Comparison of non-catalytic and catalytic fast pyrolysis of corncob in a fluidized bed reactor

Material Sciences 材料科学, 2011, 1, 10-16doi:10.4236/ms.2011.11003 Published Online April 2011 (/journal/ms/)Recent Progress in Catalytic Materials for Catalytic Combustion of Chlorinated Volatile OrganicCompounds#Xuehua Yang1, Aidong Tang1*, Xianwei Li21School of Chemistry and Chemical Engineering, Central South University, Changsha2Institute of Environment and Resource, Baosteel Co., Ltd., ShanghaiReceived: Mar. 14th, 2011; revised: Apr. 15th, 2011; accepted: Apr. 20th, 2011.Abstract: The research progress in the catalytic combustion of Cl-VOCs(Chlorinated Volatile Organic Compounds) is reviewed. In this review, the effects of the active species, catalyst support, water vapor and coking on the catalytic combustion reaction were summarized. The research related to noble metal catalysts mainly focuses on developing new supports and dual noble catalysts. The research on non-noble metal cata-lysts concentrate on the development of transition metal mixed oxide, perovskites and spinel catalysts; The chlorination of active species is regarded as an important reason for catalyst deactivation. Besides, the effects of water vapor and coking deactivation on the catalytic combustion process are discussed with considering the practical application. This review will be helpful in choosing an appropriate catalyst and the optimal reac-tion conditions for the removal of Cl-VOCs by catalytic combustion with high activity and high stability. Keywords:Catalyst; Noble metal; Metal oxide; Chlorinated Volatile Organic Compounds; Deactivation催化燃烧Cl-VOCs催化材料的研究进展#杨学华1，唐爱东1*，李咸伟21中南大学化学化工学院，长沙2宝钢股份研究院环境与资源研究所，上海收稿日期：2011年3月14日；修回日期：2011年4月15日；录用日期：2011年4月20日摘 要：从催化剂活性组分、催化剂载体、催化剂失活三个方面，对近年来催化燃烧含氯挥发性有机物(Cl-VOCs)催化剂的研究进行了总结。

1 目的与意义TiO2是一种良好的半导体光催化材料，它以光催化效率高、无二次污染、使用范围广、无毒无害、价格低廉等特点，在光催化领域受到了广泛的关注与研究[1-5]。

但其光生电子-空穴易复合，粒子易团聚，不利于光催化反应持久稳定地进行，研究者通过掺杂与负载两种方法来提高其光催化活性[5-10]。

银是一种良好的杀菌剂，通常高价银离子的杀菌效果比较好[11-12]，最新的研究报道指出，纳米单质银粒子拥有比高价银离子更好的杀菌性能，银粒子还可以作为杂质而被引入到TiO2粒子中，来提高催化剂的催化活性[12-15]。

碳纳米管拥有多层管壁和纳米级管腔结构，有较大的比表面积、较高的表面结合能、良好的导电性、较好的化学稳定性以及高机械强度，是一种良好的催化剂载体材料。

采用碳纳米管对TiO2进行负载改性处理，可以提高其分散性，从而进一步提高催化活性。

2 银纳米粉体的制备与表征[16]2.1实验参数的选择通常只要具有微弱的还原性即可将银离子还原，因而采用化学还原法制备纳米银粉，可选用的还原剂有很多。

常用的还原剂有NaBH4, H202、柠檬酸钠、抗坏血酸((VC)、水合脐、葡萄糖等。

在一定条件下，随还原剂还原能力的降低，银颗粒的成核、长大速度降低，所获得纳米粒子的尺寸减小。

如果还原能力过低，则反应十分缓慢，成核过程变的困难，反应的进行主要靠颗粒的长大来完成，颗粒粒径增大。

由于葡萄糖在酸性和中性条件下还原能力较弱，与银离子的反应十分缓慢，不利于获得细小的颗粒。

但是提高溶液的pH值和温度，可以提高葡萄糖的还原能力，从而可以通过调整pH值和温度的方式来获得适中的还原能力以制备纳米银粉。

本实验采用葡萄糖为还原剂以制备纳米银粉，并采用升高溶液的pH值和反应温度来改善葡萄糖的还原能力。

为了保证银离子完全被还原，还原剂的用量应保持适当过量，本实验采用葡萄糖与银离子的摩尔比为2:1。

为了获得分散性良好的纳米粒子，通常会在颗粒的制备过程中加入分散剂对颗粒进行保护以阻止其长大和团聚。

第2部分重要问答题总结1.细胞学说的内容有哪些? The content of the cell theory have?①一切动植物都由细胞发育而来，即生物是由细胞和细胞产物所组成。

②所有细胞在结构和组成上基本相似。

③生物体是通过其细胞的活动反映其功能的。

④新细胞由已存在的细胞分裂而来；⑤生物的疾病是因为其细胞机能失常导致的。

2.早期主要有哪些试验证实了DNA是遗传物质？1944，Avery 肺炎球菌转化小鼠试验；1952，Hershey噬菌体侵染细菌实验。

4.通常所说的分子生物学的三条基本原则是什么？举例说明之。

Usually say of molecular biology of three basic principles is what? The examples.①构成生物体的各类有机大分子的单体在不同的生物中都是相同的。

②生物体内一切有机大分子的建成都遵循共同的规则。

③某一生物体所拥有的核酸及蛋白质分子决定了它的属性。

5.现代分子生物学的主要研究领域有哪些？列举不少于三条。

Modern molecular biology major research fields have? List of not less than three.① DNA重组技术②基因表达调控研究③生物大分子的结构和功能④基因组、功能基因组与生物信息学研究6.简述DNA的化学组成。

This chemical composition of DNA.DNA由单体核苷酸首尾相接，以3′,5′-磷酸二酯键链接而成。

每个核苷酸由脱氧核苷和磷酸组成，而脱氧核苷由脱氧核糖和碱基ATCG组成。

7.染色体具有哪些作为遗传物质载体的特征？Chromosome, which have the characteristics of genetic material as a carrier?DNA分子结构应具有多样性和相对稳定性并能准确地自我复制。

