Mass Flow and Metabolic Pathway of Nonaeration Greywater Treatment in an Oxygenic Microalgal-Bacterial Biofilm.

A symbiotic microalgal-bacterial biofilm can enable efficient carbon (C) and nitrogen (N) removal during aeration-free wastewater treatment. However, the contributions of microalgae and bacteria to C and N removal remain unexplored. Here, we developed a baffled oxygenic microalgal-bacterial biofilm reactor (MBBfR) for the nonaerated treatment of greywater. A hydraulic retention time (HRT) of 6 h gave the highest biomass concentration and biofilm thickness as well as the maximum removal of chemical oxygen demand (94.8%), linear alkylbenzenesulfonates (LAS, 99.7%), and total nitrogen (97.4%). An HRT of 4 h caused a decline in all of the performance metrics due to LAS biotoxicity. Most of C (92.6%) and N (95.7%) removals were ultimately associated with newly synthesized biomass, with only minor fractions transformed into CO2 (2.2%) and N2 (1.7%) on the function of multifarious-related enzymes in the symbiotic biofilm. Specifically, microalgae photosynthesis contributed to the removal of C and N at 75.3 and 79.0%, respectively, which accounted for 17.3% (C) and 16.7% (N) by bacteria assimilation. Oxygen produced by microalgae favored the efficient organics mineralization and CO2 supply by bacteria. The symbiotic biofilm system achieved stable and efficient removal of C and N during greywater treatment, thus providing a novel technology to achieve low-energy-input wastewater treatment, reuse, and resource recovery.

pH-dependent microbial niches succession and antibiotic resistance genes distribution in an oxygen-based membrane biofilm reactor treating greywater.

System pH is found to crucially affect biofilm growth and microorganisms' activity in the biofilm-based wastewater treatment system. This study investigated the pH-dependent pollutants removal, microbial niches succession and antibiotic resistance genes (ARGs) accumulation in an oxygen-based membrane biofilm reactor treating greywater. Results indicated that neutral conditions achieved the highest biofilm concentration and living cells, which enabled the highest pollutants removal rates; multifarious functional groups in biofilm enabled pollutants adsorption, which favored its continuous bio-removal. Microbial communities under acidic condition (pH = 5.0) were significantly different with that under other conditions (p < 0.05). The neutral and alkaline niches (pH = 7.0 and 9.0) were predominant by organics biodegradation and nitrogen reduction bacteria (e.g. Sphingobacteriales, Pseudomonas, Flavobacterium and Phenylobacterium), but which were significantly dropped under acidic conditions, leading to the declined reactor performance. ARGs in biofilm (predominant by korB, intI-1, sul1 and sul2) were much higher than that in the cell-free liquid and the target ARGs accumulation (korB, intI-1, blaCTX-M, qnrS) had nearly linear positive relationships (R2 > 0.95, P < 0.01) with biofilm-attached linear alkylbenzene sulfonate (LAS). LAS stimulate ARGs proliferation in functional microorganisms (korB, sul-1 and intI-1 were significantly associated with related microbial genus) and biofilm played a key role in ARGs dissemination. The relatively low ARGs in both biofilm and effluent under neutral conditions suggested that pH controlling can be an effective strategy to inhibit ARGs dissemination and proliferation in the system.

Response of antibiotic resistance genes expression and distribution on extracellular polymeric substances and microbial community in membrane biofilm during greywater treatment.

This study evaluated how organic loading affects antibiotic resistance genes (ARGs) expression and distribution in the membrane biofilm. Organic surface loading rate of 4.65 g chemical oxygen demand (COD)/m2·d achieved the maximum biofilm thickness, concentration and linear alkylbenzene sulfonate (LAS) removal ratio of 136.9 ± 4.7 µm, 5.4 ± 0.1 g VSS/m2 and 99.4 %, respectively. Extracellular polymeric substances (EPS), EPS-attached LAS, and ARGs gradually increased in the membrane air inlet, middle and air outlet. AGRs and Intl1 were abundant in biofilm. LAS promoted EPS secretion, biofilm growth and ARGs proliferation. EPS, protein and carbohydrate were significantly correlated with most of biofilm ARGs, but not corrected with liquid-based ARGs. Microbial community structure impacted ARGs proliferation and transfer in the system. The findings indicated that EPS and microbial community play a crucial role in ARGs proliferation, spread and distribution, which lay the foundation for front-end control of ARGs during biofilm-based wastewater treatment.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture.

