Screen of O_2-tolerate Phenotype of Klebsiella oxytoca HP1 Mutants with High H_2-evolving Activity by Selection with MNZ Combination O_2

氢酶是生物制氢的关键酶,大多数氢酶因对氧极敏感而易失活,因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1%甲基磺酸乙酯对Klebsiella oxytoca HP1进行了两轮诱变,经40mmol/L甲硝唑和21%氧联合处理1h(第一轮诱变)或2h(第二轮诱变)进行筛选。所得突变菌株经产氢测试,结果在15%氧浓度条件下,第一代突变菌株HP1-A15产氢活性为出发菌株Klebsiella oxytoca HP1的3.70倍,在21%氧浓度条件下第二代突变菌株HPA15-37产氢活性为HP1-A15菌株的2.75倍,是出发菌株的11倍。突变菌株HP1-A15和HPA15-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。Hydrogenases are key enzyme for bio-hydrogen production, most of them were rapidly inacti-vated by oxygen. It is important to bio-hydrogen production and hydrogen application that improve the O2-tolerance of hydrogenase. In this experiment, the hydrogen producing strain Klebsiella oxytoca HP1 was treated with 1% ethyl methanesulfonate(EMS) , the mutants with high O2-toleration ability were screened with 40mmol/L metronidazole (MNZ) and 21% oxygen. The H2-evolving activity of the first generation mu-tant HP1-A15 was increased 3.70 times than that of the wild-type (WT) under 15% oxygen. The H2-evolving activity of the second generation mutant HPA15-37 was enhanced 11 times than that of WT under the condi-tion of 21% oxygen. The mutants HP1-A15 and HPA15-37 had steady heredity. These results suggest that MNZ and in addition oxygen is a good way to screen of O2-tolerate phenotype of facultativeanaerobe withhigh H2-evolving activity.福建省青年科技人才创新项目(No.12005J003);; 厦门市科技项目(No.13502Z20041070

Klebsiella oxytoca HP1 adhE基因插入失活法构建产氢重组菌

乙醇是产酸克雷伯氏菌(Klebsiella oxytoca)HP1厌氧发酵产H2的主要副产物,每生成1.0 mol的乙醇需要消耗2.0 mol NAD(P)H,从而降低了H2的产量.本研究以编码乙醇脱氢酶系(含乙醛脱氢酶和乙醇脱氢酶活性)的adhE基因为改造目标,利用同源重组技术获得了以提高产氢为目标的K.oxytoca重组菌.构建工作包括:根据adhE基因保守序列框克隆K.oxytoca HP1 adhE基因片段,以质粒pMHE6为模板进行链霉素抗性基因表达盒的扩增,表达链霉素抗性的aadA基因片段和adhE基因片段分别与载体pMD18-T相连构建重组质粒,同源整合质粒pTA-Str的构建,以链霉素作为筛选标记筛选重组菌.菌落PCR鉴定结果表明,aadA基因表达盒通过质粒pTA-Str的介导已定点插入K.oxytoca HP1基因组中,成功地构建了adhE基因部分片段缺失的重组菌.葡萄糖发酵实验结果表明,相同发酵条件下,重组菌比野生菌的产氢量提高了16.07%,乙醇产量下降了70.47%.利用基因工程技术提高产氢初步获得成功.国家自然科学基金(批准号:30470395);; 福建省重点科技项目(批准号:2005I106

Hydrogen Production by Chromatium vinosum with Fermentation Waste Produced by Klebsiella oxytoca

研究了酒色着色菌(Chromatiumvinosum DSM185)利用产酸克雷伯氏菌(Klebsiellaoxytoca HP1)发酵产氢废液进行光发酵和暗发酵产氢的可行性,以达到对产氢底物的充分利用和对产氢废液的进一步处理。研究结果表明C.vinosum可以利用K.oxytoca的发酵废液进行光发酵产氢和暗发酵产氢。C.vinosum发酵产氢后废液中残余还原糖和主要有机酸(丁酸)的含量明显降低,发酵产氢的最佳pH为6.5,添加0.1%(W/W)NH4Cl能促进产氢。在光照条件下丁酸利用率可达54.38%,产氢量达36.97mL/mg;在黑暗条件下丁酸利用率可达36.01%,产氢量达37.50mL/mg。Photosynthetic bacteria(PSB) showed great promise in biohydrogen production. Chromatium vinosum was able to utilize the fermentation waste of Klebsiella oxytoca for both photo-fermentative and dark-fermentative hydrogen production. The content of residual sugars and main organic acids decreased obviously after hydrogen production by C.vinosum. The maximal hydrogen production of C.vinosum was obtained at pH 6.5 adding extra 0.1%(W/W) NH_4Cl. Under photo-fermentative conditions, the content of butyric acid decreased by 54.38%, and the maximal hydrogen yield was 36.97 mL/mg cell. Under dark-fermentative conditions, the content of butyric acid decreased by 36.1% and the maximal hydrogen production was achieved as 37.50 mL/mg cell.国家863计划项目(No.2006AA05Z111);; 福建省科技项目(No.2006H0091);; 厦门大学创新项目(No.2002xjkt033)

