Isolation of Denitrifying Bacteria in Sea Sediment and Simulated Experiment of Removing Nitrate-N from Seawater

The objective of this research was to isolate denitrifying bacteria from sea sediment and simulate the removal efficiency of nitrate-N by denitrifying bacteria from seawater. The result showed that the isolated denitrifying bacteria could effectively remove nitrate-N from seawater. About 90 % of nitrate-N was removed by denitrifying bacteria from seawater within a week in the simulated experiment I （the initial concentration of nitrate-N was 100 mg/L）. The removal efficiency of nitrate-N reached about 70 % within one day in the simulated experiment Ⅱ （initial concentration of nitrate-N was 1 mg/L）. The final removal efficiency was about 98 % and 85 % in the simulated experiments Ⅰ and Ⅱ, respectively. It was found that there was positive correlation between the concentration of nitrate-N and the number of denitrifying bacteria in seawater. Lots of denitrifying bacteria would disappear and the seawater would become transparent once the process of bioremediation was completed.

Impact of Long-Term Fertilization on Community Structure of Ammonia Oxidizing and Denitrifying Bacteria Based on amoA and nirK Genes in a Rice Paddy from Tai Lake Region,China

Ammonia oxidizing （AOB） and denitrifying bacteria （DNB） play an important role in soil nitrogen transformation in natural and agricultural ecosystems. Effects of long-term fertilization on abundance and community composition of AOB and DNB were studied with targeting ammonia monooxygenase （amoA） and nitrite reductase （nirK） genes using polymerase chain reaction- denaturing gradient gel electrophoresis （PCR-DGGE） and real-time PCR, respectively. A field trial with different fertilization treatments in a rice paddy from Tai Lake region, centre East China was used in this study, including no fertilizer application （NF）, balanced chemical fertilizers （CF）, combined organic/inorganic fertilizer of balanced chemical fertilizers plus pig manure （CFM）, and plus rice straw return （CFS）. The abundances and riehnesses of amoA and nirK were increased in CF, CFM and CFS compared to NF. Principle component analysis of DGGE profiles showed significant difference in nirK and amoA genes composition between organic amended （CFS and CFM） and the non-organic amended （CF and NF） plots. Number of amoA copies was significantly positively correlated with normalized soil nutrient richness （NSNR） of soil organic carbon （SOC） and total nitrogen （T-N）, and that of nirK copies was with NSNR of SOC, T-N plus total phosphorus. Moreover, nitrification potential showed a positive correlation with SOC content, while a significantly lower denitrification potential was found under CFM compared to under CFS. Therefore, SOC accumulation accompanied with soil nutrient richness under long-term balanced and organic/inorganic combined fertilization promoted abundance and diversity of AOB and DNB in the rice paddy.

Denitrifying Bacteria in Paddy Soils of the Taihu Lake Basin,China

This study attempted to determine the characteristics of the communities, the ecological factors, and the denitrifying enzyme activity for denitrifying bacteria found in the paddy soils of the Taihu Lake Basin, China. Samples of the six main soil types of the basin were taken from paddy fields with different fertilities. The total numbers of bacteria and denitrifying bacteria in the high fertility soils were much more than those in low fertility soils, and the number of denitrifying bacteria accounted for 49% to 80% of the total number of bacteria. The O2 content was an important ecological factor that affected denitrification. Of test the strains isolated from the paddy soils in the Taihu Lake Basin, some (e.g., Pseudomonas spp.) grew well under low oxygen partial pressure, while others (e.g., Bacillus spp.) had no strict predilection with O2 content. Another critical ecological factor was the nitrogen concentration. Three selected denitrifying bacteria grew better in aculture medium with 135 instead of 276mg L^-1 nitrogen. At the same time 67% of the test strains were able to reduce NO3^- to NO2^- and 56% had N2O reductase.

Community structure and assembly of denitrifying bacteria in epiphytic biofilms in a freshwater lake ecosystem

