Differential impact of acidic and alkaline conditions on hydrothermal pretreatment, fermentation and anaerobic digestion of sludge.

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 CH4/g total chemical oxygen demandadded) was found to occur at pH 10. Therefore, alkaline sludge used for fermentation has shown technical and economic feasibility for sludge carbon recovery.

Evaluating CO2 emissions from continuous flow and batch growth systems under autotrophic mode: Implications for GHG accounting of biological nutrient removal.

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-CO2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO2 emissions should be considered distinct from the biogenic CO2 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 CO2 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 CO2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO2 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 CO2 emission rates. A linear relationship between ammonia removal and CO2 emissions was demonstrated and the quantified CO2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR.

Recent advancements in the biological treatment of high strength ammonia wastewater.

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.

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

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.

Influence of wave action on the partitioning and transport of unattached and floc-associated bacteria in fresh water.

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.

Integration and proliferation of Pseudomonas aeruginosa PA01 in multispecies biofilms.

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.

Form and function of Clostridium thermocellum biofilms.

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.

Nitrospira dominant pin-point flocs with granule-like settleability in stirred tank reactors with oxic/hypoxic/oxic zones.

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 SVI30 in RI gradually improved to 29 mL/g MLSS, and the corresponding SVI30/ SVI10 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.

Fate of sloughed biomass in integrated fixed-film systems.

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.

Impact of solid content on hydrothermal pretreatment of municipal sludge prior to fermentation and anaerobic digestion.

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 CH4/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.

A routine accredited method for the analysis of polychlorinated biphenyls, organochlorine pesticides, chlorobenzenes and screening of other halogenated organics in soil, sediment and sludge by GCxGC-µECD.

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.

Interaction between CO2-consuming autotrophy and CO2-producing heterotrophy in non-axenic phototrophic biofilms.

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.

Combined hydrothermal and free nitrous acid, alkali and acid pretreatment for biomethane recovery from municipal sludge.

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 CH4/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 CH4/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.

Growth kinetics of Hyphomicrobium and Thiobacillus spp. in mixed cultures degrading dimethyl sulfide and methanol.

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.

Metabolic differentiation in biofilms as indicated by carbon dioxide production rates.

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.

Linking performance to microbiology in biofilters treating dimethyl sulphide in the presence and absence of methanol.

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.

Cellular biopolymers and molecular structure of a secondary pulp and paper mill sludge verified by spectroscopy and chemical extraction techniques.

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.

Autotrophic Fixed-Film Systems Treating High Strength Ammonia Wastewater.

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 BioCordTM] 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 BioCordTM 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 BioCordTM CSTR; whereas, ammonia removal was relatively lower in MBBR CSTR (20-35%) and BioCordTM 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.

A Novel System for Real-Time, In Situ Monitoring of CO2 Sequestration in Photoautotrophic Biofilms.

Climate change brought about by anthropogenic CO2 emissions has created a critical need for effective CO2 management solutions. Microalgae are well suited to contribute to efforts aimed at addressing this challenge, given their ability to rapidly sequester CO2 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 CO2 sequestration. Utilizing a CO2-permeable membrane and a tube-within-a-tube design, the CO2 sequestration monitoring system (CSMS) was able to reliably detect slight changes in algal biofilm CO2 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 CO2 sequestration.

Species Interaction and Selective Carbon Addition During Antibiotic Exposure Enhances Bacterial Survival.

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 CO2 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.