Study on Hydrogen Production Activity of Klebsielal oxytoca HP1 and Stability of Its Soluble Hydrogenase under Atmosphere with O_2

目的:考察产酸克雷伯氏菌(Klebsielal oxytoca HP1)耐氧产氢特性及其可溶性氢酶的氧耐受特性。方法:研究K.oxytoca HP1在不同气相氧浓度条件下利用葡萄糖(1%,m/v)、丙酮酸钠(0.5%,m/v)及甲酸(0.1%,v/v)等底物产氢活性的以及K.oxyto-ca HP1可溶性氢酶在空气及氧饱和溶液中催化产氢活性。结果:K.oxytoca HP1在葡萄糖(1%,m/v)底物中具有较高耐氧产氢活性,6h内在气相氧浓度为5%、10%和21%条件下的氢产量分别为厌氧条件下的20.9%、13.7%、8.3%;K.oxytoca HP1可溶性氢酶在空气中孵育12h后,其活性残余85.4%,在氧饱和溶液中活性损失一半约3h。结论:试验结果提示K.oxytoca HP1具有耐氧产氢特性,其可溶性氢酶具有较高氧耐受性,在氢能源的开发中具有潜在的应用前景。Objective: To study the hydrogen production activity of K. oxytoca HP1 under condition with oxygen stress and the oxygen tolerance ability of its soluble hydrogenase. Method: The hydrogen production activity of K. oxytoca HP1 incubated in glucose (1%,m/v), sodium pyruvate (0.5%,m/v)or formate (0.1%,v/v) under atmosphere with 0%,5%,10% and 21% O2 were studied on, and the remain activity of its soluble hydrogenase incubated in air or pure O2 during 0-12h were studied on. Result: The results show that K. oxytoca HP1 has high hydrogen production activity in glucose (1%,m/v) substrate under atmosphere with oxygen. Compared to in anaerobic condition, the hydrogen production of K. oxytoca HP1 under atmosphere with 5%, 10% and 21% oxygen was 20.9%,13.7% and 8.3% respectively in 6h. The soluble hydrogenase from K. oxytoca HP1 exhibits considerable oxygen toleration ability, the remain activity was 85.4% after the hydrogenase exposure to air 12h and its activity lost half after the hydrogenase exposed to oxygen 3h. Conclusion:These results suggest that K. oxytoca HP1 and its soluble hydrogenase have potential to be applied in hydrogen production and application.福建省青年科技人才创新项目资助(No.12005J003

Isolation and Characterization of a H2 -producing Strain Enterbacter sakazakii HP

通讯作者 Tel 0592-2185731, E-mail： longmn@ xmu.edu.cn[中文文摘]在自然环境中分离到一株具有高产氢活性的微生物菌株,经细菌鉴定仪及16S rRNA基因序列分析,鉴定该菌株为Enterbacter sakazakii HP。分析了起始pH值、反应温度、碳源、起始糖浓度、起始氧浓度及菌体密度等因素对菌株产氢活性的影响。研究表明,该菌株发酵产氢较适合的条件为:以葡萄糖为产氢底物,起始pH值8.0,菌体密度OD600=0.7,反应温度35℃,糖浓度为0.1mol/L,氧浓度为0%的条件下,此时产氢菌株的最高产氢活性为5.34μmolH2/h.mgdw,氢的得率为1.94molH2/mol葡萄糖。[英文文摘]A H2 -producing bacterial strain was newly isolated and identified as Enterbacte sakazakii HP by 16S rDNA sequence analysis and detection by BBLCRYSTAL AUTOREADER.Various factors,including substrates and its concentration,initial pH,temperature and oxygen,on the hydrogen production of E.sakazakii HP have been studied extensively.Among several sugars,glucose was the favorite substrate for hydrogen production.The optimum condition for hydrogen production by Enterbacte sakazakii HP was achieved as:initial pH8.0,cell density OD600 =0.7,temperature 35℃,glucose concentration 0.1 mol/L,oxygen concentration 0%.Under batch fermentative hydrogen production conditions,the maximal hydrogen production activity and hydrogen yield were obtained as 5.34μmol H2 /h·mg dw and 1.94mol H2 /mol glucose,respectively.The research results suggest that Enterbacter sakazakii HP is an ideal candidater for biological hydrogen production.国家自然科学基金项目(No30470395);厦门市科技项目(No3502Z20041070);厦门大学新世纪优秀人才计划项目;福建省青年科技人才创新项目(No2005J003

Isolation and Characterization of H_2-producing Strains Enterobacter sp. and Clostridium sp.