Denitrifying bacteria are a crucial component of aquatic ecosystem in nitrogen cycle.However,the denitrifying bacterial community dynamics and structure in epiphytic biofilms remain unexplored.The abundance of denitrification gene(nir)and structure of nirS-denitrifying bacterial community in the epiphytic biofilms collected in July and November of 2018 from a typical plateau lake(Caohai Wetland,Guizhou,China)were studied by Real-time Quantitative Polymerase Chain Reaction(qPCR)and highthroughput sequencing.Results show that the gene abundance of nirK was higher than that of nirS(P<0.05),and it was significantly different during the growth period(July)than the decline period(November).The denitrifying bacterial species was similar in the two months and shared 76.18%of OTUs.Proteobacteria(56.55%±22.15%)was the dominant phylum in all the samples.Epiphytic biofilms between growth period and decline period displayed significantly different microbial community structures due to differences in species abundance.Water temperature was the crucial factor that affected the denitrifying microbial community structure in our study.Environmental factors explain only partially the dynamic characteristics of denitrifying microbial communities,implying that the stochastic processes affected the construction of denitrifying microbial communities.As the null model analysis results show,dispersal limitation(stochastic)and undominated processes significantly influenced the assembly of denitrifying microbial communities.This study broadened our understanding of the denitrifying bacterial community structure and its function on epiphytic biofilms in freshwater ecosystems with new information provided.

Temporal characteristics of algae-denitrifying bacteria co-occurrence patterns and denitrifier assembly in epiphytic biofilms on submerged macrophytes in Caohai Lake,SW China

Denitrifying bacteria in epiphytic biofilms play a crucial role in nitrogen cycle in aquatic habitats.However,little is known about the connection between algae and denitrifying bacteria and their assembly processes in epiphytic biofilms.Epiphytic biofilms were collected from submerged macrophytes(Patamogeton lucens and Najas marina L.)in the Caohai Lake,Guizhou,SW China,from July to November 2020 to:(1)investigate the impact of abiotic and biotic variables on denitrifying bacterial communities;(2)investigate the temporal variation of the algae-denitrifying bacteria co-occurrence networks;and(3)determine the contribution of deterministic and stochastic processes to the formation of denitrifying bacterial communities.Abiotic and biotic factors influenced the variation in the denitrifying bacterial community,as shown in the Mantel test.The co-occurrence network analysis unveiled intricate interactions among algae to denitrifying bacteria.Denitrifying bacterial community co-occurrence network complexity(larger average degrees representing stronger network complexity)increased continuously from July to September and decreased in October before increasing in November.The co-occurrence network complexity of the algae and nirS-encoding denitrifying bacteria tended to increase from July to November.The co-occurrence network complexity of the algal and denitrifying bacterial communities was modified by ammonia nitrogen(NH_(4)^(+)-N)and total phosphorus(TP),pH,and water temperature(WT),according to the ordinary least-squares(OLS)model.The modified stochasticity ratio(MST)results reveal that deterministic selection dominated the assembly of denitrifying bacterial communities.The influence of environmental variables to denitrifying bacterial communities,as well as characteristics of algal-bacterial co-occurrence networks and the assembly process of denitrifying bacterial communities,were discovered in epiphytic biofilms in this study.The findings could aid in the appropriate understanding and use of epiphytic biofilms denitrification function,as well as the enhancement of water quality.

Effect of Zinc Oxide Nanoparticles on Denitrification and Denitrifying Bacteria Communities in Typical Estuarine Sediments

For revealing the effects of increasing of zinc oxide nanoparticles(ZnO NPs)on denitrification and denitrifying bacteria communities in estuarine sediments,the surface sediments of two typical estuaries(the Yangtze River Estuary and the Yellow River Estuary)were added with medium concentration(170mgL−1)and high concentration(1700mgL−1)of ZnO NPs for anaerobic cul-ture in laboratory.The concentration of NO_(3)^(−)and NO_(2)^(−),the reductase activity and denitrification rate were measured by physico-chemical analysis,nirS gene abundance and denitrifying bacteria communities by molecular biological methods.The results showed that ZnO NPs inhibited NO_(3)^(−), NO_(2)^(−)reduction process and NO_(3)^(−), NO_(2)^(−)reductase activity,and a stronger inhibition effect resulting from the higher ZnO NPs concentration.ZnO NPs decreased nirS gene abundance and community diversity of denitrifying bacteria.In addition,the inhibition degree of ZnO NPs on the denitrification process of sediments in different estuaries was different.These results were of great significance for evaluating the potential ecological toxicity and risks of nanomaterials in estuaries.

