Biofilm is a complex microbial community that irreversibly attaches to surfaces or each other, enveloped in a matrix of extracellular substances. It exhibits altered growth rates and gene expression and affects the settlement of algae and attachment of marine organisms in the marine environment. However, biofilm development can lead to biofouling and corrosion, causing economic losses. Therefore, it is crucial to discover non-toxic strategies using marine natural products to inhibit bacterial colonization and combat biofilm formation and biofouling. This study explores the anti-biofilm activity of biosurfactant-producing marine sediment-associated bacteria against marine microfoulers isolated from steel plates, aiming to identify a potent eco-friendly anti-biofilm agent. Previous studies have highlighted the anti-microbial activity and therapeutic potential of biosurfactants, further supporting their effectiveness against biofilms. The samples undergo various assays, including biosurfactant tests, antagonistic assays, minimum inhibition concentration tests, anti-biofilm assays, and eukaryotic cell toxicity assays, revealing promising results. Among the five dominant marine microfouler, isolate SW2R shows the highest biofilm-forming activity and identified as Vibrio neocaledonicus. Four out of 78 marine sediment-associated bacteria isolates exhibits antagonistic and biosurfactant activity against V. neocaledonicus. Notably, the bacterium TS6, identified as Bacillus amyloliquefaciens displayed significant antagonistic and biosurfactant activity, with the ability to produce surfactin as secondary metabolite. The minimum inhibition concentration of the crude extract of B. amyloliquefaciens effectively inhibited the growth of V. neocaledonicus at 12.5 mg/mL. For anti-biofilm activity, the extract demonstrated 96% inhibition of biofilm at the same concentration. Importantly, the extract showed low to no toxicity towards Artemia salina at concentration of 0.1 mg/mL to 0.001 mg/mL. In summary, this research enhances our understanding of biofilm dynamics and contributes to the development of innovative and environmentally friendly strategies to mitigate the negative impacts of biofilm formation and biofouling in marine environments, utilizing marine natural products.

Our study explores bacterioplankton communities in the southern East China Sea, employing simultaneous 16S rDNA and rRNA sequencing. Distinct taxonomic groups exhibit varied activities, influencing ecosystem functioning. Despite differences in taxonomic composition between rDNA and rRNA assessments, both reveal similar seasonal variations. Notably, the rRNA/rDNA ratio, while highlighting active taxa, may not consistently indicate their metabolic activity. Key findings include class-level dominance shifts, with Nitrososphaeria and Acidimicrobiia prevalent in rDNA, while Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia dominate in rRNA. Environmental preferences vary among bacterioplankton classes. Our multi-indicator approach underscores the complexity of marine ecosystems, emphasizing the need for comprehensive assessments to predict functional changes in bacterioplankton communities.

In recent years, the plastic crisis has escalated, leading to significant marine pollution. Plastic products, especially those made of polyethylene, play a substantial role in this environmental issue. Scientists are actively exploring solutions to address plastic crisis, and employing microorganisms for plastic degradation stands out as a promising avenue. In a previous study conducted in 2022, Oceanimonas pelagia has been reported to have the ability to degrade environmental pollutants include low-density polyethylene (LDPE). Therefore, this study conducted an initial screening using the Clear Zone test to observe the LDPE degradation potential of Oceanimonas species, including Oceanimonas pelagia. The study observed that Oceanimonas pelagia and Oceanimonas marisflavi exhibited a significant Clear Zone area, indicating their potential for LDPE degradation. Subsequently, the study compared the changes in bacterial counts of Oceanimonas pelagia and Oceanimonas marisflavi after 21 days of separate cultivation with LDPE as the sole carbon source. In addition, the study use scanning electron microscopy, weighing methods, and biofilm staining to analyze the weight loss and the biofilm attachment on LDPE surfaces after separate co-cultivation with Oceanimonas pelagia and Oceanimonas marisflavi. Through these studies, we aimed to understand the LDPE degradation capabilities of Oceanimonas pelagia and Oceanimonas marisflavi.

