How denitrifiers defense ciprofloxacin: Insights from intracellular and extracellular stress response.

Overuse of antibiotics has led to their existence in nitrogen-containing water. The impacts of antibiotics on bio-denitrification and the metabolic response of denitrifiers to antibiotics are unclear. We systematically analyzed the effect of ciprofloxacin (CIP) on bio-denitrification and found that 5 mg/L CIP greatly inhibited denitrification with a model denitrifier (Paracoccus denitrificans). Nitrate reduction decreased by 32.89 % and nitrous oxide emission increased by 75.53 %. The balance analysis of carbon and nitrogen metabolism during denitrification showed that CIP exposure blocked electron transfer and reduced the flow of substrate metabolism used for denitrification. Proteomics results showed that CIP exposure induced denitrifiers to use the pentose phosphate pathway more for substrate metabolism. This caused a substrate preference to generate NADPH to prevent cellular damage rather than NADH for denitrification. Notably, despite denitrifiers having antioxidant defenses, they could not completely prevent oxidative damage caused by CIP exposure. The effect of CIP exposure on denitrifiers after removal of extracellular polymeric substances (EPS) demonstrated that EPS around denitrifiers formed a barrier against CIP. Fluorescence and infrared spectroscopy revealed that the binding effect of proteins in EPS to CIP prevented damage. This study shows that denitrifiers resist antibiotic stress through different intracellular and extracellular defense strategies.

Efficient aerobic denitrification without nitrite accumulation by Pseudomonas mendocina HITSZ-D1 isolated from sewage sludge.

A highly efficient aerobic denitrifying microbe was isolated from sewage sludge by using a denitrifier enrichment strategy based on decreasing carbon content. The microbe was identified as Pseudomonas mendocina HITSZ-D1 (hereafter, D1). Investigation of the conditions under which D1 grew and denitrified revealed that it performed good growth and nitrate removal performance under a wide range of conditions. In particular, D1 rapidly removed all types of inorganic nitrogen without accumulation of the intermediate products nitrite and nitrous oxide. Overall, D1 showed a total nitrogen removal efficiency >96% at a C/N ratio of 8. The biotransformation modes and fates of three typical types of inorganic nitrogen were also assessed. Moreover, D1 had significantly higher denitrification efficiency and enzyme activities than other aerobic denitrifying microbes (Paracoccus denitrificans, Pseudomonas aeruginosa, and Pseudomonas putida). These results suggest that D1 has great potential for treating wastewater containing high concentrations of nitrogen.

Biofilm inhibition based on controlling the transmembrane transport and extracellular accumulation of quorum sensing signals.

The regulation of bacterial quorum sensing (QS) has been used to inhibit biofouling in wastewater treatment plants and the formation of biofilms. In contrast to traditional QS regulation strategies, this study aimed to obstruct the transmembrane transport process of QS signals to decrease their extracellular accumulation. Three phytochemicals, astragaloside IV, eugenol, and baicalin were selected, their effects on biofilm formation by Pseudomonas aeruginosa PA14 were studied, and the mechanisms determined. The inhibition efficiency of biofilm formation by 50 mg/L astragaloside IV, 1 mg/L eugenol, and 1 mg/L baicalin were 37%, 26%, and 26%, respectively. Confocal laser scanning microscopy and analysis of extracellular polymeric substances indicated that the three inhibitors affected the structure and composition of the biofilms. Furthermore, bacterial motility and a variety of QS-related virulence factors were suppressed by the inhibitor treatment due to changes in bacterial QS. Notably, the three inhibitors all decreased the extracellular concentration of the QS signaling molecule 3-oxo-C12-homoseine lactone by affecting the function of efflux pump MexAB-OprM. This indirectly interfered with the bacterial QS system and thus inhibited biofilm formation. In conclusion, this study revealed the inhibitory effects and inhibition mechanism of three phytochemicals on efflux pump and QS of P. aeruginosa and realized the inhibition on biofilm formation. We update the efflux pump inhibitor library and provide a new way for biofilm contamination control.

How Nitrogen and Sulfur Doping Modified Material Structure, Transformed Oxidation Pathways, and Improved Degradation Performance in Peroxymonosulfate Activation.

