Biochemical Engineering Journal最新文献_第3页

Amine-functionalization of hierarchical calcium phosphate microflowers enhances the reusability of immobilized β-galactosidase 分级磷酸钙微花的氨基功能化提高了固定化β-半乳糖苷酶的可重复利用性

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-05 DOI: 10.1016/j.bej.2025.109921

Elizabet Moreno-Reyes, Julie M. Goddard

{"title":"Amine-functionalization of hierarchical calcium phosphate microflowers enhances the reusability of immobilized β-galactosidase","authors":"Elizabet Moreno-Reyes, Julie M. Goddard","doi":"10.1016/j.bej.2025.109921","DOIUrl":"10.1016/j.bej.2025.109921","url":null,"abstract":"<div><div>Metal phosphate-based hierarchical microflowers have been reported for enzyme immobilization; however, they suffer from enzyme desorption due to the lack of stable interactions between enzymes and the support. Herein, polyamines were used as surface modifiers of calcium phosphate microflowers to enable covalent immobilization of β-galactosidase. Calcium phosphate microflowers showed a mean diameter of 12.6 ± 3.4 μm, pore size of 439 ± 159 nm, and sheet thickness of 196 ± 51 nm, confirming its hierarchical architecture. Microflowers were modified using non-modified and crosslinked polyallylamine variants (molar ratios of 1:0.25, 1:0.5 and 1:1), and their performance was compared to that of physically adsorbed and free β-galactosidase. Polyallylamine increased immobilized enzyme by ∼60 % compared to enzyme immobilization via adsorption on non-modified microflowers. In terms of substrate conversion, k<sub>cat</sub> increased only when the lowest level of crosslinking was used, but the catalytic efficiency of the enzyme decreased without exception. In most cases, covalently immobilized β-galactosidase showed improved activity retention at a pH range of 5.5–8.5 and temperatures below 60 °C. Additionally, covalently immobilized β-galactosidase showed higher residual activity (10–40 % higher) and lower enzyme loss (≤10 vs 43.3 μg) compared to adsorbed β-galactosidase for 10 days under constant agitation at 20 °C. Therefore, this work demonstrates how hierarchically structured hybrid materials can be used in enzyme immobilization systems to enhance reusability and enzymatic activity performance.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109921"},"PeriodicalIF":3.7,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145045594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strategic optimization of β-galactosidase binding performance in packed bed ion exchange chromatography 填充床离子交换色谱中β-半乳糖苷酶结合性能的策略优化

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-04 DOI: 10.1016/j.bej.2025.109926

Chien-Hsing Chiang , Nguyen The Duc Hanh , Chanin Panjapornpon , Manop Charoenchaitrakool , Kandis Sudsakorn , Kulpavee Jitapunkul , Bing-Lan Liu , Si-Yu Li , Kuei-Hsiang Chen , Yu-Kaung Chang

{"title":"Strategic optimization of β-galactosidase binding performance in packed bed ion exchange chromatography","authors":"Chien-Hsing Chiang , Nguyen The Duc Hanh , Chanin Panjapornpon , Manop Charoenchaitrakool , Kandis Sudsakorn , Kulpavee Jitapunkul , Bing-Lan Liu , Si-Yu Li , Kuei-Hsiang Chen , Yu-Kaung Chang","doi":"10.1016/j.bej.2025.109926","DOIUrl":"10.1016/j.bej.2025.109926","url":null,"abstract":"<div><div>This study engineered <em>Pichia pastoris</em> to express β-galactosidase (β-gal), and its downstream purification was systematically optimized. Growth conditions were optimized in shake flask and fermenter cultures, with the fermenter achieving an OD₆₀₀ of approximately 45 after 24 h. The biomass was disrupted by high-pressure homogenization (30 kpsi, six cycles, 4 °C) to produce a clarified suspension (25 %, w/v) containing 2.02 × 10³ U/mL β-gal activity and 17.2 mg/mL total protein. STREAMLINE DEAE, a high-density ion-exchange adsorbent, was used to assess β-gal adsorption across pH 4–12, with optimal binding at pH 6 and a maximum static capacity of 5.6 × 10⁵ U/mL. One-factor-at-a-time (OFAT) experiments in packed bed mode evaluated the flow rate, bed height, feedstock concentration, and residence time effects on the 5 % dynamic binding capacity (DBC). Further optimization using fractional factorial design (FFD) and response surface methodology (RSM) produced a second-order predictive model relating DBC to flow rate (F), clarified feed concentration (C₀), and bed height (H). At optimal conditions (F: 2.96 mL/min; C₀: 45 % w/v; H: 16.6 cm), the predicted DBC (1.15 × 10⁵ U/mL) closely matched the experimental value (1.13 × 10⁵ U/mL). This work demonstrates a practical, model-driven approach to optimize high-density enzyme production and purification using packed bed ion-exchange chromatography.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109926"},"PeriodicalIF":3.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145045598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Isolation and characterization of oil degrading bacteria isolated from artificially crude oil-contaminated soil 人工原油污染土壤中石油降解细菌的分离与鉴定