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。Two hydrogen-producing bacterial strains were newly isolated and identified as Enterobacter sp. Z-16 and Clostridium sp. C-32 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen production of strain Z-16 were achieved as: initial pH7.0, temperature 35℃, sucrose as the favorite substrate. In comparison, The optimum condition for hydrogen production of strain C-32 were obtained as: initial pH8.0, temperature 35℃, maltose as the favorite substrate . Under batch fermentative hydrogen production conditions, the maximal hydrogen conversion rate for strain Z-16 and strain C-32 were 2.68 mol H_2/mol sucrose and 2.71mol H_2/mol maltose, respectively. Using glucose as substrate, the hydrogen conversion rate of strain Z-16 and strain C-32 were 2.35 and 2.48 mol H_2/mol glucose, respectively. This research suggest a good application potential of strain Z-16 and C-32 in the future biological hydrogen production.福建省重点科技项目(No.2005I016);; 厦门市科技项目(No.3502Z20041070)资助~

Purification and characterization of hydrogenase from Synechococcus sp. PCC 7942

报道了室温、空气环境下聚球藻Synechococcus sp.PCC 7942氢酶的分离纯化。经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46 U·mg-1蛋白。纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa。该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH 8.0。Hydrogenase from Synechococcus sp.PCC 7942 was purified to close homogeneity aerobically at room temperature.The hydrogenase-containing crude extract was collected after ultrasonic disruption and removal of cell debris by ultracentrifugation.Subsequently,three steps of column chromatographies(anion exchange,hydrophobic interaction and gel filtration)were performed.Hydrogenase was purified about 218-fold with a yield of 6.5% finally.The purified enzyme has a specific activity for hydrogen evolution of 1.46 U.mg-1 protein.SDS-PAGE gel of the purified enzyme revealed five predominant protein bands with estimated molecular weights of 83,60,47,30 and 27 kDa,respectively.The enzyme is a soluble bidirectional hydrogenase and shows maximum activity while using reduced methyl viologen as an electron donor.The optimum temperature and pH value for hydrogen evolution catalyzed by the purified hydrogenase are 50 ℃ and pH 8.0.福建省自然科学基金(C0410002)资

Enhanced hydrogen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1

Ethanol is the main byproduct of anaerobic H-2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD(P)H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3'-adenyltransferase (aadA) gene. As expected, the vector includes the insertion 5'-adhE-aadA-adhE-3'. The amplified DNA fragment 5'-adhE-aadA-adhE-31 from pTA-Str was transformed into K. oxytoca, HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H-2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H-2-production

基于递归系统模型的宫颈癌患者住院费用影响因素分析

目的分析宫颈癌患者住院费用的影响因素,为控制住院费用提供依据。方法收集厦门市某三甲医院2012 -; 2014年入院接受治疗且信息完整的宫颈癌患者的一般情况和住院费用等资料,通过递归系统模型分析各因素的直接与间接效应,计算总效应,找出费用控制的关; 键性因素。结果共纳入524例宫颈癌患者,其中医保患者占49.8%,首诊年龄的中位数为48岁,住院天数的中位数为28天,总费用的中位数为43841; .06元。支付方式、转移情况对总费用仅有直接效应,同时治疗其他疾病、MRI检查仅有间接效应;; 临床分期、治疗方案、手术方式既有直接效应又有间接效应;; 治疗方案的总效应值最大。结论宫颈癌住院费用受到多个因素的直接效应、间接效应、直接和间接效应的影响,其中以治疗方案的影响最大。可通过规范化制定治疗; 方案控制宫颈癌的住院费用。厦门大学大学生创新项目基