Current research has widely applied heteroatom doping for the promotion of catalyst activity in peroxymonosulfate (PMS) systems; however, the relationship between heteroatom doping and stimulated activation mechanism transformation is not fully understood. Herein, we introduce nitrogen and sulfur doping into a Co@rGO material for PMS activation to degrade tetracycline (TC) and systematically investigate how heteroatom doping transformed the activation mechanism of the original Co@rGO/PMS system. N was homogeneously inserted into the reduced graphene oxide (rGO) matrix of Co@rGO, inducing a significant increase in the degradation efficiency without affecting the activation mechanism transformation. Additionally, S doping converted Co3O4 to Co4S3 in Co@rGO and transformed the cooperative oxidation pathway into a single non-radical pathway with stronger intensity, which led to a higher stability against environmental interferences. Notably, based on density functional theory (DFT) calculations, we demonstrated that Co4S3 had a higher energy barrier for PMS adsorption and cleavage than Co3O4, and therefore, the radical pathway was not easily stimulated by Co4S3. Overall, this study not only illustrated the improvement due to the heteroatom doping of Co@rGO for TC degradation in a PMS system but also bridged the knowledge gap between the catalyst structure and degradation performance through activation mechanism transformation drawn from theoretical and experimental analyses.

Why the cooperation of radical and non-radical pathways in PMS system leads to a higher efficiency than a single pathway in tetracycline degradation.

Current research focused on developing multiple active species in peroxymonosulfate (PMS) system to degrade contaminants, but deepening concern lacks over why cooperation of those active species facilitated a faster degradation. Here, we employed Co3O4, rGO and Co3O4@rGO composite to activate PMS for tetracycline (TC) degradation, and detected crucial factors toward highest performance of Co3O4@rGO/PMS system. Batch experiments exhibited a satisfactory TC degradation efficiency under Co3O4@rGO/PMS, complete degraded 50 mg/L TC within 20 min. Analytical tests discovered that radical active species generated by Co3O4/PMS and non-radical species by rGO/PMS were successfully co-existed in Co3O4@rGO/PMS system, significantly improving the performance of TC removal. Subsequently, a combination of density functional theory (DFT) calculation and intermediates analysis revealed that, in Co3O4@rGO/PMS system, the cooperation rather than independent effect of radical and non-radical active species expanded TC degradation pathways, enhancing the degradation performance. Furthermore, decent adaptability, stability, and recyclability toward affecting factors variation of Co3O4@rGO/PMS demonstrated it as a potent and economical system to degrade TC. Overall, this study developed a novel Co3O4@rGO/PMS system with a cooperative oxidation pathway for highly efficient TC removal, and managed to clarify why this oxidation pathway achieved high efficiency through a combination of theoretical and experimental method.

Effect of metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS) on Bacillus subtilis: biofilm formation, flocculability and surface characteristics.

In order to understand the inhibitory mechanism of metabolic uncoupler in biofilm, this study investigated the effect of TCS on B. subtilis biofilm formation, flocculability, surface characteristics and thermodynamic properties. An optimal concentration of TCS, a metabolic uncoupler, was observed to substantially inhibit biofilm formation and the secretion of extracellular polymeric substances (EPS). The effect of TCS on the zeta potential and flocculability of bacterial suspension implied the addition of 100 µg L-1 TCS increased the net negative charge of cell surface which induced the reduction of B. subtilis flocculability. Meanwhile, the effects of TCS on bacterial surfacial thermodynamic properties were analyzed by the Derjaguin-Landau-Verwey-Overbeek (DLVO) and extend DLVO (XDLVO) theories. As DLVO and XDLVO predicted, the primary energy barrier between bacterial cells incubated with 100 µg L-1 TCS were increased compared to that of control, indicating that B. subtilis incubated with 100 µg L-1 TCS must consume more energy to aggregate or form biofilm.

Enhancing sludge biodegradability and volatile fatty acid production by tetrakis hydroxymethyl phosphonium sulfate pretreatment.

A new pretreatment method based on tetrakis hydroxymethyl phosphonium sulfate (THPS) biocide was tried to enhance sludge disintegration, and improved sludge biodegradability and subsequent volatile fatty acid (VFA) production. Sludge activity decreased to less than 10% after 2 days pretreatment using 20mg/g-TSS THPS, which also obviously destroyed EPS and cell membrane, and dissolved more biodegradable substances (48.8%) than raw sludge (19.7%). Moreover, 20mg/g-TSS THPS pretreatment shortened fermentation time to 4days and improved VFA production to 2778mg COD/L (4.35 times than that in control). Therein, the sum of n-butyric, n-valeric and iso-valeric acids unexpectedly accounted for 60.5% of total VFA (only 20.1% of that in control). The more high molecular weight VFAs (C4-C5) than low molecular VFAs (C2-C3) resulted from THPS pretreatment benefited to subsequent medium-chain volatile acids (C6-C12) generation to realize the separation and recovery of organic carbon more efficiently.

