Yong-Ze Lu

Removal of antibiotic resistance genes from wastewater treatment plant effluent by coagulation

Antibiotic resistance genes (ARGs), as emerging environmental contaminants, have become a threat to human health. Recent studies have demonstrated that the effluent from wastewater treatment plants is a significant point source of ARGs released into the environment. In this study, we investigated the effectiveness of coagulation technology in the removal of ARGs from treated wastewater. Specifically, we measured the removal of five ARGs (two sulfonamide resistance genes, sulI and sulII, and three tetracycline resistance genes, tetO, tetW and tetQ) and the class 1 integron intI1 gene via the application of two coagulants: FeCl3 and polyferric chloride (PFC). Moreover, the removal of dissolved organic carbon (DOC), NH3N and total phosphorus (TP) in the coagulation process was investigated. The coagulation process effectively removed ARGs from the effluent with 0.5-log to 3.1-log reductions. Significant removal correlations were observed between dissolved NH3N and DOC, intI1 and sulI, sulII and tetO, sulII and tetW, and tetO and tetW, implying that the co-removal of DOC, dissolved NH3N, the intI1 gene and different ARGs played an important role in ARG loss during coagulation with Fe-based coagulants. These results indicate that coagulation may play a promising role in ARG reduction in wastewater treatment plants.

Design and evaluation of universal 16S rRNA gene primers for high-throughput sequencing to simultaneously detect DAMO microbes and anammox bacteria.

To develop universal 16S rRNA gene primers for high-throughput sequencing for the simultaneous detection of denitrifying anaerobic methane oxidation (DAMO) archaea, DAMO bacteria, and anaerobic ammonium oxidation (anammox) bacteria, four published primer sets (PS2-PS5) were modified. The overall coverage of the four primer pairs was evaluated in silico with the Silva SSU r119 dataset. Based on the virtual evaluation, the two best primer pairs (PS4 and PS5) were selected for further verification. Illumina MiSeq sequencing of a freshwater sediment and a culture from a DAMO-anammox reactor using these two primer pairs revealed that PS5 (341b4F-806R) was the most promising universal primer pair. This pair of primers detected both archaea and bacteria with less bias than PS4. Furthermore, an anaerobic fermentation culture and a wastewater treatment plant culture were used to verify the accuracy of PS5. More importantly, it detected DAMO archaea, DAMO bacteria, and anammox bacteria simultaneously with no false positives appeared. This universal 16S rRNA gene primer pair extends the existing molecular tools for studying the community structures and distributions of DAMO microbes and their potential interactions with anammox bacteria in different environments.

Cr(VI) reduction coupled with anaerobic oxidation of methane in a laboratory reactor

The process of anaerobic oxidation of methane (AOM) is globally important because of its contribution to the carbon cycle in the environment. Besides, microorganisms play important roles in the environmental fate of chromium. However, there have been no studies to date on the interaction between methane and chromium in batch reactor systems. In this study, biological Cr(VI) reduction was investigated using methane as the sole electron donor. Isotopic (13)CH4 in the batch experiments and long-term performance in the reactor demonstrated that Cr(VI) reduction is coupled with methane oxidation. High-throughput sequencing of the 16S rRNA genes demonstrated that the microbial community had changed substantially after Cr(VI) reduction. The populations of ANME-2d archaea were enhanced, and they became the only predominant AOM-related microbe. Interestingly, other bacteria with significant increases in abundance were not reported as having the ability to reduce Cr(VI). According to these results, two mechanisms were proposed: 1) Cr(VI) is reduced by ANME-2d alone; 2) Cr(VI) is reduced by unknown Cr(VI)-reducing microbes coupled with ANME-2d. This study revealed the potential relationship between Cr(VI) reduction and CH4 oxidation, and extended our knowledge of the relationship between the AOM process and biogeochemical cycles.

Multiple response optimization of the coagulation process for upgrading the quality of effluent from municipal wastewater treatment plant.