[[alternative]]臺灣大學生使用英文補語結構能力之探討

博士[[abstract]]本研究旨在探討台灣大學生使用英文補語結構的能力。共有一百五十八位台灣北部四所大學的學生參與本研究。參與者依照外語能力測驗的分數分成三組。本研究的測試工具為七十題的英文補語結構測驗。本研究探討的英文補語結構有三：一為單賓語及物動詞補語結構(monotransitive complementation)，二為雙賓語及物動詞補語結構(ditransitive complementation)，三為複雜的及物動詞補語結構(complex transitive complementation)。本研究結果顯示，學習者使用英文補語結構的能力與其英文程度有正相關性。在三類英文補語結構表現上，受測者在雙賓語及物動詞表現最好，在單賓語及物動詞表現次之，而在複雜的及物動詞表現最差。受測者在英文補語結構異體的(variants)表現上不一，與異體的語法結構複雜度、動詞所允許的補語數量、動詞與其補語結構間語意相關性，以及學習者的語法能力與注意力容納力有關。[[abstract]]The recognition and the use of English complementation is among the most frequent and problematic types of grammatical errors seen in EFL learners. In an attempt to account for the different performance patterns of verb complementation among Taiwanese university students, this study aimed to investigate Taiwanese university learners’ production of English complementation. A total of one hundred and fifty-eight students participated in the study. The students were divided into three groups according to their scores in Foreign Language Proficiency Test. A total of seventy-item test, covering various aspects of verb complementation, was used in the study. Results of the study revealed that there was a positive relationship between the learners’ English proficiency and their competence in producing and using English complementation. In addition, the learners as a whole performed best in ditransitive complementation; second in monotransitive complementation; and worst in complex transitive complementation. But they performed differently in variants of different types of English complementation in accordance with the complexity of the structures, the number of complementation that a verb takes, and the syntactic and semantic categories of a matrix verb. Besides, the learners as a whole performed better in retrospective verbs than in aspectual verbs that allow both to-infinitive and gerund constructions.[[tableofcontents]]Table of Contents Acknowledgements i Chinese Abstract iii English Abstract iv Table of Contents v List of Tables ix List of Figures xii List of Appendices xiii Chapter One Introduction 1 1.1 Statement of the Problem 3 1.2 Purpose of the Study 5 1.3 Research Questions 6 1.4 Definitions of Terms 7 1.5 Significance of the Study 9 Chapter Two Review of the Literature 11 2.1 The Concept of Complementation 11 2.2 Development of the Approach to English Complementation 17 2.2.1 The Traditional Approach 17 2.2.2 The Structuralist Approach 19 2.2.3 The Transformational-Generative Approach 20 2.2.4 Semantic/Functional Approach 22 2.3 Empirical Studies on the L2 Acquisition of English Complementation 28 2.4 Problems of Grammar Textbooks in Describing English Complementation 36 2.5 Second Language Learning and Systems 38 2.5.1 Error Analysis 38 2.5.2 Focus on Form versus Focus on Forms 41 2.5.3 The Role of Attention in Language Learning 42 2.5.4 The Role of Negative Evidence 44 2.6 The Theory of Markedness 46 2.6.1 Markedness and Second Language Acquisition 47 2.7 Grammatical Knowledge in Language Learning 51 2.7.1 What is Grammar 51 2.7.2 The Role of Grammatical Competence 53 2.8 The Role of Form-focused Instruction 56 2.8.1 Form-focused Instruction and the Development of L2 Proficiency 57 2.9 Purpura’s Theory of Grammatical Knowledge 60 2.10 A Theory of Second Language Production 61 2.11 Theoretical Framework of the Study 62 Chapter Three Methodology 63 3.1 Participants 63 3.2 Participants’ English Proficiency 64 3.3 Grouping 66 3.4 Types of Verb Complementation Used in the Study 67 3.5 Variants of Three Types of Verb Complementation Investigated in the Study 68 3.6 Materials 70 3.6.1 Task Design and Theoretical Constructs 70 3.7 Procedures 75 3.8 Data Collection 75 3.9 Scoring of the English Complementation Test 76 3.10 Statistical Analysis 81 Chapter Four Results 83 4.1 Learners’ English Proficiency vs. Competence in the Use of Verb Complementation 83 4.2 Learners’ Performance of the Six Tasks 84 4.3 Learners’ Performance of the Three Types of Verb Complementation 89 4.4 Learners’ Performance in Variants of Three Types of Verb Complementation 93 4.4.1 Subsection A: Results of the Variants of Monotransitive Complementation 93 4.4.2 Subsection B: Results of the Variants of Complex Transitive Complementation 101 4.4.3 Subsection C: Results of the Variants of Ditransitive Complementation 105 4.5 