In today's society, water consumption is a crucial aspect of life. However, with the ongoing development of society, the generation of wastewater has increased significantly, highlighting the growing importance of wastewater treatment. Currently, approximately 5% of global wastewater contains high levels of salt, posing various challenges. High-salinity wastewater has significant implications for the biological treatment in wastewater processing. Biological treatment involves anaerobic microorganisms conducting anaerobic digestion to break down large organic pollutants in water into usable methane. The elevated salinity, though, exerts substantial stress on the entire biological treatment process, leading to a decrease in overall wastewater treatment efficiency. Despite the importance of addressing high-salinity wastewater, there is currently no low-energy and cost-effective treatment method. Additionally, research on how anaerobic microorganisms respond to salinity stress is relatively limited. Thus, filling the gap in this field may be crucial for enhancing anaerobic digestion under high-salinity conditions. This study establishes four laboratory-scale reactors and, through continuous time-series sampling, seeks to understand the interactions between salinity stress, reactor performance, and the microbial community. The research aims to unravel who influences these factors and, in turn, who is influenced by them. Through subsequent bioinformatics analysis, the study provides insights into patterns exhibited by anaerobic microorganisms when facing salinity stress, with the hope of further experimental validation in the future.

The Sunflower coral (Tubastraea aurea) is an invasive marine species originating from the Indo-Pacific Ocean. In this study, corals were collected from hydrothermal vents on Guishan Island, Taiwan, and other non-hydrothermal areas. A previous investigation revealed substantial variations in bacterial communities between extreme and normal habitats, demonstrating a prevalence of diverse symbiotic bacteria, notably the Vibrio genus. The presence of bacteria in the coral tissue was explored through 16S rRNA and whole genome sequencing, leading to the identification of a novel strain named NTOU-C2. This study further characterizes NTOU-C2 through morphological, physiological, and biochemical features, aiming for a comprehensive understanding of its microbiological traits. Results indicate that NTOU-C2 exhibits Gram-negative, non-motile morphology with flagella. Physiologically, it is a facultative anaerobic strain, s howcasing growth at temperature range of 15 – 40℃(optimal at 25 ℃) tolerance to a pH range of 6 - 10 (optimal at pH 8), and salinity tolerance of 1 - 9% (optimal at 2%). Biochemically, NTOU-C2 is oxidase-positive, catalase-negative, and displays specific enzymatic activities, including the reduction of nitrate to nitrite, indole production, gelatin and aesculin hydrolysis, and utilization of specific carbon sources (Tween 40, Tween 80, D-Cellobiose). Phylogenetic analysis reveals a close relationship between NTOU-C2 and Vibrio sinaloensis, Vibrio caribbeanicus, and Vibrio japonicus. Noteworthy distinctions of NTOU-C2 from other Vibrio species include its rod-shaped morphology, ability to thrive in more acidic and warmer environments, and unique capacity to utilize malate as a carbon source without the need for NaCl. This study provides valuable insights into the diversity and characteristics of bacteria associated with the Sunflower coral, emphasizing the distinctive features of the newly identified strain NTOU-C2.