Identification and Metabolic Mechanism of Non-fermentative Short-cut Denitrifying Phosphorus-removing Bacteria

To investigate the characteristics and metabolic mechanism of short-cut denitrifying phospho- rus-removing bacteria （SDPB） that are capable of enhanced biological phosphorus removal （EBPR） using nitrite as an electron acceptor, an aerobic/anoxic sequencing batch reactor was operated under three phases. An SDPB-strain YC was screened after the sludge enrichment and was identified by morphological, physiological, biochemical properties and 16S rDNA gene sequence analysis. Denitrifying phosphorus-removing experiments were conducted to study anaerobic and anoxic metabolic mechanisms by analyzing the changes of chemical oxygen demand （COD）, phosphate, nitrite, poly-fl-hydroxybutyrate （PHB）, and glycogen. The results show that strain YC is a non-fermentative SDPB similar to Paracoccus denitrificans. As a kind of non-fermentative bacteria, the energy of strain YC was mainly generated from phosphorus release （96.2%） under anaerobic conditions with 0.32 mg P per mg synthesized PHB. Under anoxic conditions, strain YC accumulated 0.45 mg P per mg degraded PHB, which produced most of energy for phosphate accumulation （91.3%） and a little for glycogen synthesis （8.7%）. This metabolic mechanism of strain YC is different from that of traditional phosphorus-accumulating organisms （PAOs）. It is also found that PHB, a kind of intracellular polymer, plays a very important role in denitrifying and accumulating phosphorus by supplying sufficient energy for phosphorous accumulation and carbon sources for denitrification. Therefore, monitoring AP/APHB and ANO2 -N/APHB is more necessary than monitoring AP/ACOD, ANO2 -N/ACOD, or AP / ANO2 -N.

Operation of three parallel AN/AO processes to enrich denitrifying phosphorus removing bacteria for low strength wastewater treatment

Three parallel anaerobic-anoxic/anaerobic-aerobic （AN/AO） processes were developed to enrich denitrifying phosphorus removal bacteria （DPB） for low strength wastewater treatment. The main body of the parallel AN/AO process consists of an AN （anaerobic-anoxic） process and an AO （anaerobic-aerobic） process. In the AO process, the common phosphorus accumulating organisms （PAOs） was dominate, while in the AN process, DPB was dominate, The volume of anaerobic zone（Vana）：anoxie zone（Vano） ： aerobic zone （Vaer） for the parallel AN/AO process is 1：1：1 in contrast with a Vana：Vaer and Vano：Vaer of 1：2 and 1：4 for a traditional biological nutrient removal process （BNR）. Process 3 excels in the 3 processes on the basis of COD, TN and TP removal. For 4 month operation, the effluent COD concentration of process 3 did not exceed 60 mg/L; the effluent TN concentration of process 3 was lower than 15 mg/L; and the effluent TP concentration of process 3 was lower than 1 mg/L.

Effect of carbon source and nitrate concentration on denitrifying phosphorus removal by DPB sludge

Effect of added carbon source and nitrate concentration on the denitrifying phosphorus removal by DPB sludge was systematically studied using batch experiments, at the same time the variation of ORP was investigated. Results showed that the denitrifying and phosphorus uptake rate in anoxic phase increased with the high initial anaerobic carbon source addition. However once the initial COD concentration reached a certain level, which was in excess to the PHB saturation of poly-P bacteria, residual COD carried over to anoxic phase inhibited the subsequent denitrifying phosphorus uptake. Simultaneously, phosphate uptake continued until all nitrate was removed, following a slow endogenous release of phosphate. High nitrate concentration in anoxic phase increased the initial denitrifying phosphorus rate. Once the nitrate was exhausted, phosphate uptake changed to release. Moreover, the time of this turning point occurred later with the higher nitrate addition. On the other hand, through on-line monitoring the variation of the ORP with different initial COD concentration, it was found ORP could be used as a control parameter for phosphorus release, but it is impossible to utilize ORP for controlling the denitrificaion and anoxic phosphorus uptake operations.

Denitrifying phosphorous removal in anaerobic/anoxic SBR system with different startup operation mode

To achieve stable and efficient nitrogen and phosphorus removal and to investigate the characteristics of the A/A SBR enriched with denitrifying phosphorus removal bacteria(DPB),the whole course of startup was studied with two reactors operated in different mode.The reactor I was operated under anaerobic/settling/anoxic/settling mode,and the reactor II was operated under anaerobic/anoxic/settling mode.Differences between the two reactors in removal efficiency of COD,nitrogen and phosphorus were examined.The results indicated that efficient performance could be achieved in both reactors with different startup operation mode,while the phosphorus removal efficiency was improved sooner in reactor I than in reactor II,which suggested that reactor I would supply a more favorable condition for DPB proliferation.Meanwhile,it was observed that the amount of organic substrates consumption had a linear correlation to that of phosphorus release in anaerobic phase when DPB was accumulated in the A/A SBR denitrifying phosphorus removal system.

