Insights into the Response in Digestive Gland of Mytilus coruscus under Heat Stress Using TMT-Based Proteomics.

Ocean warming can cause injury and death in mussels and is believed to be one of the main reasons for extensive die-offs of mussel populations worldwide. However, the biological processes by which mussels respond to heat stress are still unclear. In this study, we conducted an analysis of enzyme activity and TMT-labelled based proteomic in the digestive gland tissue of Mytilus coruscus after exposure to high temperatures. Our results showed that the activities of superoxide dismutase, acid phosphatase, lactate dehydrogenase, and cellular content of lysozyme were significantly changed in response to heat stress. Furthermore, many differentially expressed proteins involved in nutrient digestion and absorption, p53, MAPK, apoptosis, and energy metabolism were activated post-heat stress. These results suggest that M. coruscus can respond to heat stress through the antioxidant system, the immune system, and anaerobic respiration. Additionally, M. coruscus may use fat, leucine, and isoleucine to meet energy requirements under high temperature stress via the TCA cycle pathway. These findings provide a useful reference for further exploration of the response mechanism to heat stress in marine mollusks.

Comparative transcriptomic analysis revealed changes in multiple signaling pathways involved in protein degradation in the digestive gland of Mytilus coruscus during high-temperatures.

As a result of global warming, the Mytilus coruscus living attached in the intertidal zone experience extreme and fluctuating changes in temperature, and extreme temperature changes are causing mass mortality of intertidal species. This study explores the transcriptional response of M. coruscus at different temperatures (18 °C, 26 °C, and 33 °C) and different times (0, 12, and 24 h) of action by analyzing the potential temperature of the intertidal zone. In response to high temperatures, several signaling pathways in M. coruscus, ribosome, endocytosis, endoplasmic reticulum stress, protein degradation, and lysosomes, interact to counter the adverse effects of high temperatures on protein homeostasis. Increased expression of key genes, including heat shock proteins (Hsp70, Hsp20, and Hsp110), Lysosome-associated membrane glycoprotein (LAMP), endoplasmic reticulum chaperone (BiP), and baculoviral IAP repeat-containing protein 7 (BIRC7), may further mitigate the effects of heat stress and delay mortality in M. coruscus. These results reveal changes in multiple signaling pathways involved in protein degradation during high-temperature stress, which will contribute to our overall understanding of the molecular mechanisms underlying the response of M. coruscus to high-temperature stress.

A novel simultaneous partial nitritation, denitratation and anammox (SPNDA) process in sequencing batch reactor for advanced nitrogen removal from ammonium and nitrate wastewater.

This study presented a novel simultaneous partial nitritation (PN), denitratation and anammox (SPNDA) process for treating ammonium and nitrate wastewater. Results indicated that SPNDA could achieve a great total nitrogen (TN) removal of 97.6 ± 0.5%, leading to effluent TN concentration of only 3.4 mg/L. Mass balance indicated that nitrogen removal rates via anammox, simultaneous nitrification and denitrification were 96.7% and 3.3%, respectively. Extended aerobic duration (12 h) and low dissolved oxygen (DO) concentration (0.15 mg/L) could improve ammonia-oxidizing bacteria (AOB) activity and maintain PN stability. The stable suppression of nitrite-oxidizing bacteria activity was attributed to the low DO (0.15 mg/L) and high free ammonia (3.63 mg/L) in SPND. Besides, the nitrogen conversion mechanisms for SPNDA were revealed based on a typical operational cycle. Microbial analysis showed that AOB (Nitrosomonas) and partial denitrifying bacteria (Thauera and Denitratisoma) coexisted with anammox bacteria (Candidatus Brocadia and Candidatus Anammoxoglobus) in the mixotrophic bio-community.

Hydroxylamine metabolism in mainstream denitrifying ammonium oxidation (DEAMOX) process: Achieving fast start-up and robust operation with bio-augmentation assistance under ambient temperature.