Enhanced volatile fatty acid production from excess sludge by combined free nitrous acid and rhamnolipid treatment.

VFA production from excess sludge (ES) was greatly enhanced by a low-cost and high-efficient treatment: 0.67mg/L free nitrous acid (FNA) pretreatment combined with 0.04g/g TSS rhamnolipid (RL) addition (FNA+RL), which significantly shortened fermentation time to 3days and increased VFA production to 352.26mgCOD/g VSS (5.42 times higher than raw ES). Propionic and acetic acids were the two leading components (71.86% of the total VFA). Mechanism investigation manifested FNA+RL improved the biodegradability of ES, achieved positive synergetic effect on solubilization, hydrolysis and acidification efficiencies, and inhibited methanation. Microbial community distribution further explained the above phenomena. The bacteria related to polysaccharides/protein utilization and VFA generation, including Clostridium, Megasphaera and Proteiniborus, were mainly observed in FNA+RL, whereas gas-forming bacteria Anaerolineae and acid-consuming bacteria Proteobacteria were assuredly suppressed. Besides, Propionibacterineae associated with propionic acid generation was exclusively enriched in sole RL and FNA+RL.

Removal of cephalosporin antibiotics 7-ACA from wastewater during the cultivation of lipid-accumulating microalgae.

The aim of this study is to evaluate the feasibility of using lipid-accumulating microalgae to remove cephalosporin antibiotics 7-amino cephalosporanic acid (7-ACA) from wastewater with the additional benefit of biofuels production. Three isolated microalgal strains (namely, Chlorella sp. Cha-01, Chlamydomonas sp. Tai-03 and Mychonastes sp. YL-02) were cultivated under 7-ACA stress and their biomass productivity, lipid production and N-NO3- consumption were monitored. It was found that 7-ACA had slight inhibition effects on the microalgal growth at the ratio of 12.0% (Cha-01), 9.6% (YL-02), 11.7% (Tai-03). However, lipid accumulation in the three microalgae was not influenced by the presence of 7-ACA. The investigation on the 7-ACA removal mechanisms during microalgal growth shows that 7-ACA was mainly removed by microalgae adsorption as well as hydrolysis and photolysis reactions. This study demonstrates that using microalgae to treat antibiotic-containing wastewater is promising due to the potential of simultaneous antibiotic removal and biofuel production.

Enhancement of volatile fatty acid production by co-fermentation of food waste and excess sludge without pH control: The mechanism and microbial community analyses.

The study provided a cost-effective and high-efficiency volatile fatty acid (VFA) production strategy by co-fermentation of food waste (FW) and excess sludge (ES) without artificial pH control. VFA production of 867.42mg COD/g-VS was obtained under the optimized condition: FW/ES 5, solid retention time 7d, organic loading rate 9g VS/L-d and temperature 40°C. Mechanism exploration revealed that the holistic biodegradability of substrate was greatly enhanced, and proper pH range (5.2-6.4) was formed by the high buffering capacity of the co-fermentation system itself, which effectively enhanced hydrolysis yield (63.04%) and acidification yield (83.46%) and inhibited methanogenesis. Moreover, microbial community analysis manifested that co-fermentation raised the relative abundances of hydrolytic and acidogenic bacteria including Clostridium, Sporanaerobacter, Tissierella and Bacillus, but suppressed the methanogen Anaerolineae, which also facilitated high VFA production. These results were of great guiding significance aiming for VFA recovery from FW and ES in large-scale.

Economical evaluation of sludge reduction and characterization of effluent organic matter in an alternating aeration activated sludge system combining ozone/ultrasound pretreatment.

An ozone/ultrasound lysis-cryptic growth technology combining a continuous flow anaerobic-anoxic-microaerobic-aerobic (AAMA+O3/US) system was investigated. Techno-economic evaluation and sludge lyses return ratio (r) optimization of this AAMA+O3/US system were systematically and comprehensively discussed. Economic assessment demonstrated that this AAMA+O3/US system with r of 30% (AAMA+O3/US2# system) was more economically feasible that can give a 14.04% saving of costs. In addition to economic benefits, a 55.08% reduction in sludge production, and respective 21.17% and 5.45% increases in TN and TP removal efficiencies were observed in this AAMA+O3/US2# system. Considering the process performances and economic benefits, r of 30% in AAMA+O3/US2# system was recommended. Excitation-emission matrix and Fourier transform infrared spectra analyses also proved that less refractory soluble microbial products were generated from AAMA+O3/US2# system. Improvement in 2,3,5-triphenyltetrazolium chloride electron transport system (TTC-ETS) activity in AAMA+O3/US2# further indicated that a lower sludge lyses return ratio stimulated the microbial activity.