氮掺杂碳负载镍基催化剂的设计合成及其电催化性能研究摘要：随着环境污染问题的日益严重，能源转化领域的研究备受关注。

设计、合成高效的催化剂是此领域的研究热点之一。

本研究采用杂化碳纳米材料为载体，将镍纳米材料与氮掺杂结合，制备出氮掺杂碳负载镍基催化剂。

通过SEM、TEM、XRD等表征手段对催化剂进行表征。

结果表明，氮掺杂对载体的晶格结构、表面电子结构和电荷密度分布有显著的影响，能够显著提升催化剂的电催化活性。

在酸性条件下，该催化剂对硫酸盐电解液中的氢气进行电催化还原反应时，表现出优异的电化学催化活性，且稳定性较好。

因此，该催化剂有望在能源转化领域中得到应用。

关键词：氮掺杂碳；镍纳米材料；催化剂；电催化性能；能源转化Introduction:随着全球对清洁能源需求的增加，能源转化领域的研究成为了研究的热点之一。

设计、合成高效的催化剂对于促进氢燃料电池和电化学制氢的发展至关重要。

传统的催化剂主要有Pt、Pd、Ru等，但是它们的使用受到了价格和供应的限制。

因此，寻找替代高效催化剂成为了研究重点之一。

杂化碳纳米材料因其特殊的晶体结构、优良的化学稳定性和高的导电性能，成为了作为载体制备催化剂的一种优良选择。

此外，将含氮组分掺杂到碳材料中，不仅可以增强催化剂对氧化物的吸附能力，还可以优化电子亲和力和电子密度分布等催化剂的属性，从而提升其催化活性。

本研究通过掺杂氮元素改变载体表面电荷分布和电子亲和力，将镍纳米材料均匀负载在氮掺杂碳载体上，制备出氮掺杂碳负载镍基催化剂。

采用SEM、TEM、XRD等表征手段对催化剂进行表征，并研究催化剂的电催化性能。

Results and discussion:实验结果表明，将镍纳米材料与氮掺杂碳载体结合，制备出氮掺杂碳负载镍基催化剂后，其表面结构得到了显著的改善。

此外，氮掺杂也使得催化剂电子密度分布更加均匀，并增强了界面导电性能。

这些因素促进了活性位点在表面形成，优化了催化剂的催化活性。

Circulation JournalOfficial Journal of the Japanese Circulation Society http://www.j-circ.or.jpReleased online December 8, 2012Mailing address: Scientific Committee of the Japanese Circulation Society, 8th Floor CUBE OIKE Bldg., 599 Bano-cho, Karasuma Aneyakoji, Nakagyo-ku, Kyoto 604-8172, Japan. E-mail: meeting@j-circ.or.jpThis English language document is a revised digest version of Guidelines for Clinical Cardiac Electrophysiologic Studies reported at the Japanese Circulation Society Joint Working Groups performed in 2010 (website: http://www.j-circ.or.jp/guideline/pdf/JCS2011_ogawas_d.pdf).Joint Working Groups: The Japanese Circulation Society, The Japanese Society of Pediatric Cardiology and Cardiac Surgery, The Japanese College of Cardiology, The Japanese Society of Electrocardiology, The Japanese Heart Rhythm Society ISSN-1346-9843 doi: 10.1253/circj.CJ-66-0055All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cj@j-circ.or.jpTable of ContentsIntroduction of the Revised Guidelines ···············498I E quipment, Technology and KnowledgeRequired in Electrophysiologic Studies ···········498 1. Equipment, Technology and Knowledge Required in Electrophysiologic Studies ....................................498 2. Radiation Exposures ................................................498II Sinus Node Function .............................................499III Atrioventricular Block (500)IV B undle Branch Block and IntraventricularConduction Disturbance ·····································501V Preexcitation Syndrome ······································502VI S upraventricular Tachycardia Other Than Atrioventricular ReciprocatingTachycardia ····························································502VII Atrial Flutter ··························································503VIII Atrial Fibrillation ·················································503IX Premature Ventricular Contraction ·················504 1. Methods of Electrophysiologic Studies ·····················504 2. Clinical Significance ··················································505X Nonsustained Ventricular Tachycardia ···········505XI Sustained Ventricular Tachycardia ·················505 1. Definition and the Mechanism ··································505XII Brugada Syndrome ·············································506 1. Indications for Electrophysiologic Studies in Patients With Brugada Syndrome ····························507XIII Idiopathic Ventricular Fibrillation ··················507 1. Idiopathic Ventricular Fibrillation Triggered by Right Ventricular Outflow Tract Premature Ventricular Contraction/Ventricular Tachycardia ······507 2. Early Repolarization Syndrome ································507 3. Short-Coupled Variant of Torsade de Pointes ·········508 4. Short QT Syndrome··················································508 5. Idiopathic Ventricular Fibrillation With No History of ECG Abnormalities ...............................................508XIV Long QT Syndrome . (509)XV V entricular Fibrillation Associated WithStructural Heart Diseases ·································509 1. Introduction ·······························································509 2. Indications for Electrophysiologic Studies ················509 3. Methods of Electrophysiologic Studies ·····················510 4. Clinical Significance ··················································510 5. Criteria for Implementation of Electrophysiologic Studies ······································································510 6. Indications for Treatment Using Electrophysiologic Studies········································510XVI Syncope of Unknown Etiology ·······················510 1. Indications for Electrophysiologic Studies ················510 2. Methods of Electrophysiologic Studies ·····················510 3. Criteria for Electrophysiologic Diagnosis of Syncope (510)XVII P atients After CardiopulmonaryResuscitation ·····················································511XVIII E valuation of Antiarrhythmic DrugEfficacy ·······························································511XIX Surgical Treatment of Arrhythmias ··············512XX Arrhythmias in Children ····································512XXI Cardiac Resynchronization Therapy ············512 1. Introduction ·······························································512 2. Indications for Electrophysiologic Studies ················512 3. Methods of Electrophysiologic Studies ·····················513 4. Clinical Significance ··················································513 5. Criteria for Indication of Cardiac Resynchronization Therapy Based on Electrophysiologic Studies Findings ·······················································513 6. Indications for Cardiac Resynchronization Therapy.....................................................................513XXII Cardiac Pacemaker Implantation .................513XXIII Catheter Ablation .............................................514References .. (514)(Circ J 2013; 77: 497 – 518)JCS GUIDELINES498JCS Joint Working GroupTreatment strategies for severe arrhythmias have evolved sig-nificantly based on the recent development of non-pharmaco-logical procedures such as catheter ablation, implantable car-dioverter defibrillator (ICD), and cardiac resynchronization therapy (CRT). Because of the invasiveness of these proce-dures, their indications should be carefully determined after thorough consideration of their short and long-term effects. Since the results of large-scale clinical studies in Europe and the United States have suggested that ICD/CRT be used for more patients for the primary prevention of sudden death in Japan, appropriate risk stratification is required to determine indications of these procedures.The history of clinical electrophysiologic studies (EPS) began when Scherlag et al. established His bundle electrogram in 1969. Use of His bundle electrogram dominated in the evalu-ation of sinus node function and atrioventricular (AV) conduc-tion during that era. In addition to the His bundle electrogram, the premature stimulation technique significantly contributed to the understanding of the pathogenesis of arrhythmias, and was established as an important method to evaluate the effi-cacy of antiarrhythmic drugs. This method was also used to identify arrythmic foci, which stimulated the development of ablation therapy.Under these circumstances, “the Guidelines