To meet the high quality standard of receiving water, the coagulation process using polyferric chloride (PFC) was used to further improve the water quality of effluent from wastewater treatment plants. Uniform design (UD) coupled with response surface methodology (RSM) was adopted to assess the effects of the main influence factors: coagulant dosage, pH and basicity, on the removal of total organic carbon (TOC), NH4+-N and PO43−-P. A desirability function approach was used to effectively optimize the coagulation process for the comprehensive removal of TOC, NH4+-N and PO43−-P to upgrade the effluent quality in practical application. The optimized operating conditions were: dosage 28 mg/L, pH 8.5 and basicity 0.001. The corresponding removal efficiencies for TOC, NH4+-N and PO43−-P were 77.2%, 94.6% and 20.8%, respectively. More importantly, the effluent quality could upgrade to surface water Class V of China through coagulation under optimal region. In addition, grey relational analysis (GRA) prioritized these three factors as: pH > basicity > dosage (for TOC), basicity > dosage > pH (for NH4+-N), pH > dosage > basicity (for PO43−-P), which would help identify the most important factor to control the treatment efficiency of various effluent quality indexes by PFC coagulation.

Tracking the activity of the Anammox-DAMO process using excitation–emission matrix (EEM) fluorescence spectroscopy

Coupling of anaerobic ammonium oxidation (anammox) and denitrifying anaerobic methane oxidation (DAMO) microorganisms in a hollow-fiber membrane biofilm reactor (HfMBR) is a potential strategy for simultaneous anaerobic removal of nitrogen and methane in wastewater streams. In these systems, effluents contain dissolved organic substances from anammox and DAMO microorganisms, but their characteristics and relationships have not been investigated. In the present study, excitation-emission matrix (EEM) fluorescence spectroscopy was used to characterize effluent dissolved organic matter (EfDOM) from an Anammox-DAMO HfMBR. Four component types (Component 1-4) were identified by parallel factor analysis (PARAFAC) of EEM data. Component 1 was produced when anammox and DAMO microorganisms simultaneously starved, whereas Component 4 was only generated through the starving period of DAMO microorganisms, and the longer the starving period, the higher the fluorescence intensity of the components. Components 2 and 3 were generated via active and starving periods of co-cultures. More efficient nitrogen removal was accompanied by a higher fluorescence intensity and microbial activity. Compared to measuring both influent and effluent nitrogen concentrations, monitoring EfDOM can obtain other information about the reactor, such as nitrogen removal activity of the reactor, status of the microbes and the duration of starving period the reactor suffered, which therefore offers a complementary but direct tool for assessing reactor performance in complex co-culture systems.

Hollow fiber membrane bioreactor affects microbial community and morphology of the DAMO and Anammox co-culture system

Denitrifying anaerobic methane oxidation (DAMO) and Anammox co-culture system was investigated in hollow fiber membrane bioreactor (HfMBR) for the change of microbial community morphology and proportion. NO3--N and NH4+-N removal rates reached 85.33 and 37.95mg/L/d on 193d. The inoculum microorganisms were flocs and the proportion of DAMO archaea, DAMO bacteria and Anammox bacteria was 11.0, 24.2 and 0.4%, respectively, but it changed to 74.3, 11.8, 5.6% in HfMBR, respectively. Interestingly, microorganisms formed biofilms on fibers surface and the biofilms included two layers: inner layer was thin and dominated by DAMO bacteria and Anammox bacteria; while the outer layer was thick made up of granules with 100-200μm diameter and dominated by DAMO archaea. The spatial distribution of microorganisms in HfMBR was different from simulation results in the literature. Likely, HfMBR changed the interaction between DAMO and Anammox microorganisms, and the reactor configuration was beneficial for DAMO archaea growth.

Investigation of Cr(VI) reduction potential and mechanism by Caldicellulosiruptor saccharolyticus under glucose fermentation condition

This study examined the microbial reduction of hexavalent chromium [Cr(VI)] by an extremely thermophilic bacterium, Caldicellulosiruptor saccharolyticus, under glucose fermentation conditions at 70°C. Experimentation with different initial Cr(VI) concentrations confirmed that C. saccharolyticus had the ability to reduce Cr(VI) and immobilize Cr(III). At a concentration of 40mg/L, Cr(VI) was completely reduced within 12h, and 97% of the reduction product Cr(III) precipitated on the cell surface. Cr(VI) reduction was accelerated by the addition of neutral red (NR, an electron mediator), resulting in the reduction time shortened to 1h. The addition of CuCl2, a Ni-Fe hydrogenase inhibitor, also enhanced Cr(VI) reduction. Additionally, analysis of the relationship between Cr(VI) reduction and glucose fermentation suggested that different electron sources acted during CuCl2 and NR conditions. Hydrogen served as an electron donor under normal fermentation and NR conditions with the catalysis of Ni-Fe hydrogenase. However, when the activity of Ni-Fe hydrogenase was inhibited by CuCl2, C. saccharolyticus directly used reduction equivalents during glucose fermentation for intracellular Cr(VI) reduction. Therefore, our findings demonstrated high Cr(VI) reduction ability and different electron transfer pathways during Cr(VI) reduction by C. saccharolyticus.