Diesel is one of the world's most commonly used petroleum products, with widespread use in transportation, agriculture, and industry. However, accidental spills or leaks of diesel fuel can have serious environmental consequences, including soil and water contamination, air pollution, harm to wildlife, and long-term ecosystem disruption as diesel is a complex mixture of hydrocarbons that can persist in the environment for long periods. In this study, we isolated one diesel-degrading bacteria from oil-contaminated seawater, in Keelung Harbor, Taiwan. The bacterial isolates were invented by enriching bacteria from a 10mL seawater sample in Marine Broth Medium (MBM) supplemented with 5% (v/v) of diesel as a carbon source. Emulsification and oil-spreading tests have been used to screen the potential diesel-degrading bacteria. From the screening, isolate NTOU-VMBA5.3 showed a high potential in degrading diesel which has significant result in oil spreading test. The bacterium was identified based on 16S rRNA and whole genome sequence (Oxford Nanopore Technologies), and they showed similarity with Oceanimonas baumannii GB6 (NCBI BLAST-99.31%), and similarity with Oceanimonas baumannii DSM1559 (ANI-95.56%) respectively. A morphological, physiological, and biochemical characterization of this strain were also conducted to further investigate and compare the microbiological characteristics with closely related Oceanimonas genus. The hydrocarbon-degrading capabilities of the bacterium was tested using diesel as sole carbon source, and the results showed the diesel was degraded around 59% in minimal salt medium (MSM) at 30oC for 14 days. The capabilities in degrading aromatic hydrocarbon of diesel will be further analyzed using GC-MS to determine the concentration of the n-alkane in diesel. The research will contribute to the ongoing efforts to harness microbial resources for sustainable environmental solutions. 

氫能的優點為熱質高（141.8 MJ kg−1），且燃燒後的產物無二氧化碳、SOx及NOx等副產物造成環境汙染，是近年備受矚目的未來能源。為了以永續且有效的方式產氫，我們提出微藻與菌種共培養於電解槽產電的概念。在電解槽中陽極上所培養的微藻可進行光合作用，產生供產電菌生長的基質（如醣類等），同時亦可吸收CO2進行減碳，產電菌則提供胞外電子傳遞機制，最後在陽極表面產生電流，最終在陰極與質子結合產生氫氣。本實驗選用小球藻Chlorella sorokiniana SU-1與鐵還原菌Shewanella decolorationis NTOU1共生培養在碳氈陽極上。最初實驗將C. sorokiniana SU-1單獨培養於陽極碳氈上，希望透過微藻產電來達到電解產氫的效果，但實驗結果發現若不添加氧化還原物質，如2,6-dimethyl-1,4-benzoquinone與ferricyanide等作為電子傳遞媒，從藻類細胞較內部的光合電子傳遞鏈中提取電子，則不會有顯著電流產生；第二階段嘗試添加產電菌S. decolorationis NTOU1取代氧化還原物質，也是在此時形成藻菌共生的觀念，但可能是光合作用系統中產生太多氧氣，因此造成在陽極上產生很多陰極氧還原反應，造成無淨電流產生，在此階段也不得不依靠電子傳遞媒的幫助來產生電流；第三階段，則嘗試在共生系統中添加葡萄糖提供還原能，而不再添加氧化還原物質，就可以因此於陽極產生顯著的電流，且此電流的消長會受光照有無調控。為了了解第三階段中藻菌共生的產電機制，我們將S. decolorationis NTOU1單獨培養於陽極碳氈上，並提供27.8 mM的葡萄糖作為基質。結果發現，植菌兩小時後，在3小時內得到約0.5 mA的電流產生，且在第30及70小時分別得到2及4 mA的最大電流；在與C. sorokiniana SU-1共生產電的實驗中，得到最大電流則為6.3 mA，有顯著的增加。在另一個實驗中以38 mM的乳酸作為基質產電，在植入菌種後立即得到約1.5 mA的電流，接著電流產出皆在1 mA左右；在藻菌共生產電的實驗中，電流產出約為0.8 mA左右。由上述結果可知，額外添加高濃度基質可以有效產出電流，且以醣類作為基質，其產電量較高。在未來光合生物電解產氫的研發路程中，期望可以用不添加電子傳遞媒的方式達到永續產氫的目的。

