and 42 kPa (0.25 and 0.41 atm), with a porous structure at the membrane interface at the COD loading of 11.3 kg COD/1000 m2/d. Biofilm formation was improved at a higher COD loading. It is proposed that the loss of extracellular polymeric substances at the biofilm bottom is the cause for the biofilm detachment subjected to a higher oxygen partial pressure.

The presence of methanol (MeOH) improves DMS removal (up to 11-fold) by enhancing biomass growth in inorganic biofilters. Although the overall effect is positive, prolonged growth on methanol also negatively affects DMS degradation as a result of competition with DMS. The objectives of this study were to explore the potential to optimize DMS removal with methanol addition and to develop and experimentally validate a mathematical model describing the biofiltration of DMS in the presence of MeOH. Continuous experiments using three bench-scale biofilters packed with inorganic material were performed to examine the removal of DMS under different MeOH addition rates ranging from 0 to 140 g/m3/h. For a constant DMS loading of 3.5 g/m3/h, a maximum DMS removal rate of 1.8 g/m3/h was achieved at a MeOH addition rate of 20 g/m3/h in the inorganic biofilters. A steady-state model incorporating the competitive and activation effects of MeOH on DMS biodegradation was developed, and the modeled results on DMS and MeOH removal were in close agreement with experimental data. Both the experimental data and model simulation suggest that there is an optimum MeOH addition rate for a given DMS loading. A step-feeding strategy for MeOH addition was proposed and tested by the model to optimize DMS removal. The model-predicted results demonstrate that six-step feeding of MeOH enhances DMS treatment by 46% in the biofilters when compared to conventional feeding (one-step) of MeOH at the same total mass loading.

The biofiltration of dimethyl sulfide (DMS) and other reduced sulfur compounds (RSC) results in acidification of biofilters due to the accumulation of the sulfuric acid in packing material. This may lead to a decrease in biofilter performance due to a drop in pH. Results obtained from continuous experiments using three bench-scale biofilters packed with inorganic material mixed with limestone show that methanol (MeOH) alleviates the pH drop and enhances the stability of biofilter performance and DMS removal. The pH drop in the biofilters treating DMS with MeOH is 4 fold slower than that in the control biofiler treating DMS only. For the biofilters with MeOH addition, the pH of the biofilters drops more gradually (0.044 pH units per day) when compared to the MeOH suspension periods when MeOH is not added (0.23 pH units per day). MeOH addition consumes oxygen and results in a lower conversion ratio of sulfide to sulfuric acid due to the formation of elemental sulfur, reducing acidification in the biofilters. Nitrification was found to be actively taking place in the control biofilter treating DMS without MeOH addition, contributing to the significant pH drop in the reactor. It is proposed that MeOH prevents acid production from nitrification likely by limiting oxygen and nutrients to nitrifying bacteria in the MeOH-fed biofilters.

The effect of limiting P in activated sludge was investigated in laboratory scale sequencing batch reactors (SBRs) fed effluent from a container board mill. Floc characterization included measurement of hydrophobicity, surface charge, and analysis of extracellular polymeric substances (EPS). Reactor performance was assessed by monitoring COD and inorganic P removal, MLSS, and sludge settleability (SVI and batch settling flux) over a period of eight months. Control reactors (BOD:N:P of 100:5:1) were compared to reactors run under P-limited conditions (100:5:0.3; 100:5:0.1). Reactor performance at lower temperatures (14 degrees C; control = 26 degrees C) was also studied to assess the impact of P-limitation. Changes in floc structure and the composition of EPS occurred within 1 to 3 days following a reduction in P levels. There was an insignificant increase in SVI; however, gravitational settling velocity and batch settling flux values for low P floc were consistently higher than for floc generated under control conditions. Lower temperatures (14 degrees C) resulted in a deterioration in floc settling properties at a BOD:N:P of 100:5:1. This impact on settling was significantly reduced at a BOD:P of 100:0.1. Reducing P concentrations in the wastewater treatment system has the potential to improve sludge settleability and reduce final P discharges.

Air emissions from the pulp and paper industry frequently contain reduced sulfur compounds (RSC), such as dimethyl sulfide (DMS) mixed with volatile organic compounds (VOC) (e.g., methanol, MeOH) and it is desirable to treat either one or both of these groups of compounds. The objective of this study was to assess the effects of VOC (MeOH) on the biofiltration of DMS. Results obtained from continuous experiments using three bench-scale biofilters packed with inorganic material clearly show that MeOH has a positive effect (11-fold increase) on the biofiltration of DMS. Further experiments indicate that MeOH addition enhances biomass concentration and viability (threefold) in the biofilters. However, a suspension of MeOH addition causes a rapid significant increase (twofold) in the removal rate of DMS, suggesting that the presence of MeOH also has a competitive effect on DMS biodegradation. This negative effect was also confirmed in batch experiments. The decrease of biofilter performance with time for a long-term suspension of MeOH addition indicates that MeOH addition is necessary to sustain a high removal rate of DMS in inorganic biofilters. Results on metabolic products of DMS biodegradation demonstrate that DMS is almost completely converted to sulfate in the absence of MeOH, while it is partially oxidized to elemental sulfur in the presence of MeOH. This study suggests that there exists an optimum mix of DMS and MeOH for the treatment of DMS emissions in inorganic biofilters., ((c) 2006 Wiley Periodicals, Inc.)

