Achieving Long-Term Stability of Partial Nitrification and Autotrophic Denitrification in an MABR via Sulfide Dosing.

While partial nitrification (PN) has the potential to reduce energy for aeration, it has proven to be unstable when treating low-strength wastewater. This study introduces an innovative combined strategy incorporating a low rate of oxygen supply, pH control, and sulfide addition to selectively inhibit nitrite-oxidizing bacteria (NOB). This strategy led to a stable PN in a laboratory-scale membrane aerated biofilm reactor (MABR). Over a period of 260 days, the nitrite accumulation ratio exceeded 60% when treating synthetic sewage containing 50 mg NH4+-N/L. Through in situ activity testing and high-throughput sequencing, the combined strategy led to low levels of nitrite-oxidation activity (<5.5 mg N/m2 h), Nitrospira species (relative abundance <1%), and transcription of nitrite-oxidation genes (undetectable). The addition of sulfide led to simultaneous PN and autotrophic denitrification in the single-stage MABR, resulting in over 60% total inorganic nitrogen removal. Sulfur-based autotrophic denitrification consumed nitrite and inhibited NOB conversion of nitrite to nitrate. The combined strategy has potential to be applied in large-scale sewage treatment and deserves further exploration.

Novel Sulfate Reduction Coupled to Simultaneous Nitrification and Autotrophic Denitrification Process for Removing Nitrogen and Organics from Saline Wastewater.

Highly efficient sulfate reduction coupled to autotrophic denitrification plus nitrification is demonstrated by integrating an anaerobic membrane bioreactor (AnMBR) with a membrane aerated biofilm reactor (MABR). Concurrent chemical oxygen demand (COD) removal and sulfate reduction were accomplished in the AnMBR, while simultaneous nitrification and autotrophic denitrification were carried out in the MABR. Separate operation of the MABR achieved >90% total nitrogen (TN) removal when the N/S ratio was controlled at 0.4 gN/gS. The integrated AnMBR-MABR system efficiently resisted influent variability, realizing >95% COD removal in the AnMBR and >75% TN removal in the MABR when the influent COD/N ratio was above 4 gCOD/gN. Membrane fouling did not happen during ∼170 days of operation. Due to sulfide oxidation, a large amount of elemental sulfur (S0) accumulated in the MABR biofilm, where it served as an electron donor for denitrification. Microbial community analysis indicated that Nitrospira and Thiobacillus played key roles in nitrification and sulfide-driven denitrification, respectively, and that they occurred in different layers of the biofilm. This novel process offers advantages of a small land-area footprint, modular operation, and high efficiency electron-donor and oxygen utilizations, particularly for wastewater with a low COD/N ratio.

Microbial Stratification Affects Conversions of Nitrogen and Methane in Biofilms Coupling Anammox and n-DAMO Processes.

A methane-based membrane biofilm reactor (MBfR) has a suitable configuration to incorporate anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) processes because of its high gas-transfer efficiency and efficient biomass retention. In this study, the spatial distribution of microorganisms along with the biofilm depth in methane-based MBfRs was experimentally revealed, showing the dominance of anammox bacteria, n-DAMO bacteria, and n-DAMO archaea in the outer layer, middle layer, and inner layer of biofilms, respectively. The long-term and short-term experimental investigations in conjunction with mathematical modeling collectively revealed that microorganisms living in the outer layer of biofilms tend to use substrates from wastewater, while microorganisms inhabiting the inner layer of biofilms tend to use substrates originating from biofilm substratum. Specifically, anammox bacteria dominating the biofilm surface preferentially removed the nitrite provided from wastewater, while n-DAMO bacteria mostly utilized the nitrite generated from n-DAMO archaea as these two methane-related populations spatially clustered together inside the biofilm. Likewise, the methane supplied from the membrane was mostly consumed by n-DAMO archaea, while the dissolved methane in wastewater would be primarily utilized by n-DAMO bacteria. This study offers novel insights into the impacts of microbial stratification in biofilm systems, not only expanding the fundamental understanding of biofilms and microbial interactions therein but also providing a rationale for the potential applications of methane-based MBfRs in sewage treatment.