Preliminary investigation on the role of microorganisms in the production of phosphine

The relationships between the phosphine content and various microbial populations,activities of different enzymes were investigated firstly.The results indicated that the phosphine content of samples from various environments was positively related to total anaerobic microorganisms,organic phosphate compound-dissolving bacteria,denitrifying bacteria,and the activities of alkaline phosphatase and dehydrogenase,with correlation coefficients (R^2) up to 0.93,0.90,0.69,0.79,and 0.82,respectively.Results also sh...

Isolation and characteristics of Arthrobacter sp.strain CW-1 for biodegradation of PAEs

Isolation of new bacterial strains and recognition of their metabolic activities are highly desirable for sustainability of natural ecosystems. Biodegradation of dimethyl phthalate （DMP） under anoxic conditions has been shown to occur as a series of sequential steps using strain CW-1 isolated from digested sludge of Sibao Wastewater Treatment Plant in Hangzhou, China. The microbial colony on LB medium was yellowish, 3-5 mm in diameter, convex in the center, and embedded in mucous externally. The individual cells of strain CW-1 are irregular rods, measuring （0.6-0.7）×（0.9-1.0） pm, V-shaped, with clubbed ends, Gram positive and without any filaments. 16S rDNA （ 1438 bp） sequence analysis showed that the strain was related to Arthrobacter sp. CW-1 and can degrade PAEs utilizing nitrate as electron acceptor, but cannot mineralize DMP completely. The degradation pathway was recommended as： dimethyl phthalate （DMP）→monomethyl phthalate （MMP）--,phthalic acid （PA）. DMP biodegradation was a first order reaction with degradation rate constant of 0.3033 d 1 and half-life 2.25 d. The DMP conversion to PA by CW-1 could be described by using sequential kinetic model.

Rapid aerobic granulation using biochar for the treatment of petroleum refinery wastewater

Aerobic granular sludge technology has great potential for the treatment of petroleum refinery wastewater.However,strategies to shorten the granulation time and improvement the stability still need to be developed.In this work,biochar was prepared from waste petroleum activated sludge(biochar-WPS) and used in a sequencing batch reactor for the treatment of petroleum refinery wastewater.Biochar-WPS presented the surface area of 229.77 m2/g,pore volume of 0.28 cm3/g,H/C and O/C atomic ratios of 0.42 and 0.21,respectively.The porous structure and a high degree of hydrophilicity were found to facilitate microbial colonization and adhesion as well as particle aggregation.Application of biochar-WPS resulted in the formation of more substantial and stable aerobic granules(~66% of granules> 0.46 mm diameter) 15 days earlier compared with the control.The addition of biochar-WPS enhanced the average removal efficiency of chemical organic demand(~3%),oil(~4%)and total nitrogen(~10%) over the control.Increased microbial richness and diversity were observed within the formed granules and had an increased(~4%) proportion of denitrifying bacteria.These results indicate that an aerobic granulation mechanism using biochar-WPS is a feasible option for the treatment of petroleum refinery wastewater.

Characteristics of anoxic phosphors removal in sequence batch reactor

The characteristics of anaerobic phosphorus release and anoxic phosphorus uptake were investigated in sequencing batch reactors using denitrifying phosphorus removing bacteria （DPB） sludge. The lab-scale experiments were accomplished under conditions of various nitrite concentrations （5.5, 9.5, and 15 mg/L） and mixed liquor suspended solids （MLSS） （1844, 3231, and 6730 mg/L）. The results obtained confirmed that nitrite, MLSS, and pH were key factors, which had a significant impact on anaerobic phosphorus release and anoxic phosphorus uptake in the biological phosphorous removal process. The nitrites were able to successfully act as electron acceptors for phosphorous uptake at a limited concentration between 5.5 and 9.5 mg/L. The denitrification and dephosphorous were inhibited when the nitrite concentration reached 15 mg/L. This observation indicated that the nitrite would not inhibit phosphorus uptake before it exceeded a threshold concentration. It was assumed that an increase of MLSS concentration from 1844 mg/L to 6730 mg/L led to the increase of denitrification and anoxic P-uptake rate. On the contrary, the average P-uptake/N denitrifying reduced from 2.10 to 1.57 mg PO4^3--P/mg NO3^--N. Therefore, it could be concluded that increasing MLSS of the DEPHANOX system might shorten the reaction time of phosphorus release and anoxic phosphorus uptake. However, excessive MLSS might reduce the specific denitrifying rate. Meanwhile, a rapid pH increase occurred at the beginning of the anoxic conditions as a result of denitrification and anoxic phosphate uptake. Anaerobic P release rate increased with an increase in pH. Moreover, when pH exceeded a relatively high value of 8.0, the dissolved P concentration decreased in the liquid phase, because of chemical precipitation. This observation suggested that pH should be strictly controlled below 8.0 to avoid chemical precipitation if the biological denitrifying phosphorus removal capability is to be studied accurately.