Nitrogen removal from mainstream wastewater via DEnitrifying AMmonium OXidation (DEAMOX) is often challenged by undulated actual temperature and high loading rate. Here, we discovered NH2OH addition (HA) and bio-augmentation (BA) tactics on start-up and operation performance of DEAMOXs (R1 and R2) under ambient temperature (11.3-31.7 °C). Over 340-day operation suggested that R2 received 10 mg/L HA and 1:25 BA ratio (v/v, anammox/partial denitrification sludge) achieved desirable nitrogen removal efficiency (NRE) of 97.22% after 145-day, while R1 under higher BA ratio of 1:12.5 without HA obtained lower NRE (90.86%) after 184-day. Batch tests revealed that nitrate-nitrite transformation ratio reached 98.64% at low COD/NO3--N of 2.6 with HA. Significantly, compared with R2, R1 recovered quickly with satisfactory effluent total nitrogen of 4.21 mg/L despite nitrogen loading rate greater than 0.15 kg N/m3/d and temperature decreased to 14.6 °C. The abundant narG represented high nitrate reduction potential, hzsA and hdh were extensively detected as the symbolisation of anammox metabolism. Thauera, Denitratisoma and unclassified f Comamonadaceae dominated nitrite accumulation. Ca. Brocadia as the dominant anammox bacteria, and its population maintained stable against low temperature and load shocks by NH2OH intensification. Overall, this study offers an opportunity for the wide-applications of DEAMOX treating mainstream wastewater.

Development of a novel denitrifying phosphorus removal and partial denitrification anammox (DPR + PDA) process for advanced nitrogen and phosphorus removal from domestic and nitrate wastewaters.

A novel energy-efficient DPR + PDA (denitrifying phosphorus removal and partial denitrification anammox) process for enhanced nitrogen and phosphorus removal was developed in the combined ABR-CSTR reactor. After 220 days operation, excellent total inorganic nitrogen (TIN) and phosphorus removal (97.57% and 95.66%, respectively) were obtained under external C/NO3--N of 0.7, with the effluent TIN and PO43--P concentrations of 3.51 mg/L and 0.28 mg/L, respectively. At the steady period, DPR contributed major TN removal (58.65%), while PDA mediated an increasingly considerable impact and finally achieved 37.07%, in which anammox accounted for a significant percentage. Batch tests demonstrated that efficient PD with nitrate-to-nitrite transformation ratio of 97.67% supplying stable nitrite for anammox, and phosphorus was mainly removed using nitrate as electron acceptor via DPR with the ideal phosphorus release/uptake rate (7.73/22.17 mgP/gVSS/h). Accumulibacter (6.24%) dominated high phosphorus removal performance, while Thauera (8.26%) and Candidatus Brocadia (2.57%) represented the superior nitrogen removal performance.

A novel denitrifying phosphorus removal and partial nitrification, anammox (DPR-PNA) process for advanced nutrients removal from high-strength wastewater.

This study developed a novel DPR-PNA (denitrifying phosphorus removal, partial nitrification and anammox) process for sustaining high-strength wastewater treatment in a modified continuous flow reactor without external carbon source. After 259-days operation, a synchronous highly-efficient total inorganic nitrogen, PO43--P and CODcr removal efficiencies of 88.5%, 89.5% and 90.1% were obtained, respectively even influent nitrogen loading rate up to 3.2 kg m-3 d-1. Batch tests revealed that denitrifying phosphorus accumulating organisms (DPAOs) using NO3--N as electron acceptors significantly enriched (74% in total PAOs), which emerged remarkable positive impacts on deep-level nutrient removal as the key limiting factor. Furthermore, the NO2--N inhibitory threshold value (∼20.0 mg L-1) for DPAOs was identified, which demonstrated as an inhibitory component in excessive recycling NOx--N. From the molecular biology perspective, Dechloromonas-DPAOs group (18.59%) dominated the excellent dephosphatation performance, while Nitrosomonas-AOB (ammonia oxidizing bacteria) group (16.26%) and Candidatus_Brocadia-AnAOB (anammox bacteria) group (15.12%) were responsible for the desirable nitrogen loss process. Overall, the present work highlighted the novel DPR-PNA process for nutrients removal is a promising alternation for wastewater of high nitrogen but low carbon.

[Rapid Start-up and Stability of Partial Denitrification Based on Different Waste Sludge Sources].