Long-Term Succession Shows Interspecies Competition of Geobacter in Exoelectrogenic Biofilms.

Geobacter spp. are well-known exoelectrogenic microorganisms that often predominate acetate-fed biofilms in microbial fuel cells (MFCs) and other bioelectrochemical systems (BESs). By using an amplicon sequence variance analysis (at one nucleotide resolution), we observed a succession between two closely related species (98% similarity in 16S RNA), Geobacter sulfurreducens and Geobacter anodireducens, in the long-term studies (20 months) of MFC biofilms. Geobacter spp. predominated in the near-electrode portion of the biofilm, while the outer layer contained an abundance of aerobes, which may have helped to consume oxygen but reduced the relative abundance of Geobacter. Removal of the outer aerobes by norspermidine washing of biofilms revealed a transition from G. sulfurreducens to G. anodireducens. This succession was also found to occur rapidly in co-cultures in BES tests even in the absence of oxygen, suggesting that oxygen was not a critical factor. G. sulfurreducens likely dominated in early biofilms by its relatively larger cell size and production of extracellular polymeric substances (individual advantages), while G. anodireducens later predominated due to greater cell numbers (quantitative advantage). Our findings revealed the interspecies competition in the long-term evolution of Geobacter genus, providing microscopic insights into Geobacter's niche and competitiveness in complex electroactive microbial consortia.

Buffer capacity regulates the stratification of anode-respiring biofilm during brewery wastewater treatment.

Improving the buffer capacity of the electrolyte can enhance the anode performance in bioelectrochemical systems (BESs). To elucidate the mechanism underlying the facilitated BESs performance, this study used three different anode biofilms cultured with different concentrations of phosphate buffer (5, 50 and 100 mM) to investigate the biofilm response, in terms of the spatial structure of metabolic activity and microbial community, to different buffer capacities. Results showed that the electrochemical activities of the anode biofilms positively correlated with the buffer concentration. The spatial stratification of metabolic activity and microbial community of the anode biofilms were regulated by the buffer capacity, and the spatial microbial heterogeneity of the anode biofilm decreased as the buffer concentration increased. With increasing buffer capacity, Geobacter spp. were enriched in both the inner and outer layers of the biofilm, and the inhibition of methanogens growth improved the COD removal attributed to anode respiration. Additionally, the stimulation of EPS production in biofilms played a role in increasing the electrochemical performance of biofilms by buffer improvement. This study first revealed the regulation of buffer capacity on the stratification of anode biofilm during brewery wastewater treatment, which provided a deep insight into the relation of biofilm structure to its electrochemical properties.

Computer simulation study of nutrient-driven bacterial biofilm stratification.

Here, employing computer simulation tools, we present a study on the development of a bacterial biofilm from a single starter cell on a flat inert surface overlaid by an aqueous solution containing nutrients. In our simulations, surface colonization involves an initial stage of two-dimensional cell proliferation to eventually transition to three-dimensional growth leading to the formation of biofilm colonies with characteristic three-dimensional semi-ellipsoids shapes. Thus, we have introduced the influence of the nutrient concentration on bacterial growth, and calculated the cell growth rate as a function of nutrient uptake, which in turn depends on local nutrient concentration in the vicinity of each bacterial cell. Our results show that the combination of cell growth and nutrient uptake and diffusion leads to the formation of stratified colonies containing an inner core in which nutrients are depleted and cells cannot grow or divide, surrounded by an outer, shallow crust in which cells have access to nutrients from the bulk medium and continue growing. This phenomenon is more apparent at high uptake rates that enable fast nutrient depletion. Our simulations also predict that the shape and internal structure of the biofilm are largely conditioned by the balance between nutrient diffusion and uptake.

Biofilm stratification and autotrophic-heterotrophic interactions in a structured bed reactor (SBRIA) for carbon and nitrogen removal.