The effect of solids retention time (SRT) (4-20 d) on sludge floc structure, size distribution and morphology in laboratory-scale sequencing batch reactors receiving a glucose-based synthetic wastewater was studied using image analysis in a long-term experiment over one year. Floc size distribution (>10 microm) could be characterized by a log-normal model for no bulking situations, but a bi-modal distribution of floc size was observed for modest bulking situations. In each operating cycle of the SBRs, the variation in food /microorganisms ratio (0.03-1.0) had no significant influence on floc size distribution and morphology. The results from a long-term study over one year showed that no clear relationship existed between SRT and median floc size based on frequency. However, sludge flocs at the lower SRTs (4-9 d) were much more irregular and more variable in size with time than those at higher SRTs (16 and 20 d). The level of effluent-suspended solids at lower SRTs was higher than that at higher SRTs.

The effect of limiting phosphorus (P) in activated sludge was investigated in laboratory-scale sequencing batch reactors (SBRs). Correlative microscopy revealed that P-limitation (COD:N:P = 100:5:0.05) leads to morphological changes in floc structure and the composition of extracellular polymeric substances (EPS). This was found to be accompanied by expression of quorum-sensing in an acyl homoserine lactone bioassay. Differential gene expression in relation to P-limitation was examined in a global profile using the Affymetrix Escherichia coli antisense genomic microarray. Three separate experiments were conducted where the impact of P-limitation was examined under batch conditions and in SBRs at stable operating conditions and within 3-7 days following a down-shift in P. Significant changes in open reading frames (ORF) and intergenic regions based on the E. coli microarray were observed. Several genes associated with cell structure, including slt, wbbH, fimH, amB, rfaJ and slp were found to be expressed. Quorum regulated genes were also found to be expressed including psiF which is known to be induced by P-starvation (92% confidence level; 1.45 log ratio).

The potential impact on a variety of bioassay organisms when pulp-mill biosolids from a thermomechanical pulp mill (western Canada) were applied to a reference soil has been investigated in a laboratory setup. The current research assessed acute, chronic, and reproductive impacts using a battery of terrestrial and aquatic organisms. Terrestrial organisms were exposed to soil amended with different concentrations of biosolids, while aquatic organisms were used to assess the impact of biosolids' runoff into receiving waters. The former bioassays showed that an application rate of 20 tonneshectare(-1) (tha(-1)) "bone-dry" biosolids applied to reference soil produced no observable adverse impact on the terrestrial organisms. In the latter assays, undiluted (100%) and 50% diluted biosolids' runoff into receiving water had a detrimental impact on the aquatic organisms. However, concentrations not exceeding 25% (environmentally relevant concentrations) had neither an acute nor chronic impact compared to reference populations. The organisms' abilities to reproduce were also unaltered. While this study only examined the biosolids from one mill, there is the potential that land-application of characteristically well-defined pulp mill biosolids may constitute an acceptable way of disposing of pulp and paper mill biosolid residues. However, the biosolids coming from different mills, with differing processes, must be dealt with on a case-by-case situation. Each series of biosolids must be rigorously tested for toxicological impact in the laboratory under tightly controlled conditions. Subsequently, field experimentation must be conducted before definitive conclusions can be made.

Industrial and municipal processes may produce and release endocrine-disrupting compounds (EDCs) into the environment, but the exact nature of their effects is difficult to investigate. EDCs typically exert their effect by affecting gene expression aberrantly. To determine if gene expression profiles could be used to detect and distinguish estrogenic EDCs, an estrogen receptor positive human breast cancer cell line (MCF-7) was exposed to known estrogenic compounds, suspected EDCs, and extracts from three effluent samples. A set of specifically estrogen-regulated genes was identified by microarray analysis. Nine estrogen up-regulated genes (IGFBP4, HSPA8, B4GALT1, XBP1, KRT8, GTPBP4, HNRPAB, SLC2A1, and CALM1) and two estrogen down-regulated genes (ID2 and ZNF217) were consistently detectable in response to estrogen and other estrogenic compounds. Gene expression patterns in cells that were exposed to effluent sample extracts were compared to gene expression patterns in cells that were exposed to known endocrines. Using this technique, two of the effluent samples were shown to have estrogenic activity. This approach could easily be extended to screen for other types of receptor-mediated endocrine disruption. For example, cells expressing androgen or aryl hydrocarbon receptors could be used in gene expression profiling assays to detect androgenic effects or for the presence of bioactive aromatic hydrocarbons. Gene expression profiling is emerging as a sensitive and specific method to screen complex samples for endocrine disrupting activity.