To explore the feasibility of the rapid start-up of partial denitrification and the stable accumulation of NO2--N in different waste sludge sources, three identical SBR reactors (S1, S2, and S3) were inoculated respectively with sludge discharged from a laboratory municipal wastewater denitrifying phosphorus removal system, surplus sludge from a municipal wastewater treatment plant, and river sediment sludge. The characteristics of the partial denitrification start-up and NO2--N accumulation were compared, and the partial denitrification activity of the system or NO3--N→NO2--N transformation performance were investigated by analyzing the characteristics of the functional bacteria genera of the reactor from the perspective of microbiology. The results showed that all three SBR partial denitrification reactors could be launched successfully in a short time with sodium acetate as the sole carbon source, under a high alkalinity, and by using a suitable COD/NO3--N ratio. The average NO3--N→NO2--N transformation ratio of the system was ranked as:S1 > S2 > S3 (75.92% > 73.36% > 69.90%). It was found that S1 and S2 had different degrees of partial denitrification performance deterioration under a continuous low temperature, but that S3 could maintain a good NO2--N accumulation performance. High throughput sequencing showed that Proteobacteria and Bacteroidetes were dominant in the partial denitrification system, and that the abundance of Thauera was significantly different in the three PD reactors:S3 > S1 > S2 (25.09% > 4.71% > 3.60%), thus indicating that S3 had stable and efficient NO2--N accumulation performance and that a high abundance of Thauera might play a significant role in maintaining low temperature partial denitrification activity.

Enhancement of nitrite production via addition of hydroxylamine to partial denitrification (PD) biomass: Functional genes dynamics and enzymatic activities.

This study investigated the activity of partial denitrification (PD) biomass/key enzymes, functional gene expressions in response to 0 ~ 50 mg/L hydroxylamine (NH2OH) addition. Results indicated that NH2OH contributed to nitrite (NO2--N) production, facilitating the maximum increase of nitrate (NO3--N) to NO2--N transformation ratio to 80.47 ± 2.82%, leading to 2.56-fold NO2--N higher than those of control. The observed transient inhibitory effect on NO3--N reduction process was attributed by high-level NH2OH (35 ~ 50 mg/L). Enzymatic assays revealed the enhanced activity of both NO3--N and NO2--N reductase while the former showed obvious superiority which led to high NO2--N accumulation. These results were further confirmed by the corresponding functional genes (narG, napA, nirS and nirK). Besides, negative influence of NH2OH addition was limited to PD aggregates, due to the increasing secretion of extracellular polymeric substances (EPS) as well as proteins/polysaccharides ratios in tightly-bound structure of EPS.

[Start-up of an Integrated Process of Denitrifying Phosphorus Removal Coupled with Partial Nitritation and Anaerobic Ammonium Oxidation].

An integrated process uses an anaerobic baffled reactor combined with a fully mixed reactor (ABR-CSTR) as a test carrier for low-carbon, high-ammonia nitrogen (NH4+-N ≥ 200 mg·L-1) wastewater under continuous flow operating conditions; the normal anaerobic sludge in different compartments is subjected to domestication and cultivation to realize denitrifying phosphorus removal, partial nitritation, and anaerobic ammonium oxidation, thereby achieving the coupling effect of the three. Partial nitritation was successfully achieved in the A4 (CSTR) section by the strategy of limited oxygen (dissolved oxygen DO=0.8 mg·L-1) and intermittent aeration (exposure ratio=30 min:30 min) after 30 days. Subsequently, a strategy of shortening the hydraulic retention time (HRT) was adopted to achieve a stable operation of partial nitritation, and a stable influent substrate of NO2--N/NH4+-N 1.0-1.1 was provided for anaerobic ammonium oxidation. The anaerobic ammonium oxidation function was achieved after 154 days in the A5 and A6 compartments. The removal rates of NH4+-N and NO2--N were 94% and 97%, respectively, and the NO3--N concentration in the effluent was stable at 22 mg·L-1. The denitrifying phosphorus removal function was successfully achieved in the A1-A3 compartments by using NOx--N in the reflux as an electron acceptor. The removal rate of PO43--P was 77%. The integrated process was successfully coupled through 175 days, achieving simultaneous removal of C, N, and P.

New insights on biological nutrient removal by coupling biofilm-based CANON and denitrifying phosphorus removal (CANDPR) process: Long-term stability assessment and microbial community evolution.

It was difficult to obtain a stable and efficient biological nutrient removal for high-strength wastewater treatment, the possibility of exploiting innovative CANDPR process, integrating biofilm-based completely autotrophic nitrogen removal over nitrite (CANON) with denitrifying phosphorus removal (DPR) was evaluated to resolve the difficulty. Results revealed that the excellent NH4+-N, PO43--P and COD removal efficiencies of 96%, 96% and 91%, were achieved respectively under a high nitrogen loading rate (0.79 kg·m-3·d-1) without adding organic matters during 320 days operation. Promoting NOx--N recirculation demonstrated as an efficient strategy for further nutrient depletion, facilitating the enhanced NO3--N removal to 100% with the considerably high P-uptake performance. Batch tests confirmed that denitrifying phosphorus accumulating organisms (DPAOs) using NO3--N as electron acceptors accounting for 68% in total PAOs. Dechloromonas was identified as dominating genus in DPR, while Nitrosomonas (1.31%), Candidatus_Kuenenia (5.53%) and Candidatus_Brocadia (1.77%) contributed to the desirable nitrogen removal, indicating that cooperative consortia of DPAOs, AOB and AnAOB were harvested during long-term operation. The CANDPR process was verified to be energy-saving and treatment-reliable for renovating of existing plants.