for Clinical Car-diac Electrophysiologic Studies” was published in 2006 (2004–2005 Joint Working Groups Report, Chair: Iwao Yamaguchi) to provide guidance to physicians in Japan regarding the use of cardiac EPS in the diagnosis, selection of treatment strate-gies, and prediction of prognosis in patients with arrhythmias.1 The present edition of the guidelines was prepared as a partial revision that is undertaken once every five years. For the pres-ent edition, a new Joint Working Groups was organized and has worked for one year with the following considerations in mind: 1) Contents of this guideline should be consistent with those of “the Guidelines for Non-Pharmacotherapy of Cardiac Arrhythmias” and “the Guidelines for Indications and Proce-dures of Catheter Ablation” which were under preparation by the Japanese Circulation Society, and overlap with these guide-line documents should be avoided; 2) the authors who wrote chapters for the previous edition revised, and, if not possible, specialists with expertise in the relevant therapeutic areas revised each chapter; 3) substantial modifications were made for the chapters regarding therapeutic areas where new understand-ings of a disease have changed its diagnosis and treatment during the past five years. For example, regarding “Ventricu-lar Fibrillation” a new section “Idiopathic Ventricular Fibril-lation” was added to separate it from “Ventricular Fibrillation Associated with Structural Heart Diseases” because new con-cepts such as early repolarization syndrome and short QT syn-drome have been incorporated in the field. On the other hand, for Brugada syndrome and some other conditions, the indica-tions for EPS remained almost unchanged although new authors were involved since no consensus was achieved regarding the significance of clinical EPS in determining treatment strate-gies despite large amounts of new data were added. The sec-tion for the identification of ablation sites for the treatment of chronic atrial fibrillation (AF) was modified by the author who wrote the section in the previous version to reflect the change in indications for EPS to identify ablation sites in the treatment of chronic AF for which catheter ablation has become increas-ingly common.Indications for EPS (indications by class) are listed in the same format as before in this revision. However, for the points where the members of Joint Working Groups could not reach agreement due to the lack of sufficient evidence, the present view was included upon the discussion among the members for which further revision may be necessary.It is difficult to decide which should be included in a guide-line document to be revised every five years regarding those therapeutic areas that are rapidly evolving. The present guide-lines are the accumulation of wisdom of the members of the Joint Working Groups, who are top experts actively engaged in the area of arrhythmias and cardiac electrophysiology. Readers should use this guideline accordingly.1. Equipment, Technology and KnowledgeRequired in Electrophysiologic Studies2Although many types of electrode catheters are available for cardiac EPS, all electrode catheters share the same basic func-tion of recording intracardiac electrical potentials and stimu-lating heart muscles. Physicians should select appropriate elec-trode catheters according to the purpose of the test. The test requires an X-ray machine, a recorder to record surface elec-trocardiography (ECG) and intracardiac electrical potentials, and a stimulator to determine refractory periods and induce tachycardia (Table 1).3 A defibrillator and emergency resus-citation equipment should be prepared for use. EPS are per-formed by a team of physicians, nurses, radiology technicians and/or clinical engineers.Physicians who perform EPS should have expertise in insert-ing and manipulating electrode catheters and have extensive knowledge of clinical electrophysiology (especially on cardiac conduction and refractory period) (Table 2). The stimulator is used to induce arrhythmias, and the results are used to diag-nose arrhythmias and develop treatment strategies for each case. EPS must be performed carefully by preparing for rare but serious complications.2. Radiation ExposuresEPS may cause radiation exposure not only to the patient but also to the staff members in the laboratory. The amount of radiation exposure increases with the time spent during the test. Usually, the total exposure time rarely exceeds 10 minutes, and the amount of radiation exposure during EPS is smaller than that during coronary angiography.4 Since a posterior-anterior projection is commonly used in EPS, erythema and necrosis of the skin of the back may develop.5 The threshold level of radiation dose that causes radiation-induced skin injuries is 2 Gy, and the absorbed dose in the skin reaches to 2 Gy when the patient received X-rays from a conventional X-ray machine for a total of about 60 minutes.6 An EPS requires at most 10499JCS Guidelines for Clinical EPS minutes of radiation exposure, and the radiation exposure to the skin will not cross the threshold.Staff members are also exposed to X-rays in the laboratory due to X-ray scattering in every direction. Therefore, staff mem-bers should wear protective clothing which also covers their back. The operator who handles electrode catheters should wear a neck guard and goggles as well, and should be careful not to expose his/her hands and arms directly to X-rays. Radiationexposure to the patient and staff members should be mini-mized using appropriate X-ray techniques (e.g., narrowing the radiation field, reducing X-ray exposure time, avoiding mag-nified imaging, and holding the image intensifier close to the patient), appropriate equipment setting (e.g., using pulse radi-ation, a low image acquisition rate, and non-radiological devices), and appropriate protection measures.7(Indications by class are listed in Table 3.)Sinus node function is influenced by three factors, i.e., sinus node automaticity, sinoatrial conductivity, and the activity of the autonomic nerves affecting sinus node. The electrical impulse generated in the sinus node travels through the sino-atrial junction to the atria.Sick sinus syndrome (sinus node dysfunction) is character-ized by bradycardia due to abnormal sinus node automaticity or conductivity that causes central nervous system symptoms such as syncope and blackouts.Increased sinus rate caused by enhanced sinus node auto-maticity may be observed in patients with sympathicotonia, fever, hyperthyroidism and inappropriate sinus tachycardia (IST) or other conditions.Sick sinus syndrome is a clinical term used to describe signsand symptoms of sinus node dysfunction. In order to diagnose the condition, the relationship between sinus bradycardia and the symptoms must be confirmed. Patients in whom the rela-tionship between bradycardia and symptoms can be confirmed by long-term Holter ECG monitoring or other tests are not indi-cated for EPS. For patients who are suspected to have sinus node dysfunction but in whom the relationship between ECG findings and symptoms is not clear, noninvasive, long-term monitoring such as frequent use of Holter ECG monitoring, wireless ECG monitoring, and ambulatory ECG is performed to obtain ECG findings during the