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

Microalgal extracellular polymeric substances (EPS) and their roles in cultivation, biomass harvesting, and bioproducts extraction.

Microalgae extracellular polymeric substances (EPS) are complex high-molecular-weight polymers and the physicochemical properties of EPS strongly affect the core features of microalgae cultivation and resource utilization. Revealing the key roles of EPS in microalgae life-cycle processes in an interesting and novelty topic to achieve energy-efficient practical application of microalgae. This review found that EPS showed positive effect in non-gas uptake, extracellular electron transfer, toxicity resistance and heterotrophic symbiosis, but negative impact in gas transfer and light utilization during microalgae cultivation. For biomass harvesting, EPS favored biomass flocculation and large-size cell self-flocculation, but unfavored small size microalgae self-flocculation, membrane filtration, charge neutralization and biomass dewatering. During bioproducts extraction, EPS exhibited positive impact in extractant uptake, but the opposite effect in cellular membrane permeability and cell rupture. Future research on microalgal EPS were also identified, which offer suggestions for comprehensive understanding of microalgal EPS roles in various scenarios.

Biofilm bioactivity affects nitrogen metabolism in a push-flow microalgae-bacteria biofilm reactor during aeration-free greywater treatment.

Non-aeration microalgae-bacteria biofilm has attracted increasing interest for its application in low cost wastewater treatment. However, it is unclear the quantified biofilm characteristics dynamics and how biofilm bioactivity affects performance and nitrogen metabolisms during wastewater treatment. In this work, a push-flow microalgae-bacteria biofilm reactor (PF-MBBfR) was developed for aeration-free greywater treatment. Comparatively, organic loading at 1.27 ± 0.10 kg COD/(m3⋅d) gave the highest biofilm concentration, density, specific oxygen generation (SOGR) and consumption rates (SOCR), and pollutants removal rates. Contributed to low residual linear alkylbenzene sulfonates and bioactivity, reactor downstream showed low bacteria and protein concentrations and SOCR (12.8 mg O2/g TSS·h), but high microalgae, carbohydrate, biofilm density, SOGR (49.4 mg O2/g TSS·h) and pollutants removal rates. Dissolved organic nitrogen (DON) showed higher molecular weight, CHONS and fraction with 4 atoms of N in reactor upstream. Most of nitrogen was fixed to newly synthesized biomass during assimilation process by related functional enzymes, minor contributed to denitrification due to low N2 emission. High nitrogen assimilation by microalgae showed high SOGR, which favored efficient multiple pollutants removal and reduced DON emission. Our findings favor the practical application of PF-MBBfR based on biofilm bioactivity, enhancing efficiency and reducing DON emission for low- energy-input wastewater treatment.

Distribution and dynamics of antibiotic resistance genes in a three-dimensional multifunctional biofilm during greywater treatment.

Antibiotic resistance genes (ARGs) have been identified as serious threats to public health. Despite the widespread in various systems, dynamics of ARGs in three-dimensional multifunctional biofilm (3D-MFB) treating greywater are largely undefined. This work tracked the distributions and dynamics of eight target genes (intI1, korB, sul1, sul2, tetM, ermB, blaCTX-M and qnrS) in a 3D-MFB during greywater treatment. Results showed that hydraulic retention times at 9.0 h achieved the highest linear alkylbenzene sulfonate (LAS) and total nitrogen removal rates at 99.4% and 79.6%, respectively. ARGs presented significant liquid-solid distribution feature, but non-significant with biofilm position. Intracellular ARGs (predominant by intI1, korB, sul1 and sul2) at bottom biofilm were 210- to 4.2 × 104- fold higher than that in cell-free liquid. Extracellular polymeric substances (EPS)-attached LAS showed linear relationship with most of ARGs (R2 > 0.90, P < 0.05). Sphingobacteriales, Chlamydiales, Microthrixaceae, SB-1, Cryomorphaceae, Chitinophagaceae, Leadbetterella and Niabella were tightly bound up with target ARGs. Key is that EPS-attached LAS considerably determines the occurrence of ARGs, and microbial taxa play an important role in the dissemination of ARGs in the 3D-MFB.