污水處理程序承載著人類對水資源及環境永續保護的重大責任，其中除了常規的污染物外(例如:懸浮固體、有機物、氮、磷化合物等)，非政府管制污染物（或稱新興污染物）的擴散是一個嚴重的環境及公共衛生問題。然而，在特定的生物程序當中，豐富的菌群及後端選擇壓力下，微生物會藉由多種水平基因轉移機制，使抗生素抗藥性基因(Antibiotic resistance genes, ARGs)被持續傳遞並擴散，進而可能導致人類死亡率的提升，因此，污水的生物處理程序可能是抗生素抗藥性和致病菌生存及擴散的潛在熱點。本研究假設不同材質和形狀的生物載體在處理程序中，會導致生物膜附著及細菌群落結構的變化，而影響ARGs的豐度差異，研究目標包括檢驗臭氧化醫療廢水對生物載體中ARGs的改變、材質類型對生物膜群落組成的影響，旨在選擇出最佳的生物載體材料，以最大限度地減少廢水處理程序，菌群偕帶ARGs存在的機率，從而降低抗生素抗藥性傳播風險。

養豬廢水中含有高濃度COD及高氨氮，利用藻菌共生的方式，可藉光合作用供氧促進異營菌生長之外，產生之胞外葡萄糖也可供異營菌作為基質來利用，而異營菌進行呼吸作用會釋放出CO2，微藻可以吸收CO2及氨氮，反應槽放流的藻類細胞亦可再利用生成高價值化學品（如生質柴油等）。本實驗將小球藻Chlorella sorokiniana SU-1與鐵還原菌Shewanella decolorationis NTOU1共同培養在批次瓶中，並使用含有6360 mg L−1葡萄糖的合成廢水以模擬處理養豬廢水，以四組實驗參數（分別為同時添加有機碳與無機碳，僅添加有機碳，僅添加無機碳，及無添加碳源）進行七天的實驗。由結果得知，僅添加無機碳之小球藻生長情況較佳，比生長速率達0.79 d−1，在同一條件中，S. decolorationis NTOU1從第二天生長至第四天細胞濃度從5.45×107 cell mL−1增長至2.25×108 cell mL−1，發現有顯著生長現象，但C. sorokiniana SU-1細胞濃度穩定維持在107 cell mL−1，因此可以得知菌確實有可能利用微藻所產生之胞外醣類以生長。在僅添加有機碳的組別中，化學需氧量之降解率約16%，在僅添加有機碳及有機碳與無機碳均添加的組別中，氨氮去除率分別為82%和85%。由掃描式電子顯微鏡觀測結果可得知C. sorokiniana SU-1的直徑大小約為3 𝜇m，S. decolorationis NTOU1的菌體大小約為1 𝜇m。從拍攝結果中觀察到桿狀的S. decolorationis NTOU1圍繞在光滑完整的圓球狀C. sorokiniana SU-1細胞周圍，兩者間並無發現互相吞噬或破壞對方細胞的現象。以螢光原位雜交的方式觀察，使用EUB338+6FAM探針雜交至S. decolorationis NTOU1細胞後，會發出綠色螢光，而C. sorokiniana SU-1本身會發出自體紅光，在螢光顯微鏡下能明顯看出其分佈比例。此次實驗中添加有機碳的整體去除效果是最為顯著的，證實利用藻菌共生能達到減碳及永續循環的效果。