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-04 DOI: 10.1016/j.bej.2025.109928

Aman Bisht , Mandar S. Bhagat , M. Abdul Rasheed , G. Archana

{"title":"Isolation and characterization of oil degrading bacteria isolated from artificially crude oil-contaminated soil","authors":"Aman Bisht , Mandar S. Bhagat , M. Abdul Rasheed , G. Archana","doi":"10.1016/j.bej.2025.109928","DOIUrl":"10.1016/j.bej.2025.109928","url":null,"abstract":"<div><div>The purpose of this study is to identify and isolate bacteria that degrade crude oil (CO) in an artificially polluted soil, with the aim of designing the study to better understand local bacteria adaptation to CO pollution. Soil samples were artificially contaminated with CO and then incubated for 60 days in order to promote microbial adaptability and the enrichment of populations that can break down hydrocarbons. Subsequent to the incubation period, bacterial strains were identified and their ability to degrade CO was described. Furthermore, the isolates' biosurfactant (BS) and bioemulsifier (BE) synthesis was assessed. The results showed that extended exposure to CO leads to selective enrichment of hydrocarbon-degrading bacteria, indicating adaptation to the pollutant. Some bacterial isolates, like S4 and S5 (<em>Pseudomonas indica</em>), shows high CO degradation rate (45 % and 50 % respectively) and shows high BE index (<em>Pseudomonas indica</em>, E24; 45 % and 50 %, respectively) while S1 (<em>Brevibacillus formosus</em>) and exhibits moderate CO degradation efficiency (33 %). In addition to their significant CO-degrading activity, some isolates (S4 and S5) have the ability to produce BS and BE, which indicates their potential for use in bioremediation processes. This technique offers a quick and affordable way to separate and specifically enrich bacteria that can decompose CO in a controlled environment. CO bioremediation using microbial consortia may increase with further advancements in this technique.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109928"},"PeriodicalIF":3.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145019405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Aeration effectiveness in column bioleaching: Microbial concentration responses, porosity evolution and copper sulfide leaching 柱式生物浸出的曝气效果：微生物浓度响应、孔隙演化和硫化铜浸出

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-04 DOI: 10.1016/j.bej.2025.109925

Mingqing Huang , Jiawei Li , Ming Zhang , Fenghao Lu

{"title":"Aeration effectiveness in column bioleaching: Microbial concentration responses, porosity evolution and copper sulfide leaching","authors":"Mingqing Huang , Jiawei Li , Ming Zhang , Fenghao Lu","doi":"10.1016/j.bej.2025.109925","DOIUrl":"10.1016/j.bej.2025.109925","url":null,"abstract":"<div><div>This study investigates aeration effectiveness in heap bioleaching systems through controlled column experiments. Column bioleaching tests with aeration intensities of 0–150 L/h were conducted, employing nuclear magnetic resonance for porosity evolution analysis. Results demonstrate that forced aeration increased dissolved oxygen concentrations and optimized pore structure in lower sections, shortening the bacterial transition from lag to logarithmic phase while extending stationary phases. Mineral leaching rates correlated positively with microorganism concentrations, with copper leaching exhibiting sequential lag, rapid, and stable phases. Aeration enhanced Fe³ ⁺ regeneration and microbially catalyzed oxidation, improving copper sulfide leaching. Aeration effectiveness ranged from 1.59 % to 10.4 %, inversely correlated with aeration intensity. The column-scale findings establish fundamental mechanisms for pore-microbe-mineral interactions under forced aeration, providing critical parameters for scaling to industrial heap operations where oxygen transfer limitations significantly constrain copper recovery efficiency.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109925"},"PeriodicalIF":3.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145019364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biotransformation pathway, growth inhibition, and biochemical response of Scenedesmus obliquus to brilliant green dye: Implications for bioremediation 绿光染料对斜景木的生物转化途径、生长抑制和生化反应：对生物修复的启示

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-04 DOI: 10.1016/j.bej.2025.109927