Microbial niches and dynamics of antibiotic resistance genes in a bio-enhanced granular-activated carbon biofilm treating greywater.

Accumulation and transmission of antibiotic resistance genes (ARGs) in greywater treatment systems present risks for its reuse. In this study, a gravity flow self-supplying oxygen (O2) bio-enhanced granular activated carbon dynamic biofilm reactor (BhGAC-DBfR) was developed to treat greywater. Maximum removal efficiencies were achieved at saturated/unsaturated ratios (RSt/Ust) of 1:1.1 for chemical oxygen demand (97.6 ± 1.5%), linear alkylbenzene sulfonates (LAS) (99.2 ± 0.5%), NH4+-N (99.3 ± 0.7%) and total nitrogen (85.3 ± 3.2%). Microbial communities were significantly different at various RSt/Ust and reactor positions (P < 0.05). The unsaturated zone with low RSt/Ust showed more abundant microorganisms than the saturated zone with high RSt/Ust. The reactor-top community was predominant by aerobic nitrification (Nitrospira) and LAS biodegradation (Pseudomonas, Rhodobacter and Hydrogenophaga) related genera; but reactor-bottom community was predominant by anaerobic denitrification and organics removal related genera (Dechloromonas and Desulfovibrio). Most of the ARGs (e.g., intI-1, sul1, sul2 and korB) were accumulated in the biofilm, which were closely associated with microbial communities at reactor top and stratification. The saturated zone can achieve over 80% removal of the tested ARGs at all operation Phases. Results suggested that BhGAC-DBfR can provide assistance in blocking the environment dissemination of ARGs during greywater treatment.

Removal kinetics of linear alkylbenzene sulfonate in a batch-operated oxygen based membrane biofilm reactor treating greywater: Quantitative differentiation of adsorption and biodegradation.

Oxygen-based membrane biofilm reactor (O2-MBfR) is a unique technique for high linear alkylbenzene sulfonate (LAS)-containing greywater (GW) treatment. Despite the efficient removal of LAS, the dynamics of how it is taken up and the quantitative differentiation of adsorption and biodegradation are largely undefined. In this study, we tracked the fate of LAS, chemical oxygen demand and nitrogen in various systems: GW, GW with inactivated sludge (InAS) and GW with activated sludge (AS). We determined the distribution of biodegraded-, free-, and extracellular polymeric substances (EPS)-attached LAS, and we also developed a model to simulate all the steps. Results showed that AS exhibited high live cells proportion and microbial activity, but the opposite trend for GW and InAS. Both of nitrogen and organics could be simultaneously and efficiently removed in the AS inoculated system. The two-step model for LAS uptake and biodegradation represented the experimental results well. EPS adsorption led to the fast LAS accumulation in biofilm, and biodegradation led to the continuous removal of LAS in the system. After operated for 24 h, biodegradation and EPS accumulation of LAS were 94% and 4%, respectively, and the residual soluble LAS was lower than 1%. This work lays the foundation for using O2-MBfR to treat GW and other types of wastewater, and understanding the key roles of EPS and the mathematical model of LAS removal in the system.

Assessment and optimization of the oxygen based membrane biofilm reactor as a novel technology for source-diverted greywater treatment.

The oxygen based membrane biofilm (O2-MBfR) has been proved to be a novel technology in treating greywater (GW) and response surface methodology (RSM) was used to model the removal of chemical oxygen demand (COD) and total nitrogen (TN) with operation parameters COD/TN ratio, system pH and lumen air pressure (LAP). Results indicated that the all target single factors affect GW treatment efficiency, and the regression model with central composite design (CCD) showed good agreement with the experimental results with high R2 and R2 adj values (all >0.97) for all the target responses. Statistical evaluation revealed that system pH was the most significant parameter affecting COD and TN removal, followed by COD/TN ratio and LAP. The interaction between COD/TN ratio and system pH also played an important role on the GW treatment. The optimized maximum removal of COD (96.48%) and TN (133 g N/m2-day) were achieved with the COD/TN ratio 17.76 g COD/g TN, system pH 7.10 and LAP 1.00 psi. Thus, RSM combined with CCD could be used for predicting the organics and nitrogen removal during GW treatment in the O2-MBfR.