香菇(Lentinula edodes)營養價值高且具獨特風味，為世界主要栽培的食用蕈。香菇為白腐菌，能分解不同的木質纖維素材料，因此木材和農業資材常作為香菇生產的基質。段木香菇為臺灣林下經濟重要產業之一，利用特定樹種資材進行栽培，段木栽培的香菇口感與氣味較太空包佳。根據文獻證實除了木材種類及品質，食用蕈與木材細菌的交互作用也會影響子實體的生長與品質，但目前沒有關於段木香菇與木材細菌群落的相關研究。本研究目的以臺灣苦櫧(Castanopsis formosana)段木接種不同香菇菌株，從走菌期間至出菇共採樣4次，以監測木材細菌群落動態變化及木材物理化學性質變化，菌株來源分離自高士(kus)、東源(mal)和旭海(mac)部落之香菇子實體。本研究目前已完成段木上細菌固氮效率、細菌群落結構及香菇品質分析。結果顯示接種香菇菌株之固氮效率比未接種(con)高，如CFmac1-4和CFmal1-1在出菇中(III)固氮效率為3.21 和1.24 μL C2H4 kg-1・hr-1；CFcon-2為-1.38 μL C2H4 kg-1・hr-1。CFmac1-4及CFmal1-1出菇中(III)固氮效率分別為3.21及1.24 μL C2H4 kg-1・hr-1，出菇後(IV)分別為237.79和297.12 μL C2H4 kg-1・hr-1，有增加的趨勢。段木香菇生長過程段木細菌群落結構分析共得到1,318,683條高品質序列，以Streptomyoetales、Micrococcales、Corynebacteriales、Rhizobiales為較優勢的細菌目，其中Rhizobiales是重要的固氮細菌。以屬的分類層級來看依序為Streptomyces、Burkholderia-Caballeronia-Paraburkhoderia較優勢的細菌屬。本研究香菇氣味以1,3,5-Cycloheptatriene、Terpinen-4-ol及Lenthionine等化合物為主，Lenthionine為乾燥香菇具有獨有的氣味。CCA分析Lenthionine對細菌群落結構之相關性。Taibaiella、Gryllotalpicola、Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium等細菌屬影響而產生Lenthionine。段木分解過程的物理化學性質持續分析中，進一步將試著找出影響香菇品質或氣味的重要細菌群落。

微生物燃料電池（microbial fuel cell，MFC）技術是利用微生物代謝有機物質的過程來產生電能。畜殖廢水中含有高濃度有機物，因此是MFC技術可處理並產生電力的優質物料。前期對目標廢水進行檢測，從屏東東海豐畜牧場取得之樣品，檢測結果為化學需氧量（chemical oxygen demand，COD）=19.8 g/L、NH4+= 1.0 g-N/L、總凱氏氮（total Kjeldahl nitrogen，TKN）=1.2 g-N/L、SS= 16.6 g/L、VSS= 13.8 g/L。本研究選用之菌體為Shewanella decolorationis NTOU1、Shewanella xiamenensis BC01以及畜殖污水馴養之污泥作為動力學分析對象。將微生物燃料電池連接上定電位儀，工作電極固定電壓於＋0.4 V（vs. 銀／氯化銀參考電極），連接數據紀錄器完整的記錄應答電流以供後續分析，再進一步以Nernst-Monod equation規劃求解以研究基質與比電流密度（j，mA g-cell−1）之間的關係。所得的最大產電量、電極上的電位等，為微生物電化學動力學重要的參數，可以用來判斷微生物對基質的親和性，依據所得的數據做全面性且客觀的比較，以評估產電菌的優劣。在初始廢水COD濃度為2.2 g/L（含醋酸12 mM）的實驗中，選植S. xiamenensis BC01與 S. decolorationis NTOU1進行產電試驗，由分析結果得知約90小時醋酸均無明顯降解。因此本實驗另外利用污水之污泥馴養出強勢醋酸利用菌種以利降解高醋酸污水進行產電。汙泥微生物菌的馴養過程經過兩個月，馴養過程中每經過4天即更換馴養培養液（成份包括醋酸49.9 mM、MgSO4 1.02 mM、NaOH 80 mM、NaH2PO4 100 mM、NH4Cl 27.9 mM、KCl 76.6 mM及微量元素 50 µl），在每一次的更換培養基周期中，在培養瓶中所產生的生質氣體平均可以達到40.8 mL/d。在另一次產電的實驗中，選用相同初始COD濃度的陽極液，並選植馴化完成的污泥，由分析結果可得知在90小時內COD由2.2降至1.2 g/L，降解率約為56%，但此過程中所產生電流量平均為0.0002 mA，在第六天後加入了250 μM ferricyanide做為電子傳遞媒，但仍不見植入菌種之產電活性，因此推論本次馴養策略尚無成功找出目標優質降解醋酸之產電菌種。