[Start-up and Stable Operation of CANON Coupled with Denitrifying Phosphorus Removal].

A process coupled completely autotrophic nitrogen removal over nitrite (CANON)with denitrifying phosphorus removal in a modified anaerobic baffled reactor (ABR) coupled with a membrane bioreactor (MBR), inoculated with ordinary activated sludge, was proposed for treating artificial wastewater with ammonia 200 mg·L-1 and COD/TN=1. This experiment studied the start-up of the process and its nitrogen and phosphorus removal efficiency by controlling the recycle ratio and increasing it from 50% to 200% step by step, with a temperature of (25±1)℃ and pH of 7.5±0.2. The results showed that the anaerobic part in the ABR consumed 70% COD, and resulted in a quick start-up of partial-nitrification at 21 d under low DO and high ammonia nitrogen. Then, by controlling the intermittent aeration (exposure stop ratio:2 h:2 h, DO 0.3-0.4 mg·L-1), the start-up of the CANON part in the coupling process was successfully achieved at 132 d, such that the concentration of nitrates in the electron acceptor of the ABR anoxic section increased steadily, and finally the coupling process started successfully at 160 d. With stable operation, the TN removal load in the MBR reached 0.22 kg·(m3·d)-1, and the average removal efficiency of COD, TN, and PO43--P was 87.0%, 90.4%, and 81.8%, respectively. The batch experiment estimated that the denitrifying phosphate accumulating organisms (DPAOs) using nitrates as electron acceptors in the ABR accounted for 68% of the phosphate accumulating organisms (PAOs). The DPAOs, ammonia-oxidizing bacteria (AOB), and anaerobic ammonium oxidizing bacteria (AnAOB) have been developed in the system and have good simultaneous nitrogen and phosphorus removal efficiency.

[Efficiency and Mechanism of Nitrogen and Phosphorus Removal in Modified Zeolite Wetland].

To study the efficiency and mechanism of nitrogen and phosphorus removal for decentralized rural sewage in modified zeolite wetland, the modified zeolite was applied as substrate into a combined process composed of anaerobic baffled reactor (ABR) and baffled flow constructed wetland (BFCW), providing a new way for rural sewage treatment in Suzhou City. The study was contrasted with zeolite wetland. The results showed that the modified zeolite wetland had high efficiency and stability of nitrogen and phosphorus removal, and the nitrogen and phosphorus removal quantities of modified zeolite wetland were 1.8% and 1 times higher than those of zeolite wetland during the trial. The modified zeolite wetland mainly removed nitrogen and phosphorus by substrate adsorption, and the main fractions of modified zeolite were Ca-P and Al-P. The oxygen-secretion and absorption of plants stabilized the water quality of the effluent. The substrate adsorption was the main nitrification removal pathway in front of the wetland, and nitrification and denitrification were the main nitrification removal pathways at the end of the wetland. The nitrogen and phosphorus adsorption capacities during the pilot test were much higher than those of the static test. The optimization of phosphorus adsorption capacity for modified zeolite was achieved under the synergy of multiple pathways. The effect of configuration and plant root was the main reason for the difference of nitrogen and phosphorus adsorption quantities. Nitrification intensity led to the seasonal fluctuation of nitrogen removal effect and stability in modified zeolite wetland, and the low nitrification intensity in the front of wetland was related to the strong adsorption of NH4+-N by the modified zeolite.

[Effect of Substrate Ratio on Nitrogen Removal Performance of ANAMMOX in ABR].

In order to solve the problem of low nitrogen removal caused by incomplete removal of anaerobic ammonium oxidation (ANAMMOX) substrate, The nitrogen removal performance of the ANAMMOX was investigated by controlling different influent substrate ratios in an anaerobic baffled reactor (ABR). The result showed the optimal influent NO2--N/NH4+-N was 1.34 with which the NH4+-N and NO2--N removal efficiencies were about 99.99% and the total nitrogen removal efficiency reached a peak of 87%. When the influent NO2--N/NH4+-N gradually reduced from 1 to 0.49 and increased from 1.34 to 1.62, the absolute removal of NH4+-N and NO2--N was stable in the reactor and no significant inhibition was observed in the system. Under the condition of different substrate ratios, the removal of NH4+-N and NO2--N was basically consumed in the first compartment of ABR, the change of substrate ratio did not have an obvious impact on the nitrogen removal performance of each compartment in the ABR, thus, the ABR ANAMMOX system had good stability to the change of substrate concentration.