Anoxic Biological Phosphorus Uptake in A^2O Process

A lab-scale anaerobic-anoxic-oxic (A2O) process used to treat a synthetic brewage wastewater was investigated. The objectives of the study were to identify the existence of denitrifying phosphorus removing bacteria (DPB), evaluate the contribution of DPB to biological nutrient removal and enhance the denitrifying phosphorus removal in A2O bioreactors. Sludge analysis confirmed that the average anoxic P uptake accounted for approximately 70% the total amount of P uptake, and the ratio of anoxic P uptake rate to aerobic P uptake rate was 69%. In addition, nitrate concentration in the anoxic phase and different organic substrate introduced into the anaerobic phase had significant effect on the anoxic P uptake. Compared with conventional A2O processes, good removal efficiencies of COD, phosphorus, ammonia and total nitrogen (92.3%, 95.5%, 96% and 79.5%, respectively) could be achieved in the anoxic P uptake system, and aeration energy consumption was saved 25%. By controlling the nitrate recirculation flow in the anoxic zone, anoxic P uptake could be enhanced, which solved the competition for organic substrates among poly-P organisms and denitrifiers successfully under the COD limiting conditions. Therefore, in wastewater treatment plants the control system should be applied according to the practical situation to optimize the operation.

Denitrification and Dephosphatation by Anaerobic/Anoxic Sequencing Batch Reactor

Removal of denitrifying phosphorus was verified in a laboratory anaerobic/anoxic sequencing batch reactor (A/A SBR). The results obtained demonstrated that the anaerobic/anoxic strategy can enrich the growth of denitrifying phosphorus removing bacteria (DPB) and take up phosphate under anoxic condition by using nitrate as the electron acceptor. The phosphorus removal efficiency was higher than 90% and the effluent phosphate concentration was lower than 1 mg·L-1 after the A/A SBR was operated in a steady-state. When the chemical oxygen demand(COD) of influent was lower than 180mg· L-1, the more COD in the influent was, the higher efficiency of phosphorus removal could be attained under anoxic condition. However, simultaneous presence of carbon and nitrate would be detrimental to denitrifying phosphorus removal. Result of influence of sludge retention time (SRT) on denitrifying phosphorus removal suggested that the decrease of SRT caused a washout of DPB and consequently the enhanced biological phosphorus removal decreased with 8 days SRT. When the SRT was restored to 16 days, however, the efficiency of phosphorus removal was higher than 90%.

Enhanced N2O emission rate in field soil undergoing conventional intensive fertilization is attributed to the shifts of denitrifying guilds

The emission of greenhouse gas N2O in agricultural soil is modulated by N fertilization that could be converted to N2O by denitrifiers under anaerobic condition. Nevertheless, the effect of denitrifiers on N2O emission has not been thoroughly elucidated. In this study, we explored the denitrifying gas kinetics,nitrate content, transcribed denitrifying functional genes(narG, nirS, nirK, and nosZ), and the active bacteria during anaerobic incubation of soils with conventional intensive N fertilization(CNS) and reduced N fertilization(RNS), both sampled from a vegetable greenhouse experimental site. The CNS sample showed significantly higher N2O emission rates relative to the RNS sample. However, the difference in N2O emission between the soils was neither because of the cumulative nitrate content nor the quantity of denitrifying gene transcripts. The distinct fertilization regimes shaped the significantly different bacterial communities in these soils. The absolute abundance of bacteria that produce N2O but lack the nosZ gene for N2O reduction(for example, the dominant Kaistobacter) was higher in CNS than in RNS. Meanwhile, the abundance of two operational taxonomy units(OTUs), namely Rhodanobacter,belonging to the most abundant genus in denitrifying guilds, was strongly enriched in CNS and showed significant positive correlation with N2O/(N2O +N2). The predominance of these bacterial OTUs in the CNS denitrifying guild strongly suggested that high N2O emission from the soil with long-term conventional intensive fertilization might be primarily attributed to the reshaping of distinct denitrifying guilds in their bacterial communities.