The structured-bed reactor with intermittent aeration (SBRIA) is a promising technology for simultaneous carbon and nitrogen removal from wastewater. An in depth understanding of the microbiological in the reactor is crucial for its optimization. In this research, biofilm samples from the aerobic and anoxic zones of an SBRIA were analyzed through 16S rRNA sequencing to evaluate the bacterial community shift with variations in the airflow and aeration time. The control of the airflow and aeration time were essential to guarantee reactor performances to nitrogen removal close to 80%, as it interfered in nitrifying and denitrifying communities. The aeration time of 1.75 h led to establishment of different nitrogen removal pathways by syntrophic relationships between nitrifier, denitrifier and anammox species. Additionally, the predominance of these different species in the internal and external parts of the biofilm varied according to the airflow.

Layered Design of a Highly Repeatable Electroactive Biofilm for a Standardized Biochemical Oxygen Demand Sensor.

Microbial electrochemical sensors are promising to monitor bioavailable organics in real environments, but their application is restricted by the unpredictable performance of the electroactive biofilm (EAB), which is randomly acclimated from environmental microflora. With a long-term stable EAB as a template, we successfully designed EAB (DEAB) by the sequential growth of Geobacter anodireducens and automatched microbes, achieving a reproducible high current than those naturally acclimated from wastewater (NEAB). Pre-inoculation of planktonic aerobes as oxygen bioscavengers was necessary to ensure the colonization of Geobacter in the inner layer, and the abundant Geobacter (50%) in DEAB guaranteed 4 times higher current density with a 15-fold smaller variation among 20 replicates than those of NEAB. The sensor constructed with DEAB exhibited a shorter measuring time and a precise biochemical oxygen demand (BOD) measurement with acetate, real domestic wastewater, and supernatant of anaerobic digestion. Here, we for the first time proposed an applicable strategy to standardize EABs for BOD sensors, which is also crucial to ensure a stable performance of all bioelectrochemical technologies.

Biofilm stratification in counter-diffused membrane biofilm bioreactors (MBfRs) for aerobic methane oxidation coupled to aerobic/anoxic denitrification: Effect of oxygen pressure.

Aerobic methane oxidation coupled with denitrification (AME-D) executed in membrane biofilm bioreactors (MBfRs) provides a high promise for simultaneously mitigating methane (CH4) emissions and removing nitrate in wastewater. However, systematically experimental investigation on how oxygen partial pressure affects the development and characteristics of counter-diffusional biofilm, as well as its spatial stratification profiles, and the cooperative interaction of the biofilm microbes, is still absent. In this study, we combined Optical Coherence Tomography (OCT) with Confocal Laser Scanning Microscopy (CLSM) to in-situ characterize the development of counter-diffusion biofilm in the MBfR for the first time. It was revealed that oxygen partial pressure onto the MBfR was capable of manipulating biofilm thickness and spatial stratification, and then managing the distribution of functional microbes. With the optimized oxygen partial pressure of 5.5 psig (25% oxygen content), the manipulated counter-diffusional biofilm in the AME-D process obtained the highest denitrification efficiency, due mainly to that this biofilm had the proper dynamic balance between the aerobic-layer and anoxic-layer where suitable O2 gradient and sufficient aerobic methanotrophs were achieved in aerobic-layer to favor methane oxidation, and complete O2 depletion and accessible organic sources were kept to avoid constraining denitrification activity in anoxic-layer. By using metagenome analysis and Fluorescence in situ hybridization (FISH) staining, the spatial distribution of the functional microbes within counter-diffused biofilm was successfully evidenced, and Rhodocyclaceae, one typical aerobic denitrifier, was found to survive and gradually enriched in the aerobic layer and played a key role in denitrification aerobically. This in-situ biofilm visualization and characterization evidenced directly for the first time the cooperative path of denitrification for AME-D in the counter-diffused biofilm, which involved aerobic methanotrophs, heterotrophic aerobic denitrifiers, and heterotrophic anoxic denitrifiers.