[Quick Start-up Performance of Combined ANAMMOX Reactor Based on Different Inoculated Sludge Types].

In order to determine the optimal sludge source of anaerobic ammonium oxidation (ANAMMOX) and the rapid formation of ANAMMOX granular sludge, two CAMBRs (combined ABR and MBR) were compared for ANAMMOX enrichment with different inoculated sludge types, the anaerobic granular sludge (R1) and flocculent denitrifying sludge (R2). The results showed that ANAMMOX was successfully initiated after 45 d (R1) and 60 d (R2) in both reactors, respectively. The enrichment processes are divided into three different phases, lag phase, activity elevation phase, and stationary phase but the removal rules of nitrogen in each phase were different. In the steady phase, the average removal rates of NH4+-N and NO2--N were higher than 95%. In addition, the red ANAMMOX granular sludge with the main diameter of 0.8-1.6 mm was formed in R1 while the flocculent sludge and irregular block with a low degree of granulation were mainly developed in R2. The phenomenon of red granular sludge floating in the two reactors was also observed. The quantitative relationship analysis between NH4+-N, NO2-N, and NO3--N showed the occurrence of nitrate-dependent ANAMMOX, which resulted in the oxidation of excess ammonia and the typical nitrite-dependent ANAMMOX occurred in R2.

[Quick Start-up of Anaerobic Ammonium Oxidation Process].

In order to study the quick start-up process of anaerobic ammonium oxidation (ANAMMOX), two types of reactors with different hydraclic flow state inoculated with aerobic nitrifying sludge were investigated, the membrane bioreactor (MBR) and anaerobic baffled reactor (ABR), respectively. The result showed that both reactors successfully started up ANAMMOX process. The start-up period of the MBR (90 d) was 20% shorter than the ABR (111 d). During the stable operation, the average nitrogen (NH4+-N+NO2--N) removal capacity of 0.098 kg·(m3·d)-1 in the MBR was also higher than that of 0.089 kg·(m3·d)-1 in the ABR. In addition, the differences of sludge morphology were obvious in the two reactors; flocculent sludge was developed in the MBR while ANAMMOX granular sludge was mainly formed in the first compartment of the ABR. Moreover, the quantitative relationship analysis between NH4+-N, NO2--N and NO3--N showed that the MBR system contained more kinds of bacteria with nitrogen removal function, compared to the ABR, and it was therefore more conducive to the removal of nitrogen. MBR exhibited a more excellent performance for ANAMMOX start-up.

Partial Nitrification and Denitrifying Phosphorus Removal in a Pilot-Scale ABR/MBR Combined Process.

A pilot-scale combined process consisting of an anaerobic baffled reactor (ABR) and an aerobic membrane bioreactor (MBR) for the purpose of achieving easy management, low energy demands, and high efficiencies on nutrient removal from municipal wastewater was investigated. The process operated at room temperature with hydraulic retention time (HRT) of 7.5 h, recycle ratio 1 of 200%, recycle ratio 2 of 100%, and dissolved oxygen (DO) of 1 mg/L and achieved good effluent quality with chemical oxygen demand (COD) of 25 mg/L, NH4 (+)-N of 4 mg/L, total nitrogen (TN) of 11 mg/L, and total phosphorus (TP) of 0.7 mg/L. The MBR achieved partial nitrification, and NO2 (-)-N has been accumulated (4 mg/L). Efficient short-cut denitrification was occurred in the ABR with a TN removal efficiency of 51%, while the role of denitrification and phosphorus removal removed partial TN (14%). Furthermore, nitrogen was further removed (11%) by simultaneous nitrification and denitrification in the MBR. In addition, phosphorus accumulating organisms in the MBR sufficiently uptake phosphorus; thus, effluent TP further reduced with a TP removal efficiency of 84%. Analysis of fluorescence in situ hybridization (FISH) showed that ammonia oxidizing bacteria (AOB) and phosphorus accumulating organisms (PAOs) were enriched in the process. In addition, the accumulation of NO2 (-)-N was contributed to the inhibition on the activities of the NOB rather than its elimination.