onset of symptoms. Elec-trophysiologic evaluation of sinus node function to determine sinus node recovery time (SNRT), sinoatrial conduction time, and intrinsic heart rate is beneficial when no definitive find-ings have been obtained with noninvasive assessment. DuringEPS, cardiac electrophysiologic studies.Adapted from Fujiki A. Electrophysiologic Studies: techniques and devices. In: Inoue H, Okumura K, editors. Clinical Cardiac Electrophysiologic Studies. p24. Igaku-Shion Ltd., Tokyo, 2002.3EPS, cardiac electrophysiologic studies.500JCS Joint Working GroupEPS patients should also be assessed for the presence/absence of supraventricular tachyarrhythmia, AV conduction distur-bance, and retrograde ventriculoatrial conduction to obtain information useful for the determination of treatment strate-gies, drug regimens and pacing modes. EPS are generally not necessary for patients in whom the relationship between bra-dycardia and symptoms has been confirmed with noninvasive methods such as ECG and Holter ECG monitoring and whoare not complicated with AV conduction disturbance or tachy-cardia, or have only asymptomatic sinus bradycardia.A new minimally-invasive ECG monitoring method using an implantable loop recorder implanted under the skin in the left anterior chest has been introduced to obtain detailed ECG data during syncope in patients with syncope of unknown eti-ology.(Indications by class are listed in Table 4.)AV block is defined as a delay or interruption in the transmis-sion of an impulse from the atria to the ventricles through the AV node, His bundle and His-Purkinje system. The severity of AV block is determined not only by ECG pattern but also by the sites of block. The severity is high especially among patients where the conduction is blocked within or below the Hisbundle since these blocks may induce more severe conduction block or represent unstable lower pacemakers that generate escape rhythms. It is therefore important to locate the sites of block in predicting prognosis and determining treatment strat-egies. Since there is a limit to locate the sites of block by using the standard 12-lead ECG, Holter ECG monitoring or exerciseECG, EPS is necessary for patients with AV block.AV, atrioventricular; ECG, electrocardiography; EPS, cardiac electrophysiologic studies.501 JCS Guidelines for Clinical EPS(Indications by class are listed in Table 5.)The intraventricular conduction system consists of the His bundle, right bundle branch, left bundle branch and Purkinje fibers. Although the geometry of the left bundle branch can vary greatly among individuals, it generally subdivides into the anterior and posterior fascicles in the interpretation of ECG. Table 6 lists the criteria for ECG-based diagnosis of intraven-tricular conduction disturbance.8 A blockage in either the right or left bundle of the conducting pathway is called bundle branch block. A blockage in either the left anterior or posterior fas-cicle is called fascicular block. Bifascicular (bilateral bundle branch) block shows up on the ECG as a combination of char-acteristic wave patterns of right bundle branch block in con-junction with either left anterior or posterior fascicular block.A conduction disturbance in all three fascicles is called trifas-cicular block, and shows up on the ECG as a mixture of wave-forms of bifascicular block and either first or second degree AV block. EPS using intracardiac electrogram recording andAV, atrioventricular; ECG, electrocardiography; EPS, cardiac electrophysiologic studies. ECG, electrocardiography.502JCS Joint Working Groupcardiac electrical stimulation are useful to predict the risk of AV block among patients with bundle branch block and/or intraventricular conduction disturbance.9 The presence of con-duction disturbance may be determined when at least one of the following criteria are met:1. A n HV interval of 55 msec or more for cases of bifascicular or trifascicular block.2. T he presence of HV block induced by continuous atrial stim-ulation at 150 bpm or less.103. A n effective refractory period of the His-Purkinje system of 450 msec or more.4. A t least a two-fold prolongation of the HV interval from baseline or a prolongation of the HV interval by 100 msec or more (the HV interval may prolong by 20% or less under normal condition), or induction of second or third degree block within or below the His bundle following a procain-amide loading (300 to 1,000 mg intravenous injection [I.V.]); or induction of second or third degree block by continuous ventricular stimulation at baseline or following administra-tion of procainamide or lidocaine (1 to 2 mg/kg I.V.).5. I nduction of ventricular tachycardia (VT) or ventricular fibrillation (VF) by programmed ventricular stimulation.(Indications by class are listed in Table 7.)American College of Cardiology/American Heart Association (ACC/AHA) Guidelines for Clinical Intracardiac Electrophys-iological and Catheter Ablation Procedures were published in 1995, and have been widely used as a guide for clinical EPS in patients with Wolff-Parkinson-White (WPW) syndrome.11 However, indications for EPS in this patient population may be changed when the safety of EPS is improved, the diagnos-tic criteria to specify high-risk patients are established, and the positive predictive value for sudden death is increased.12 Although the prevalence of sudden death is low among patients with WPW syndrome,13–15 it has been reported that 10% of young patients who resuscitated from cardiac arrest have WPWsyndrome.16 A broad consensus has been reached on the use-fulness of EPS in patients with WPW syndrome who have experienced cardiac arrest, syncopal attacks of unknown etiol-ogy, and/or tachycardia attacks.EPS may be indicated for symptomatic patients with Lown-Ganong-Levine (LGL) syndrome, a condition characterized by tachycardia, but are generally not indicated for asymptom-atic patients with LGL syndrome who only show a short PR interval without tachycardia.17Indications for EPS in patients with atypical WPW syndrome where the involvement of the Mahaim fiber is suspected should be in accordance with those in overt WPW syndrome.(Indications by class are listed in Table 8.)Paroxysmal supraventricular tachycardia not involving AV accessory pathways include tachycardias originated from atrial muscle (including the sinoatrial node) other than atrial flutter/AF, and tachycardias originated from the AV junction.18 Since the safety and efficacy of radiofrequency catheter ablation in the treatment of AV nodal reentrant tachycardia (AVNRT), the most common form of this type of tachycardias, have been established, patients with this type of tachycardia are generally indicated for EPS to make a definitive diagnosis prior to cath-eter ablation. EPS is especially useful for patients with uncom-mon-type AVNRT in assessing the mechanism and making differential diagnosis. Even patients in whom ablation is not planned often undergo EPS to determine the optimal drug regi-men and evaluate the efficacy of the treatment regimen. EPS is indicated for patients with atrial tachycardia (AT) when they are symptomatic and should be assessed in detail to clarify the mechanism and origin of tachycardia to select appropriate drugs.EPS, electrophysiologic studies; WPW, Wolff-Parkinson-White.。