Membrane separation technology is increasingly becoming an important innovation in biological wastewater treatment. Biofouling of the membrane is a major factor affecting the efficient and economic operation of membrane separation bioreactors (MBRs). This review summarizes the state-of-the-art progress in understanding the mechanisms and factors affecting membrane biofouling and the strategies for biofouling control. Biofouling mechanisms include the adsorption of soluble and suspended extracellular polymers on membrane surfaces and in membrane pores, the clogging of membrane pore structure by fine colloidal particles and cell debris, and the adhesion and deposition of sludge cake on membrane surfaces. Design and operating conditions of membrane modules and materials, hydrodynamic conditions in MBRs, process and environmental conditions of activated sludge systems, and the physicochemical properties of the wastewater are the dominant factors determining membrane biofouling. Current strategies to control biofouling include periodic relaxation, backwashing, chemical cleaning, and possible manipulation of hydrodynamic conditions and sludge properties. Achieving full integration of MBRs in wastewater treatment technology requires further research and development. Fundamental information on the bacteria, colloid, and membrane interaction, developed through multimethod and multiscale approaches, is particularly needed.

Estrogen receptors (ERs) are nuclear transcription factors that regulate gene expression in response to estrogen and estrogen-like compounds. Identification of estrogen-regulated genes in target cells is an essential step toward understanding the molecular mechanisms of estrogen action. Using cDNA microarray examinations, 19 genes were identified as induced by 17 beta-estradiol in MCF-7 cells, 10 of which have been reported previously to be estrogen responsive or to be linked with ER status. Five known estrogen-regulated genes, E2IG4, IGFBP4, SLC2A1, XBP1 and B4GALT1, and AFG3L1, responded quickly to estrogen treatment. A novel estrogen-responsive gene was identified and named EEIG1for early estrogen-induced gene 1. EEIG1 was clearly induced by 17 beta-estradiol within 2 h of treatment, and was widely responsive to a group of estrogenic compounds including natural and synthetic estrogens and estrogenic environmental compounds. EEIG1 was expressed in ER-positive but not in ER-negative breast cancer cell lines. EEIG1 expression was repressed by antiestrogens 4-OH-tamoxifen and ICI 182,780 but not by protein synthesis inhibitors cycloheximide and puromycin. These results provide evidence that some estrogenic compounds differentially enhance the transcription of estrogen-regulated genes and suggest a role for EEIG1 in estrogen action.

It has recently been discovered that a number of synthetic chemicals and naturally occurring compounds released into the environment can influence endocrine activity. These endocrine-disrupting compounds (EDCs) are highly varied in structure, provenance, and mode of action (MOA). Many EDCs are anthropogenic, products of the chemical industry. Others are natural compounds. Although natural hormones exist at low levels in the environment, industrial sites such as pulp and paper mills and municipal sewage treatment plants can gather large amounts of natural EDCs and release them into the environment as part of their daily operations, thus increasing background environmental concentrations. The potential consequences of endocrine disruption are serious; however, comparatively little is known about the phenomenon. There is considerable debate over the true concentrations, sources, identity, and effects of potential EDCs. Investigation of the problem is hampered by the diversity of potential EDCs, which may have synergistic as well as individual effects, and the complexity of the endocrine system itself. Effective and standardized tests to accurately detect the presence of such chemicals in the environment are not available. While many tests have been proposed, they are unsatisfactory because they have only one limited endpoint (e.g., they can detect only one of many potential hormonal responses) and do not provide any mechanistic information. In addition, results from rapid screening tests are difficult to correlate with whole organism response, while larger whole organism bioassays are very costly and time consuming to perform. There is tremendous potential in the application of DNA microarray technology to screen for EDCs. DNA microarrays provide a "snapshot" of transcriptional activity in tissue samples showing which genes were actively expressed within the cells at one point in time. By combining this technology with human cell lines grown in vitro, it should be possible to conduct relatively rapid and straightforward assays to identify EDCs by observing the changes in gene expression patterns in response to exposure.

Activated sludge floc from a wastewater treatment system was characterized, with regard to principal structural, chemical, and microbiological components and properties, in relation to contaminant-colloid associations and settling. Multiscale analytical microscopies, in conjunction with multimethod sample preparations, were used correlatively to characterize diverse colloidal matrices within microbial floc. Transmission electron microscopy, in conjunction with energy dispersive spectroscopy (EDS), revealed specific associations of contaminant heavy metals with individual bacterial cells and with extracellular polymeric substances (EPS). Floc structure was mapped from the level of gross morphology down to the nano-scale, and flocs were described with respect to settling properties, size, shape, density, porosity, bound water content, and EPS chemical composition; gross surface properties were also measured for correlation with principal floc features. Compartmentalization results based on 171 EDS analyses and representative high-resolution images showed that nano-scale agglomerations of (i) silver (100%) and (ii) zinc (91%) were confined almost entirely to EPS matrices while (iii) Pb (100%) was confined to intracellular granules and (iv) aluminum was partitioned between EPS matrices (41%) and intracellular matrices (59%). The results suggest that engineered changes in microbial physiology and/or in macromolecular EPS composition may influence metal removal efficiencies.

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.