Free acetic acid as the key factor for the inhibition of hydrogenotrophic methanogenesis in mesophilic mixed culture fermentation

The inhibition of acetate under acidic pH is an ideal way to reduce methanogenesis in mesophilic mixed culture fermentation (MCF). However, the effects of acetate concentration and acidic pH on methanogenesis remain unclear. Besides, although hydrogenotrophic methanogens can be suitable targets in MCF, they are generally ignored. Therefore, we intentionally enriched hydrogenotrophic methanogens and found that free acetic acid (FAA, x) concentration and specific methanogenic activity (SMA, y) were correlated according to the equation: y = 0.86 × 0.31/(0.31 + x) (R2 = 0.909). The SMA was decreased by 50% and 90% at the FAA concentrations of 0.31 and 2.36 g/L, respectively. The coenzyme M concentration and relative electron transport activity agreed well with the FAA concentration. Moreover, the methanogenic activity could not be recovered when the FAA concentration exceeded 0.81 g/L. These findings indicated that neither acetate nor acidic pH, but FAA was the key factor to inhibit methanogenesis in MCF.

Mass transfer affects reactor performance, microbial morphology, and community succession in the methane-dependent denitrification and anaerobic ammonium oxidation co-culture

Denitrifying anaerobic methane oxidation (DAMO) combining anaerobic ammonium oxidation (Anammox) process is a novel nitrogen removal technology. However, the roles of methane transfer (gas phase) and nitrogen transfer (liquid phase) in the heterogeneous process remain unclear. In this study, granular DAMO and Anammox co-cultures were inoculated from a hollow-fiber membrane bioreactor into a sequence batch reactor (SBR). Since the methane transfer became limited in SBR, the nitrate removal rate first decreased and then increased to 10 mg/(L∙day), while the ammonium removal rate did not recover and was around 2 mg/(L∙day). The activity of DAMO archaea and Anammox bacteria decreased noticeably. Furthermore, granular aggregates dispersed into small granules and ultimately became flocs with poor settleability in SBR. The content of extracellular polymeric substances decreased, especially that of proteins and humics. DAMO archaea decreased by 94.6% and Anammox bacteria decreased by 72%. In summary, the limitation of methane transfer affected DAMO and Anammox processes more notably than nitrogen transfer, resulting in lower nitrogen removal, granule disruption, and microbial community succession.

Degradation of organic pollutants by anaerobic methane-oxidizing microorganisms using methyl orange as example

Anaerobic oxidation of methane (AOM) microorganisms widespread in nature and they are able to utilize methane as electron donor to reduce sulfate, nitrate, nitrite, and high valence metals. However, whether persistent organic contaminants can also be degraded remains unknown. In this study, the organic pollutant methyl orange (MO) was used to address this open question. The initial concentration of MO affected its degradation efficiency and higher concentration (>100 mg/L) caused considerable inhibition. A 13CH4 isotope experiment indicated that methane oxidation was involved in MO degradation, which produced N, N-dimethyl-p-phenylenediamine, and 4-aminobenzenesulfonic acid corresponded stoichiometrically. During the long-term experiment, the maximum degradation rate was 47.91 mg/(L·d). The percentage of Candidatus Methanoperedens and Pseudoxanthomonas significantly increased after 30-d of MO degradation under CH4 conditions; moreover, Candidatus Methanoperedens dominated (46.83%) the microbial community. Candidatus Methanoperedens, either alone or in combination with Pseudoxanthomonas, utilized methane as the sole carbon source to degrade MO via direct interspecies electron transfer or the syntrophy pathway. This study will add to our understanding of the functions and applications of AOM microorganisms.