Research review paperStable isotope probing in the metagenomics era:A bridge towards improved bioremediationOndrej Uhlik a ,Mary-Cathrine Leewis b ,Michal Strejcek a ,Lucie Musilova a ,Martina Mackova a ,1,Mary Beth Leigh b ,Tomas Macek a ,⁎a Institute of Chemical Technology Prague,Faculty of Food and Biochemical Technology,Department of Biochemistry and Microbiology,Technicka 3,16628Prague,Czech Republic bInstitute of Arctic Biology,University of Alaska Fairbanks,902N.Koyukuk Dr.,Fairbanks,AK 99775-7000,USAa b s t r a c ta r t i c l e i n f o Article history:Received 20April 2012Received in revised form 17September 2012Accepted 17September 2012Available online 26September 2012Keywords:Bioremediation BiodegradationStable isotope probing MetagenomicsSequence-based screening Function-based screening Gene-targeted metagenomics High-throughput sequencing High-throughput microarrays Carbon flowMicrobial biodegradation and biotransformation reactions are essential to most bioremediation processes,yet the speci fic organisms,genes,and mechanisms involved are often not well understood.Stable isotope probing (SIP)enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers.SIP is thus applicable as a tool for the identi fication of active members of the microbial community and associated genes integral to the community functional potential,such as biodegradative processes.The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes,processes,and applications,thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.©2012Elsevier Inc.All rights reserved.Contents1.Introduction ..............................................................1552.Principles of stable isotope probing (SIP)................................................1553.The metagenomics era .........................................................1554.Integration of SIP with metagenomics ..................................................1584.1.SIP and metagenomics to study biodegradation of ecologically signi ficant C 1compounds .......................1584.2.SIP and metagenomics to study biodegradation of anthropogenic compounds ............................1585.Potential contributions of SIP and metagenomics to bioremediation ....................................1595.1.Assessing bioremediation potential ................................................1595.2.Bioaugmentation ........................................................1605.3.Understanding the mechanisms underlying biostimulation and phytoremediation technologies ....................1605.4.Carbon flow through contaminated systems ............................................1606.Limitations ..............................................................1607.New frontiers of SIP ..........................................................1618.Alternatives to SIP ...........................................................1619.Conclusions ..............................................................162Acknowledgements .............................................................162References .. (162)Biotechnology Advances 31(2013)154–165⁎Corresponding author.Tel.:+420220443140;fax:+420220445167.E-mail addresses:ondrej.uhlik@vscht.cz (O.Uhlik),mcleewis@ (M.-C.Leewis),michal.strejcek@vscht.cz (M.Strejcek),lucie.musilova@vscht.cz (L.Musilova),mbleigh@ (M.B.Leigh),tomas.macek@vscht.cz (T.Macek).1In loving memory of Prof.Martina Macková(May 7,1965–August 2,2012).0734-9750/$–see front matter ©2012Elsevier Inc.All rights reserved./10.1016/j.biotechadv.2012.09.003Contents lists available at SciVerse ScienceDirectBiotechnology Advancesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m/l o c a t e /b i o t e c ha d v1.IntroductionIn addition to their critical role in global biogeochemical cycling, microbes play an essential role in the degradation,mineralization, or transformation of environmental pollutants and thus are poten-tially capable of restoring contaminated sites(Diaz,2004;Geoffrey, 2010;Liu and Suflita,1993).The natural biodegradative processes occurring in contaminated sites are known as intrinsic bioreme-diation or natural attenuation(Jørgensen,2007;Mulligan and Yong, 2004).These microbial biodegradative processes can often be accel-erated using various strategies,known as bioremediation.Together with phytoremediation,microbially mediated bioremediation is gen-erally considered an environmentally friendly,inexpensive,publicly accepted and promising means to remove contaminants from the environment(Macek et al.,2000;Schnoor et al.,1995).When designing a strategy for the bioremediation of any contam-inated site,an understanding of the indigenous microbial community can be highly rmation integral to the success of biore-mediation strategies can include:(i)identification of the microor-ganisms present in the contaminated site,(ii)investigation of their metabolic capabilities,and(iii)understanding of potential microbial community shifts in response to changing environmental factors (Lovley,2003).Much of this information,however,has been difficult to elucidate and is rarely achieved with traditional microbiological techniques.Until recently,knowledge of microbes involved in biore-mediation processes had been based mostly on culture-dependent studies,which do not account for the fact that laboratory conditions differ substantially from the environment.Cultivation-based tech-niques have been shown to target only about1%of microbes occur-ring in the environment(Lozupone and Knight,2008;Torsvik and Øvreås,2002;Zhang and Xu,2008).More importantly,cultivation often fails to predict which microbes and which specific metabolic pathways will be active under realistic environmental conditions (Morales and Holben,2011).The advent of molecular microbial ecology enabled culture-independent phylogenetic analyses of com-munities and functional genes,however,linking contaminant trans-formation to phylogenetic identity and specific genes/enzymes of metabolically active microbes remained a major challenge and still required cultivation.The development of stable isotope probing (SIP)was instrumental in circumventing the limitations of culture based investigations of biodegradation(Friedrich,2006;Wellington et al.,2003).The coupling of SIP with the rapidly advancingfield of metagenomics is further increasing its potential benefits to thefield of bioremediation.This review aims to discuss the emerging trends in stable isotope probing techniques integrated with metagenomics, which can provide researchers with an unparalleled understanding of contaminant biodegradation and biotransformation processes in the environment.2.Principles of stable isotope probing(SIP)SIP tracks the incorporation of heavy stable isotopes,mainly13C (Chen and Murrell,2010;Dumont and Murrell,2005;Madsen, 2010;Neufeld et al.,2007b;Radajewski et al.,2003;Uhlík et al., 2009a),15N(Bell et al.,2011;Buckley et al.,2007a,b;Roh et al., 2009),or rarely18O and2H(Aanderud and Lennon,2011;Woods et al.,2011),from specific substrates into phylogenetically infor-mative biomarkers associated with microbes that assimilate the substrate.After stable isotopes have been pulsed into the environ-ment and metabolically active cells have incorporated the label into their biomass,biomarkers are recovered and analyzed(Dunford and Neufeld,2010;Neufeld et al.,2007b)(Fig.1).Therefore,SIP is an ap-proach that can identify microbial populations with a defined function. Thefirst biomarkers introduced in SIP studies were phospholipid-derived fatty acids(PLFA)(Boschker et al.,1998),followed by DNA (Radajewski et al.,2000),and rRNA(Manefield et al.,2002).Although it is the most sensitive of the three,PLFA-SIP is restricted to the clas-sification of broad taxonomic groups(Table1).Analyzing nucleic acids(RNA or DNA)can be far more informative taxonomically, with rRNA being a more responsive biomarker than DNA.This is due to the fact that the rates of rRNA synthesis are much higher than those