Ricky Rajamanickam , Rangabhashiyam Selvasembian

{"title":"Biotransformation pathway, growth inhibition, and biochemical response of Scenedesmus obliquus to brilliant green dye: Implications for bioremediation","authors":"Ricky Rajamanickam , Rangabhashiyam Selvasembian","doi":"10.1016/j.bej.2025.109927","DOIUrl":"10.1016/j.bej.2025.109927","url":null,"abstract":"<div><div>The potential of live microalgae <em>Scenedesmus obliquus</em> was tested for removing Brilliant green dye (BGD). BGD is a cationic dye that has been widely used as a colouring agent in various sectors, but this dye has been reported in wastewater due to its high solubility in water. <em>Scenedesmus obliquus</em> is one of the most widely employed microalgae species with good bioremediation potential and biochemical composition. This study reports growth inhibition of <em>Scenedesmus obliquus</em> in test runs 5 mg/L (17 %), 10 mg/L (31 %), 25 mg/L (42 %), and 50 mg/L (57 %), and the EC<sub>50</sub> value was predicted to be 33.7 mg/L using probit statistical analysis. The removal efficiency decreased from 99 % to 87 % as the BGD concentration increased, and the removal mechanism was reported in the order of biodegradation > bioaccumulation > bioadsorption. The biodegradation potential decreased from 97 % to 84 % at the BGD concentration of 50 mg/L, and the Monod kinetic study revealed that the half-saturation constant increased when the BGD concentration crossed the EC<sub>50</sub> value. Protein (498 mg/L) and lipid (728 mg/L) accumulation was highest at 5 mg/L of BDG, implying growth-stimulating effects on microalgae as the growth inhibition is less. These findings highlight <em>Scenedesmus obliquus</em> potential for effective BGD removal below EC<sub>50</sub> concentrations with identification of 6 biotransformed products and simultaneous production of value-added biomass.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109927"},"PeriodicalIF":3.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145004584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Activation of indigenous bacteria for rapid degradation of medium-chain and long-chain hydrocarbons in petroleum-contaminated soils 活化原生细菌快速降解石油污染土壤中长链和中链烃

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-09-03 DOI: 10.1016/j.bej.2025.109922

Jinlan Xu , Jiayi Wang , Chuanyu Liu , Huiwen Guan , Rankang Zhou , Xin Zhai , Qilin Shu

{"title":"Activation of indigenous bacteria for rapid degradation of medium-chain and long-chain hydrocarbons in petroleum-contaminated soils","authors":"Jinlan Xu , Jiayi Wang , Chuanyu Liu , Huiwen Guan , Rankang Zhou , Xin Zhai , Qilin Shu","doi":"10.1016/j.bej.2025.109922","DOIUrl":"10.1016/j.bej.2025.109922","url":null,"abstract":"<div><div>To explore the rapid degradation of medium-chain and long-chain alkanes (C<sub>20</sub>-C<sub>40</sub>) in petroleum-contaminated soils by activating indigenous bacteria, as well as the degradation mechanism, a 30-day biodegradation experiment was conducted by adding different proportions of activators (glucose, sodium acetate). The results showed that the removal of total petroleum hydrocarbons (TPH), medium-chain, and long-chain alkanes in the activation group reached 9753.06 <span><math><mrow><mtext>mg</mtext><mo>/</mo><mtext>kg</mtext></mrow></math></span>, 4185.85 <span><math><mrow><mtext>mg</mtext><mo>/</mo><mtext>kg</mtext></mrow></math></span>, and 3778.15 <span><math><mrow><mtext>mg</mtext><mo>/</mo><mtext>kg</mtext></mrow></math></span>, respectively, which were significantly higher than those of the inactive group. Furthermore, the microbial community of the activation group was dominated by Mycobacterium (14.55 %) and Pseudarthrobacter (13.95 %). The activation group consumed a large amount of DOC (10,052.64 <span><math><mrow><mtext>mg</mtext><mo>/</mo><mtext>kg</mtext></mrow></math></span>), activating Mycobacterium and Pseudarthrobacter to actively uptake oxygen, and significantly increased the activities of dehydrogenase and lipase,which accelerated the transformation of hydrocarbons. This may be the reason why the activation group achieved rapid TPH degradation. Furthermore, predictive modeling using three machine learning algorithms demonstrated strong concordance with experimental data, validating their utility in optimizing bioremediation strategies. This study presents a cost-effective and sustainable approach for the efficient remediation of petroleum-contaminated soils, supported by data-driven insights.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109922"},"PeriodicalIF":3.7,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144997096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluating RNA ligases to facilitate the synthesis of circular mRNA 评价RNA连接酶促进环状mRNA合成的作用

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-08-29 DOI: 10.1016/j.bej.2025.109920

Xiaoxue Wang , Yiming Wang , Jiajun Liu , Jian Dong , Yuan Lu

引用次数: 0

A novel method for quantifying enzyme immobilization in porous carriers using simple NMR relaxometry 一种用简单核磁共振弛豫法定量酶在多孔载体上固定化的新方法

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-08-29 DOI: 10.1016/j.bej.2025.109909

M. Raquel Serial , Luca Schmidt , Muhammad Adrian , Grit Brauckmann , Stefan Benders , Victoria Bueschler , Andreas Liese , Alexander Penn