畜牧業的廢水具有高有機物及高氨氮的特性，成為環境汙染的重大隱憂。因此，為了處理畜殖污水帶來的環境問題，本研究計畫預期結合懸掛浸水式植物栽種發電系統(中華民國專利I814127號）與藻類陰極，建立出新穎光合作用程序，可以在去除廢水污染物的同時發電。本研究評估的重點在化學需氧量(chemical oxygen demand，COD）與總凱氏氮的去除、蔬菜與藻類的生長狀況、以及產電量。陽極選植的蔬菜是空心菜(Ipomoea aquatica），陰極藻則選用小球藻(Chlorella sorokiniana SU-1）。空心菜從菜苗到採收之栽培週期為三週，在第一個實驗中觀察添加不同劑量(濃度分別為1 g L-1及0.67 g L-1）之市售化學肥料花寶2號 (肥進(複)字第0110023號，其成分: TN 20.0 g(內含NH3-N  4.0 g ;NO₃-N 4.0 g); P2O5 20.0 g;K2O 20.0 g）對產電的影響，結果在高及低化學肥料濃度下，在種植週期內總共可以分別產生76庫侖和60庫倫的電量。在另一組實驗中，以陰極槽中有無添加C. sorokiniana SU-1分成兩個實驗組，藻類組有在陰極槽添加藻類，市售化學肥料、及食品廢水（COD ≅ 800 mg L-1），而無藻類組則沒有添加藻類及廢水。在一天之內，藻類組電壓峰值最高即可上升至0.35 V，而無藻類組則維持在0.01 V，顯示藻類添加確實有促進產電的效果。在室內使用人工光源栽種的結果，以土壤介質操作VMFC所得之結果，有分為藻類組和無藻類組，其COD濃度在14天內，藻類組從139 mg L-1到775 mg L-1，而無藻類組從131 mg L-1 升到554 mg L-1，推論造成COD增加的原因可能有兩點，一是藻類自行產生醣類，二是土壤中有機質逸散到水中，另外氨氮濃度有藻類組與無藻類組分別在七天內降低19.04 mg-N L-1與6.16 mg-N L-1。為了消除土壤中有機物的影響，同時也希望栽種介質本身能提升陽極導電性，因此將土壤更換為活性碳。從操作結果得知，藻類組的COD濃度在七天中從295 mg L-1 降低到222 mg L-1，有下降的趨勢，氨氮的部分則降低10.8 mg-N L-1，在操作過程中，小球藻的細胞密度最高達到1.4𑛀106 cells mL-1，未來希望能持續提升懸掛浸水式植物栽種發電系統之功效，朝向永續循環經濟的目標前進。

The evolution and origin of life have always been important microbial research topics. Biological nitrogen fixation (BNF) has been an indispensable reaction of the earth's nitrogen cycle from ancient times to the present. Although many studies have studied the evolution of BNF, due to the very ancient time, there has been no good hypothesis about the reasons for the evolution and branching of nitrogen-fixing species. Atmospheric oxidation events lead to the evolution of species, the breakup of supercontinents leads to the evolution of species diversity, and meteorite impacts cause environmental changing. These major events on Earth are increasingly found to be related to biological evolution. However, due to the lack of fossil evidence, it is often impossible to infer the evolutionary history of microorganisms from a single data. This study, for the first time, used more than 1,000 concatenated nitrogen-fixing protein sequences to construct a BNF timetree, combined with BNF isotope data, earth geological data and recent research, to reveal the possible reasons for the evolution and separation of BNF on Earth. We found that the major Group I, II and III of BNF diverged ~2.2 to 1.9 billion years ago, and the Kenorland supercontinent breakup, the Great Oxidation Event, Yarrabubba/ Vredefort impacts happened at the same divergence time. In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.