of DNA,and rRNA labeling is independent of cellular rep-lication(Manefield et al.,2002,2004;Whiteley et al.,2006).In addi-tion,reverse transcription and sequence analysis of16S rRNA provides equal resolution for phylogenetic identification as sequence analysis of16S rRNA genes(DNA,Table1).Ribosomal RNA-based analyses,however,cannot provide access to the functional genes responsible for the metabolic capabilities of the community,and generally13C-labeled mRNA is challenging to isolate in sufficient quantities for SIP(Neufeld et al.,2007a;Simon and Daniel,2009). However,success has been achieved with mRNA-SIP for detection of naphthalene dioxygenase transcripts following SIP incubations with naphthalene(Huang et al.,2009).Further research employing DNA-, mRNA-,and rRNA-SIP(Dumont et al.,2011)demonstrated higher rates of labeling of functional mRNAs than their genes,and hence higher sensitivity of mRNA-SIP compared to DNA-SIP(Table1).In addition,protein-SIP has recently been developed,taking advantage of proteins as a combined indicator for a specific metabolic activity as well as for obtaining phylogenetic information(Table1,Jehmlich et al.,2008a,b,2009).To date,when functional gene analyses coupled to specific assim-ilatory processes are desired,DNA-SIP has been most often employed, due to the relative ease of detecting functional genes in13C-DNA compared to mRNA.The use of either DNA-SIP or RNA-SIP can enable both the phylogenetic identification of those microbes performing the process as well as key metabolic genes possessed by the active populations likely to be involved in the process.When coupled with the rapidly expandingfield of metagenomics,DNA-SIP has the poten-tial for a focused,in-depth analysis of the collective genomes of the community active in a particular biodegradative process.This pro-vides definitive information about the populations active in specific assimilatory processes and also requires considerably less sequencing effort than a full metagenomic analysis of the total community.3.The metagenomics eraMetagenomics(also known as ecological genomics,community genomics,or environmental genomics)is a discipline that uses geno-mic methods to analyze natural ecological communities,namely the collective genomes in an environmental community(Handelsman et al.,1998;Riesenfeld et al.,2004).The major goal of metagenomics is to explicate the genomes of uncultured microbes,thereby permit-ting investigation of the broad diversity of taxonomically and phylo-genetically relevant genes,individual catabolic genes,and whole operons(Schmeisser et al.,2007).Metagenomics itself was initially recognized for its potential to aid in discovery of novel biomolecules for biotechnological applications(Riesenfeld et al.,2004).Although the basic concept of metagenomics wasfirst introduced at the end of last century(Handelsman et al.,1998),early forms of metagenomics had begun to emerge previously,with one example being the phyloge-netic analyses of environmental microbial communities(Pace et al., 1985).The approach introduced by Handelsman et al.(1998)involves extraction of the metagenome(genomic DNA from all organisms inhabiting the environment),its fragmentation,cloning,transforma-tion,and subsequent screening of the constructed metagenomic li-brary.The primary aim is to screen environmental communities for a specific biological activity and identify genes or gene clusters asso-ciated with it;also referred to as function-based screening(Yun and Ryu,2005).The advent of high-throughput next generation DNA sequencing(e.g.454pyrosequencing,Illumina(Solexa)sequencing, SOLiD sequencing)gave rise to another approach to metagenomics,155O.Uhlik et al./Biotechnology Advances31(2013)154–165sequence-based screening.This method was first demonstrated by environmental genome shotgun sequencing of the Sargasso Sea (Venter et al.,2004),showing the potential of revealing the vast phy-logenetic and metabolic diversity of microbial communities (Yun and Ryu,2005).In addition to sequence-based screening of environmental metagenomic libraries,direct pyrosequencing of environmental com-munities is possible,bypassing cloning completely (Edwards et al.,2006).Because communities from habitats such as soils and sediments are often too diverse to permit screening in suf ficient depth,even with the use of high-throughput sequencing,gene-targeted metagenomics techniques have emerged (Iwai et al.,2011),which are based on sequencing of PCR-generated amplicons (Fig.2).In addition to high-throughput sequencing,metagenomic analy-ses have recently been performed with the use of high-throughput microarrays (Fig.2).These have been used to analyze microbial communities and monitor environmental biogeochemical processes.GeoChip microarrays (He et al.,2007,2010)currently contain 83,99250-meric sequences covering approximately 152,414genes encoding for enzymes responsible for biogeochemical (C,N,P,S)cycling,metabolic processes,heavy metal resistance,antibiotic resis-tance,degradation of pollutants,and gyrB genes (Hazen et al.,2010;Lu et al.,2012).Marker gyrB encoding for the gyrase β-subunit is used instead of the more common 16S rRNA genes as probes for 16S rRNA usually do not provide resolution below genus level.gyrBFig.1.Scheme of a DNA-based stable isotope probing (SIP)experiment with13C-labeled substrate.156O.Uhlik et al./Biotechnology Advances 31(2013)154–165can be used to differentiate even closely related species (He et al.,2010).GeoChip microarrays can be therefore used to study structure,dynamics,and potential metabolic activity of microbial communities and their variations depending on different stimuli.Another type of microarray valuable to microbial ecology and contaminant biode-gradation is the PhyloChip,which is used for high throughputTable 1Comparison of methodological considerations for DNA-SIP,RNA-SIP,PLFA-SIP,and protein-SIP.Trait Comparison of applicability of biomarkersExplanationSensitivity Protein>PLFA>RNA>DNA DNA-SIP requires 15–20%isotopic enrichment,while protein-SIP only requires 1%.RNA labeling is 6.5faster than that of DNA.Incubation time Protein>PLFA>RNA>DNA Incubation time is directly linked to sensitivity.DNA-SIP is the only technique that requires active cell division requiring the longest incubation periods potentially leading to biases.Taxonomic resolution DNA ≈RNA>protein>PLFA PLFA-SIP only distinguishes broader taxonomic groups,while DNA or RNA-SIP provides identi fication to the genus level or below.Databases for protein sequences are more limited than for 16S rRNA genes.Indication ofmetabolic activity Protein>RNA>DNA Proteins are the most explicit indicators of metabolic activity,while DNA only shows the metabolic potential.Ease of isolation DNA ≈PLFA>RNA>protein Isolation of PLFA and DNA are routinely performed in different matrices,but isolation of RNA and proteins from environmental samples can be very challenging.StabilityDNA ≈PLFA>protein>RNA DNA or PLFA are fairly stable,but proteins may denature,and mRNA is very sensitive to degradation.Application with ‘omics ’DNA>RNA>proteinThe application potential depends upon the developmental stage of the ‘omics ’methods.Currently,metagenomics is the most well-developed followed by metatranscriptomics and metaproteomics,respectively.Fig.2.Overview of metagenomic approaches that can be used to analyze stable isotope labeled metagenomes.157O.Uhlik et al./Biotechnology Advances 31(2013)154–165phylogenetic analyses of microbial communities(Brodie et al.,2006),and has been used for a variety of applications including assessing microbial community responses to petroleum contamination(DeAngelis et al., 2011;Hazen et al.,2010).With thefirst applications of metagenomic techniques,it became ap-parent that they enable the discovery of genomic and metabolic diversity that had not been previously imagined(Schloss and Handelsman,2005). As research progressed,however,the main drawbacks of metagenomics were realized:the inability to link specific functions to individual populations and to achieve full sequence coverage in more complex communities(Vieites et al.,2009).By combining metagenomic tech-niques with SIP,these limitations can be significantly reduced.SIP exper-iments are designed to provide a targeted analysis of the active populations,omitting the inactive majority that is not the focus of the study(Fig.2).4.Integration of SIP with metagenomicsOne of the main drawbacks of total community metagenomics is the unlikelihood of detecting particular genes of interest due to the extremely vast diversity and abundance of microbial genes occurring in most ecosystems,even when function-based metagenomic screen-ing is used.Targeting metagenomics to specific subpopulations that are likely to contain the genes of interest,as with SIP,may overcome this obstacle(Schloss and Handelsman,2003).This was demonstrat-ed by Schwarz et al.