Learners’ Performance of Meaning-discrepancy Task 110 4.6 Learners’ Grammatical Competence and Learners’ Performance in Verb Complementation 115 4.7 Major Findings 116 Chapter Five Discussion 122 5.1 Learners’ English Proficiency and Competence in the Use of Verb Complementation 122 5.2 Learners’ Performance in Six Tasks 123 5.3 Learners’ Performance of the Three Types of Verb Complementation 127 5.4 Learners’ Performance in Variants of Three Types of Verb Complementation 131 5.4.1 Section A: Findings of the Variants of Monotransitive Complementation 131 5.4.2 Section B: Findings of the Variants of Complex Transitive Complementation 135 5.4.3 Section C: Findings of the Variants of Ditransitive Complementation 137 5.5 Learners’ Performance of Meaning-discrepancy Task 139 5.6 Learners’ Grammatical Competence and the Performance of English Complementation 142 Chapter Six Conclusions 145 6.1 Pedagogical Implications 149 6.2 Limitations of the Study 153 6.3 Recommendations for Further Research 154 References 156 Appendices 178 List of Tables Table 3.1 Student Grouping 67 Table 3.2 Three Types of Verb Complementation 68 Table 3.3 Variants of Monotransitive Complementation Type 68 Table 3.4 Variants of Complex Transitive Complementation 69 Table 3.5 Variants of Ditransitive Complementation 70 Table 3.6 Verbs Used in Task I 71 Table 3.7 Verbs Used in Task II 72 Table 3.8 Verbs Used in Task III 72 Table 3.9 Verbs Used in Task IV 73 Table 3.10 Verbs Used in Task V 73 Table 3.11 Verbs Used in Task VI 74 Table 4.1 Correlation between Learners’ FLPT Scores and Their Verb Complementation Test Scores 84 Table 4.2 Performance of the Six Tasks 85 Table 4.3 One-way ANOVA—Comparing the Mean Scores in Verb Complementation Test of the Groups 86 Table 4.4 Cross Comparison of the Performance between Groups in Verb Complementation Test 88 Table 4.5 Mean Scores of Three Types of Verb Complementation 89 Table 4.6 One-way ANOVA—Comparing the Mean Scores of Different Groups in Different Types of Verb Complementation 90 Table 4.7 Cross Comparison of the Differences of Different Groups 91 Table 4.8 Paired-samples t-Test for Different Types of Verb Complementation 92 Table 4.9 Mean Scores of Variants of Monotransitive Complementation 93 Table 4.10 One-way ANOVA—Comparing the Mean Scores in Different Variants of Monotransitive Complementation 97 Table 4.11 Cross Comparison of the Performance between the Three Groups 98 Table 4.12 Mean Scores of the Three Variants of Complex Transitive Complementation 101 Table 4.13 One-way ANOVA—Comparing the Three Variants of Complex Transitive Complementation between the Groups 103 Table 4.14 Cross Comparison of the Three Variants of Complex Transitive Complementation between the Three Groups 103 Table 4.15 Paired-samples t-Test for Differences between Variants of Complex Transitive Complementation 105 Table 4.16 Mean Scores in the Three Variants of Ditransitive Complementation 106 Table 4.17 One-way ANOVA—Comparing the Three Variants of Ditransitive Complementation between the Groups 107 Table 4.18 Cross Comparison of the Three Variants of Ditransitive Complementation between the Three Groups 108 Table 4.19 Paired-samples t-Test for Differences between Variants of Ditransitive Complementation 109 Table 4.20 Mean Scores in the Meaning-discrepancy Test 110 Table 4.21 One-way ANOVA—Comparing the Mean Scores in the Meaning-discrepancy Test 111 Table 4.22 Paired-samples t-Test for Significance in Aspectual and Retrospective Verbs 111 Table 4.23 Mean Scores of the Verbs that Take To-infinitive or Gerund 112 Table 4.24 One-way ANOVA—Differences in the Verbs Allowing Two Constructions 113 Table 4.25 Cross Comparison between the Groups 113 Table 4.26 Paired-samples t-Test for Verbs Allowing To-infinitive or Gerund Construction 114 Table 4.27 Correlation between Learners’ Grammatical Competence Scores and Their Verb Complementation Test Scores 115 List of Figures Figure 1 Basic meaning of infinitive to 140 List of Appendices Appendix A Syntactic Structures of Variants of Monotransitive Complementation 178 Appendix B Syntactic Structures of Variants of Complex Transitive Complementation 180 Appendix C Syntactic Structures of Variants of Ditransitive Complementation 181 Appendix D English Complementation Test 182 Appendix E Consent Form for the Study 188[[note]]學號: 891010034, 學年度: 9