Enhanced nitrogen removal of the anaerobic ammonia oxidation process by coupling with an efficient nitrate reducing bacterium(Bacillus velezensis M3-1)

Bacillus velezensis M3-1 strain isolated from the sediment of Myriophyllum aquatium con-structedwetlandswas found to efficiently convert NO_(3)^(-)-N to NO_(2)^(−)-N,and the requirements for carbon source additionwere not very rigorous.Thiswork demonstrates,for the first time,the feasibility of using the synergy of anammox and Bacillus velezensis M3-1 microorganisms for nitrogen removal.In this study,the possibility of M3-1 that converted NO_(3)^(−)-N produced by anammox to NO_(2)^(−)-N was verified in an anaerobic reactor.The NO_(3)^(−)-N reduction ability of M3-1 and denitrifying bacteria in coupling system was investigated under different C/N conditions,and it was found that M3-1 used carbon sources preferentially over denitrifying bacteria.By adjusting the ratio of NH4+-N to NO_(2)^(−)-N,it was found that the NO_(2)^(−)-N con-verted from NO_(3)^(−)-N by M3-1 participated in the original anammox.The nitrogen removal efficacy(NRE)of the coupled system was increased by 12.1%,compared to the control group anammox system at C/N=2:1.Functional gene indicated that itmight be a nitrate reducing bacterium.This study shows that the nitrate reduction rate achieved by the Bacillus velezensis M3-1 can be high enough for removing nitrate produced by anammox process,which would enable improve nitrogen removal from wastewater.

Influence of carbon source and temperature on the denitrifying phosphorus removal process

To supply the valuable operating parameters for the popular usage of the new denitrifying phosphors removal process,it is essential to study the dominant biochemical reactions and the characteristics of denitrifying phosphorus removing bacteria(DPB).Thus,parallel batch experiments using DPB sludge were carried out to assess the effect of substrates(sewage,HAc,and endogenous carbon source)on denitrifying dephosphorus removal efficiency in this study.The results showed that the initial specific phosphorus release rate increased with the high concentration of the short-chain volatile fatty acids ratio in the influent,and sufficient phos-phorus was released by DPB.This improved the subsequent denitrification and phosphorus uptake efficiency.The specific endogenous denitrification mainly relies on the internal carbon source(PHB)stored by poly-P bacteria.Denitrifying phosphorus removing bacteria were very hungry when the internal PHB was consumed.Consequently,the specific endogenous denitrification rate was low and the phosphorus uptake did not happen.On the other hand,in the experiment,the denitrifying phosphorus removal performance under two temperature conditions(8-10°C and 25-26°C)was also investigated and analyzed.It was found that the lower temperature decreased the specific phosphorus release and uptake rate,but did not inhibit the denitrifying phosphorus removal completely.Therefore,the negative influence of the low temperature on the overall phosphorus removal was not significant.

Effects of herbicide butachlor application on the growth of periphytic biofilms and nitrogen loss in paddy systems

The application of butachlor as an herbicide in paddy fields is widely practiced,aiming to increase rice yield by directly or indirectly influencing the paddy environment.Periphytic biofilms,which form at the soil-water interface in paddy fields,are complex bioaggregates that play an important role in nitrogen (N) cycling.The objective of this study was to investigate the effect of butachlor on periphytic biofilm growth and N cycling under both light and dark conditions in the laboratory.The results revealed that butachlor application hindered the growth of periphytic biofilms and led to the dominance of Cyanobacteria as the primary prokaryotes,while inhibiting the development of eukaryotic Trebouxiophyceae.Furthermore,the application of butachlor reduced the richness and diversity of prokaryotes,but increased those of eukaryotes in periphytic biofilms.The light treatments exhibited higher total N loss because light favored periphytic biofilm growth and enhanced ammonium (NH_(4)^(+)) assimilation and nitrification.Additionally,butachlor application resulted in the increased retention of NH_(4)^(+)-N and nitrate (NO_(3)^(-))-N and an increase in N loss via denitrification.The abundances of functional genes encoding enzymes such as ammonia monooxygenase,nitrite reductase,and nitrous oxide reductase were increased by butachlor application,favoring nitrification and denitrification processes.Overall,the results suggest that butachlor application leads to an increase in total N loss mainly through denitrification in paddy systems,particularly in the presence of periphytic biofilms.Thus,the results may provide valuable insights into the changes in periphytic biofilm growth and N cycling induced by butachlor,and future studies can further explore the potential implications of these changes in paddy soils.