Possible causes of excess sludge reduction adding metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), in sequence batch reactors.

Two parallel sequence batch reactors (SBRs) were operated, with and without TCS addition, to research the causes of sludge reduction by uncouplers. Three possible mechanisms of sludge reduction by TCS were studied: (1) occurrence of metabolic uncoupling, (2) consumption of more energy to resist the infection of TCS, (3) promotion of lysis-cryptic growth by TCS addition. Results showed the remarkable reduction of electronic transport system (ETS) activity and specific cellular ATP (SATP) in TCS reactor, which proved the occurrence of metabolic uncoupling. The increasing amounts of extracellular polymeric substances (EPS), as measured by chemical methods and excitation-emission matrix (EEM) fluorescence spectra, implied microorganisms consumed more energy to resist TCS. The similar DNA concentrations of the effluents in two reactors indicated sludge lysis was not intensified by TCS. Therefore, uncoupler might not only cause metabolic uncoupling but also induce more energy consumption in the production of some substances to resist uncoupler.

Thermophilic hydrogen production from sludge pretreated by thermophilic bacteria: analysis of the advantages of microbial community and metabolism.

In this study, the effects of thermophilic bacteria pretreatment and elevated fermentation temperature on hydrogen production from sludge were examined. The highest hydrogen yield of 19.9mlH2g(-1) VSS was achieved at 55°C by using pretreated sludge, which was 48.6% higher than raw sludge without pretreatment, and 28.39% higher than when fermented at 35°C. To explore the internal factors of this superior hydrogen production performance, the microbial community and the metabolism analysis were performed by using high-throughput sequencing and excitation-emission matrix. The pretreated sludge showed better utilization of dissolved organic matter and less inhibition of metabolism, especially at thermophilic condition. The 454 sequencing data indicated that microbial abundance was distinctly reduced and extremely high proportion of hydrogen-producing bacteria was found in the thermophilic community (Thermoanaerobacterium accounted for 93.75%). Thus, the pretreated sludge and thermophilic condition showed significant advantages in the hydrogen production using waste sludge as substrate.

Treatability study of 3,3',4',5-tetrachlorosalicylanilide (TCS) combined with 2,4,6-trichlorophenol (TCP) to reduce excess sludge production in a sequence batch reactor.

The present study investigated the synergistic effects of a novel combined uncoupler of TCS and TCP on excess activated sludge reduction during a 60-day operation using a sequence batch reactor (SBR). Response surface methodology (RSM) was employed to obtain the optimal dosage of the combined uncoupler. The results of 60-day operation demonstrated the combined uncoupler had effectively reduced the sludge yield by approximately 52%, without serious affecting the substrate removal efficiency. The high sludge reduction rate revealed that it was feasible and effective to utilize a combined uncoupler to limit excess activated sludge. The three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy analysis of activated sludge with different metabolic uncouplers indicated that tryptophan, tyrosine protein-like substances and tryptophan, tyrosine amino-like substances were reduced by adding a combined uncoupler. Moreover, the variation of sludge components provided a better understanding of the effects of uncouplers on activated sludge reduction.

Characterizing the fluorescent products of waste activated sludge in dissolved organic matter following ultrasound assisted ozone pretreatments.

This study investigated the effects of ozone and ultrasound (US) pretreatments, both individually and combined, on waste activated sludge reduction. Batch tests were conducted first to optimize the individual ozone and US pretreatments. Maximum sludge reduction ratios of 10.89% and 23% were obtained at 0.15g O3/g total solids ozone dose and 1.5W/mL US energy density, respectively. The combined ozone and US pretreatments were studied using response surface methodology. A maximum sludge reduction ratio of 40.14% was achieved by the combined ozone/US pretreatment with an ozone dose of 0.154g O3/g total solids and an US energy density of 1.445W/mL. The analysis of the dissolved organic matter by three-dimensional excitation-emission matrix fluorescence spectroscopy showed that the combined pretreatment was superior to the individual ozone and US pretreatments, and also demonstrated the synergetic effect of these two combined pretreatments.