自來水在進到配水管網前會經後加氯程序來控制水中微生物的增生，但隨著配水時間與配水管網的長度增加，水中的自由餘氯濃度會逐漸下降，若當水中的可同化有機碳(Assimilable organic carbon, AOC)濃度過高時會造成配水管網中微生物增生或生物腐蝕現象進而導致水質惡化或消耗餘氯的增加。因此定期監測及維持生物穩定性在維護配水管網健康相當重要。目前各國自來水事業在配水管網中監測生物穩定性的常見指標為AOC，然而此方法在檢測過程中需使用塗碟以及稀釋等程序，而根據培養溫度的不同需要等待六到十二日的檢測時間耗時且費力，對於檢驗人員的要求較高，較不容易做為經常性的檢驗方法。因此本研究測試以三磷酸腺苷(adenosine triphosphate, ATP)的冷光監測方法，用於檢測自來水中AOC。先經試驗個別取得AOC代表菌株 Pseudomonas fluorescens P17 及 Spirillum NOX 所產生ATP相對於單位醋酸根碳的生產係數(yield coefficients)，再將自來水培養3天後以ATP冷光法檢測，並使用生產係數回推水中AOC濃度。由於ATP檢測方法是以冷光讀值的方式進行，簡單且快速，此方法相較於傳統的稀釋塗碟培養減少了大量的培養基製備、勞力成本及計數上的實驗誤差，本研究預期ATP檢測方法可以有機會應用在配水管網生物穩定性的監測。

This study investigates the impact of biochar amendment on methane emissions and pollutant removal in constructed wetlands (CWs). CWs, as a wastewater treatment technology, offer significant potential for carbon sequestration. However, concerns persist over greenhouse gas emissions from CWs, particularly methane, which has a global warming potential 28 times greater than carbon dioxide over a 100-year period. Biochar, a carbon-rich product derived from agricultural waste through pyrolysis, has demonstrated its capacity to modify soil geotechnical properties. Previous research suggests that biochar amendment to landfill cover soil can promote methanotrophic methane oxidation. In this study, four mesocosms of CWs were established with varying ratios of biochar (0%, 25%, 50%, 100%, CW0, CW25, CW50, CW100). Weekly water samples were collected and analyzed for pH, dissolved oxygen (DO), chemical oxygen demand (COD), total nitrogen (TN), nitrite (NO2-), nitrate (NO3-), and ammonium (NH4+). Methane concentrations were measured weekly using a static chamber method, with chambers placed at a depth of 20 cm within each mesocosm and methane concentration analyzed via GC-TCD. After 98 days of operation, non-biochar CW (CW0) exhibited a COD removal of 71.6%, while biochar-amended CWs (volume ratio of biochar: 25%, 50%, 100%, CW25, CW50, CW100) achieved COD removals of 74.6%, 74.6%, and 94.2%, respectively. CW25, CW50, and CW100 demonstrated significantly higher COD removal (p < 0.05) compared to CW0. In contrast, TN removal for CW0, CW25, CW50, and CW100 was 23.1%, 21.3%, 20.9%, and 22.8%, respectively, with no significant difference (p > 0.05) observed among the four CWs. Regarding methane emissions, CW0 exhibited higher emissions than CW25, CW50, and CW100 in all measurements. To account for initial soil volume variations, methane fluxes measured from CW25 and CW50 were normalized by their respective soil proportions. While CW25 consistently emitted less methane than CW0, normalized values revealed that under certain conditions, methane emissions per unit volume of soil in CW25 were higher than in CW0. Moreover, regardless of normalization, methane emissions from CW50 were significantly lower than those from CW0 and CW25, but higher than CW100. Overall, this study suggests that biochar amendment in constructed wetlands has the potential to reduce methane emissions while maintaining water treatment capacity.