{"title":"A novel method for quantifying enzyme immobilization in porous carriers using simple NMR relaxometry","authors":"M. Raquel Serial , Luca Schmidt , Muhammad Adrian , Grit Brauckmann , Stefan Benders , Victoria Bueschler , Andreas Liese , Alexander Penn","doi":"10.1016/j.bej.2025.109909","DOIUrl":"10.1016/j.bej.2025.109909","url":null,"abstract":"<div><div>Enzyme immobilization plays a crucial role in enhancing the stability and recyclability of enzymes for industrial applications. However, traditional methods for quantifying enzyme loading within porous carriers are limited by time-consuming workflows, cumulative errors, and the inability to probe enzymes adsorbed inside the pores. In this study, we introduce Time-Domain Nuclear Magnetic Resonance (TD-NMR) relaxometry as a novel, non-invasive technique for directly quantifying enzyme adsorption within porous carriers. Focusing on epoxy methyl acrylate carriers, commonly used in biocatalysis, we correlate changes in <span><math><msub><mrow><mi>T</mi></mrow><mrow><mi>2</mi></mrow></msub></math></span> relaxation times with enzyme concentration, leading to the development of an NMR-based pore-filling ratio that quantifies enzyme loading. Validation experiments demonstrate that TD-NMR-derived adsorption curves align closely with traditional photometric measurements, offering a reliable and reproducible alternative for enzyme quantification. The accessibility of tabletop TD-NMR spectrometers makes this technique a practical and cost-effective tool for optimizing biocatalytic processes. Furthermore, the method holds promise for real-time monitoring of adsorption dynamics and could be adapted for a wider range of carrier materials and enzymes.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109909"},"PeriodicalIF":3.7,"publicationDate":"2025-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145019365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

RNA helicase CgDBP4 improves tolerance and fermentation performance of Candida glycerinogenes and Yarrowia lipolytica from undetoxified cellulose hydrolysate RNA解旋酶CgDBP4提高了产甘油假丝酵母和解脂耶氏菌对未解毒纤维素水解产物的耐受性和发酵性能

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-08-29 DOI: 10.1016/j.bej.2025.109919

Xinyao Lu , Xiaoqing Hao , Xinran Mao , Bin Zhuge , Hong Zong

{"title":"RNA helicase CgDBP4 improves tolerance and fermentation performance of Candida glycerinogenes and Yarrowia lipolytica from undetoxified cellulose hydrolysate","authors":"Xinyao Lu , Xiaoqing Hao , Xinran Mao , Bin Zhuge , Hong Zong","doi":"10.1016/j.bej.2025.109919","DOIUrl":"10.1016/j.bej.2025.109919","url":null,"abstract":"<div><div>RNA helicase functions in RNA transcription, translation, and degradation that promotes the assembly of mRNP, affecting the cell's sugar transport process, DNA repair, and cell cycle progression. This study employed the RNA helicase CgDBP4 from <em>Candida glycerinogenes</em> as a stress-resistant genetic element for both <em>C. glycerinogenes</em> and <em>Y. lipolytica</em>. CgDBP4 affected cell tolerance of <em>C glycerinogenes</em> to NaCl, high sugar, acetic acid, and heat stresses. Under 44 ℃, the ethanol production rate of <em>C. g-antiDBP4</em> (It is a recombinant strain that moderately upregulates the transcriptional level of <em>CgDBP4</em>) increased from 1.5 g·(L·h⁻¹) to 2.0 g·(L·h⁻¹). <em>CgTy3</em> is a retrotransposon rescued the cell growth of the <em>C. g-antiDBP4</em> under heat stress. The downregulation of <em>CgTy3</em> impaired the transcription of RNA polymerase III, genomic stability, protein processing, and the activation of heat-responsive genes, consequently influencing the ability of <em>CgDBP4</em> in cell tolerate to heat stress. <em>CgDBP4</em> also enhanced the resistance of <em>Y. lipolytica</em> Po1f to multiple stresses and improved lipid titer by 44.5 % in typical media. Besides, <em>Y. l‐CgDBP4</em> produced 38.1 mg/L of lipids under 34 ℃ and 49.1 mg/L of lipids in undetoxified cellulose hydrolysates.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"225 ","pages":"Article 109919"},"PeriodicalIF":3.7,"publicationDate":"2025-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145004583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Silk fibroin hydrogel loaded with mTOR inhibitor regulates chondrocyte autophagy to improve cartilage cell damage 负载mTOR抑制剂的丝素蛋白水凝胶调节软骨细胞自噬，改善软骨细胞损伤

IF 3.7 3区生物学

Biochemical Engineering Journal Pub Date : 2025-08-26 DOI: 10.1016/j.bej.2025.109918

Chengchun Shen , Jiapei Yu , Shengtao Zou , Feng Zhang , Lei Huang

引用次数: 0