(2006)who isolated genes encoding for coen-zyme B12-dependent glycerol dehydratases.The source for this key enzyme for the anaerobic dehydration of glycerol was the enrichment cultures of13C-glycerol-fermenting microorganisms from a sediment sample.When metagenomic library construction was preceded by SIP,the frequency of clones bearing target genes was increased by almost four fold.However,expressing target genes successfully in vitro,such as in metagenomic libraries,can be very challenging in some ing high-throughput sequencing technologies for di-rect shotgun sequencing of SIP-derived metagenomes can aid in over-coming this obstacle.Selective enrichment of targeted populations, whose diversity is much less complex than that of total communities, with subsequent isolation of the particular functional metagenome of interest increases the feasibility of achieving coverage and assembly of individual genomes with significantly reduced efforts and sequencing cost.Shotgun sequencing of SIP-derived metagenomes can also help en-sure that portions of the microbial community that have low abundance but are integral to the metabolic processes of interest will not be overlooked(Wellington et al.,2003).The major drawback of this ap-proach is usually the recovery of DNA in quantities too small to be suf-ficient for shotgun sequencing.Advances in multiple displacement amplification over the last years(Binga et al.,2008)have helped to overcome this limitation.Although no bioremediation studies have yet been published performing direct shotgun sequencing of SIP-derived metagenomes,they are very likely to arise in the near future.4.1.SIP and metagenomics to study biodegradation of ecologicallysignificant C1compoundsOne of the pilot studies integrating DNA-SIP with metagenomics revealed a complete methane monooxygenase operon in forest soils (Dumont et al.,2006).Methane and other one-carbon(C1)com-pounds are of global ecological significance because they can affect climate change,influence atmospheric and marine chemistry,and im-pact cloud formation.In the context of bioremediation,methanotrophs and/or methylotrophs have been implicated in the degradation of trichloroethylene and cis-1,2-dichloroethylene(Arai et al.,1999;Little et al.,1988;Shigematsu et al.,1999;Takeuchi et al.,2005),insecticides (Topp et al.,1993),nitro-substituted explosives(Van Aken et al.,2004), methyl halides(Warner et al.,2005),methyl tert-butyl ether(Kane et al.,2007;Nakatsu et al.,2006),and other xenobiotic compounds.Therefore,investigating the metabolism of C1compounds is also poten-tially valuable for bioremediation.Dumont et al.(2006)were thefirst to apply SIP in combination with function-and sequence-based metagenomic library screening in thisfield.After the incubation of a soil sample with13CH4, 13C-DNA was used for the construction of a metagenomic library using a bacterial artificial chromosome(BAC).Subsequent screening of the library for key methylotrophy genes resulted in the discovery of a clone carrying a pmoCAB operon,encoding for the particulate methane monooxygenase.A complete sequence of the operon was determined by shotgun sequencing.The sequence of the identified pmoA gene was almost identical to a Methylocystis sp.sequence which had been previously detected in this soil(Radajewski et al., 2002).Additionally,12other putative genes were detected on the same clone(Dumont et al.,2006),3of which take part in beled methylotroph populations were also analyzed phylo-genetically by16S rRNA gene DGGEfingerprints and subsequent sequencing of dominant DGGE bands.In addition to Methylocystis, the methanotrophic genera Methylobacter and Methylocella were identified together with sequences similar to Bacteroidetes and γ-Proteobacteria.One of the main criticisms associated with SIP is considered to be in-appropriately high concentrations of labeled substrates introduced dur-ing incubations compared to concentrations that occur in situ.However, these high concentrations were necessary to achieve sufficient yields of labeled DNA for metagenomic analysis.Murrell and colleagues were thefirst to resolve this issue(Neufeld et al.,2008).Their strategy was the application of multiple displacement amplification to“bridge the gap”between the picogram quantities of labeled DNA and required mi-crogram quantities for subsequent metagenomic analyses.Their study found Methylophaga spp.to be involved in oceanic methanol cycling and detected a9-kb DNA fragment that encoded for the enzymes in-volved in methanol dehydrogenase synthesis,regulation,and assembly. Similarly,the techniques described were used for the analysis of as yet uncultured Methylocystis-related populations in acidic peatlands(Chen et al.,2008).These populations,which had been found to be dominant in the majority of acidic peatlands sampled,were further confirmed to be actively involved in oxidizing methane by SIP-based investigations. After13CH4had been assimilated,13C-labeled DNA containing the ge-nome of Methylocystis spp.was used for a construction of a metagenomic library and screened for the presence of key methylotrophic genes.Shot-gun sequencing of a clone containing methanol dehydrogenase gene permitted the researchers to assemble a gene cluster encoding polypep-tides involved in methanol utilization(mxaFJGIRSAC).These reports (Chen et al.,2008;Dumont et al.,2006;Neufeld et al.,2008)were thefirst ones to show that retrieval of targeted genetic information can be achieved with minimal sequencing effort.At the same time,the au-thors proposed early sequencing of complete genomes of microbial populations directly from the environment(Dumont et al.,2006).Not long after,a nearly complete genome was obtained of a novel uncultured methylotrophic bacterium Methylotenera mobilis from the water and sediments of Lake Washington,WA,USA(Kalyuzhnaya et al.,2008). This proof-of-principle study shows that genomes of ecologically rele-vant subpopulations can be reassembled after whole genome shotgun sequencing of stable isotopically labeled DNA.Additional results of this study revealed several clades of bacteria involved in C1substrate metab-olism;some were traditional methylotrophs such as Methylobacter, Methylomonas,Methyloversatilis,or Ralstonia,and others clustered with Verrucomicrobia,Nitrospirae,and Planctomycetes,clades not commonly associated with methylotrophs.4.2.SIP and metagenomics to study biodegradation of anthropogenic compoundsSome of the most widespread and environmentally significant xe-nobiotics are polychlorinated biphenyls(PCBs)(Breivik et al.,2002).158O.Uhlik et al./Biotechnology Advances31(2013)154–165Correspondingly,studies combining SIP and metagenomics to study PCB-degrading bacteria are common.Thefirst such study performed DNA-SIP integrated with GeoChip-mediated metagenomic analysis of bacteria in the root zone of an Austrian pine(Pinus nigra L.)grow-ing naturally in PCB-contaminated soil using13C-biphenyl,a PCB an-alogue,as a substrate(Leigh et al.,2007).Thefindings of this study pointed to novel populations of biphenyl-utilizing bacteria,including Pseudonocardia,Nocardioides,Kribella,Variovorax,and Polaromonas in addition to previously known PCB-degrading Sphingomonas spp. GeoChip analyses of13C-DNA detected30genes associated with organic contaminant degradation in the13C-DNA,majority of which were associated with the degradation of aromatics,including biphe-nyl,benzoate,catechol,protocatechuate,naphthalene,phenol,diben-zofuran,and phenylpropionate.The presence of these genes in biphenyl-labeled populations suggests that they have the potential to degrade several aromatic substrates.In addition,genes of the β-ketoadipate pathway were detected indicating potential abilities of the populations to mineralize monoaromatics once biphenyl has been transformed into monoaromatic intermediates.Only four genes,however,were detected from the biphenyl upper pathway (bph operon)associated with rhodococci and bacilli,suggesting that only a tiny fraction of the actual diversity in the upper biphenyl path-way genes had been revealed.This hypothesis was supported by PCR amplification and sequence analyses of genes encoding for aromatic ring hydroxylating dioxygenases(ARHD),all of which shared homol-ogy but were not identical to those previously deposited in GenBank. Thus,these novel genes were undetected using the microarray.More-over,some of the sequences did not cluster with any known ARHDs and represented a novel clade.Sul et al.(2009)applied metagenomics directly to isolate a novel biphenyl dioxygenase(bphA)gene from PCB-contaminated river sed-iment bacteria enriched by the incubation with13C-biphenyl.Biphe-nyl dioxygenase(BphAE),a multicomponent enzyme catalyzing the activation of biphenyl ring by insertion of two oxygen atoms,is cru-cial for biodegradation of biphenyl.Degradation of PCBs is permitted by relaxed substrate specificity of the enzyme,which has been deter-mined to be closely connected with its primary structure(Vézina et al.,2007,2008).The dioxygenase sequence detected by Sul et al. (2009)was highly similar to that in Pseudomonas sp.Cam-1and Pseudomonas pseudoalcaligenes KF707.Although in most laboratory PCB-degrading strains,genes bphAE are organized in operons with other enzymes for subsequent transformation of dihydroxylated bi-phenyl,this clone only contained bphAE genes.The authors ascribe this phenomenon to an acquisition of the genes from another micro-organism during exposure of the sediment to PCBs,possibly by hori-zontal gene transfer.This hypothesis was supported by a different G+C content of bphAE than the average for the cloned fragment. The activity of BphAE was tested after expression of the genes bphAE from the cosmid clone along with bphFGBC from Burkholderia xenovorans LB400.The spectrum of metabolized PCB congeners was similar to that of P.pseudoalcaligenes KF707,transforming only the congeners without chloro substitutions at the2,3positions.The identification of previously characterized organisms and genes by Sul et al.(2009)may be due to the long SIP incubation and enrichment of fast-growing organisms that would be amenable to detection using cultivation-based approaches.In the future,com-bining direct shotgun metagenomic sequencing or function-based screening with DNA-SIP could enable the discovery of highly novel degradative genes,rather than those with similarity to known sequences as were detected with Leigh et al.(2007)and Sul et al. (2009)using PCR-based or microarray-based detection.Although Sul et al.(2009)did not discover a dioxygenase with a broader sub-strate specificity than had been observed previously,this study con-tributes to our understanding of genomic features of degradative populations.This particular case suggests that the gene organization of bph genes in nature might be scattered rather than clustered in operons.The idea of catabolic genes being dispersed on chromosomes and plasmids was supported by another paper which discusses the organization of aromatic degradation pathway genes(Suenaga et al., 2009).Thirty-eight fosmid clones were analyzed carrying genes for extradiol dioxygenases,and only two of the clones contained complete degradation pathways that are commonly found in aromatic compound-utilizing isolates.The other clones contained only subsets of the pathway genes with novel gene arrangements.Recent results(Uhlík et al.,2012)demonstrated the ability of biphenyl-metabolizing bacteria to utilize other aromatic compounds in contaminated soil;populations of Rhodanobacter,Burkholderia, Pandoraea,Dyella and other Proteobacteria were observed to derive carbon from benzoate and naphthalene in addition to biphenyl.This study combined SIP with sequence analysis of16S rRNA gene pyrotags amplified from13C-DNA to identify taxa associated with the biodegradation of pollutants.Results of a few recently published bioremediation-related SIP studies reveal bacteria that had not been associated with utilization of the substrates before.Examples include newly associated populations of Pusillimonas or Rhodanobacter with the degradation of biphenyl(Lee et al.,2011;Uhlík et al.,2012)and Thermincola with the degradation of toluene(Pilloni et al.,2011).In addition,many unclassified16S rRNA gene sequences were retrieved from13C-DNA labeled by different substrates pointing to novel yet-to-be described bacterial taxa involved in biodegradation of biphenyl,benzoate,naphthalene,or toluene(Lee et al.,2011;Pilloni et al.,2011;Uhlík et al.,2012).5.Potential contributions of SIP and metagenomicsto bioremediationFundamental research on microbial aspects of bioremediation improves understandings of processes and could potentially improve bioremediation technologies.Thorough analysis of the labeled metagenomes of bioremediative populations can provide valuable in-formation for assessing bioremediation potential of autochthonous microorganisms as well as designing and monitoring engineered bio-remediation strategies.5.1.Assessing bioremediation potentialBefore bioremediation strategies are applied to a contaminated site,the bioremediation potential of the indigenous microflora should be assessed.In this case,SIP can be valuable for determining whether organisms capable of metabolizing the contaminant are already pres-ent at the site.If so,then biostimulation would likely be a viable bioremediation strategy.For these purposes,SIP incubations can be performed either in vitro using microcosms constructed fromfield-collected samples(Leigh et al.,2007;Uhlík et al.,2009b;Winderl et al.,2010)or directly in situ(Bombach et al.,2010;DeRito et al., 2005;Liou et al.,2008;Mahmood et al.,2005;Padmanabhan et al., 2003;Pumphrey and Madsen,2008).Metagenomic functional gene analyses of SIP studies are particularly valuable in the case of bio-degradation of xenobiotics that occur as mixtures,such as PCBs, since biphenyl dioxygenase enzymes vary widely in their capability to degrade different lower chlorinated PCB congeners(Erickson and Mondello,1993;Mondello et al.,1997).Analyzing the SIP-labeled dioxygenase gene sequence in relation to known enzymes with known substrate specificities can help predict which of the congeners are likely to be degraded by the microbial community(Barriault et al., 2002;Vézina et al.,2008).The absence or inactivity of dioxygenases with appropriate congener specificity for the PCBs on-site might indi-cate that an anaerobic treatment to promote dehalogenation would be appropriate(Smidt and de Vos,2004;Wiegel and Wu,2000).In addition,metagenomic exploration of active populations can clarify the metabolic capabilities as well as regulatory mechanisms within microbes,such as through sequence-based detection of regulatory159O.Uhlik et al./Biotechnology Advances31(2013)154–165。

高中克隆与不克隆辩论英语作文Debate on Cloning in High SchoolEnglish Response:The debate on cloning in high school is a complex and multifaceted issue that has been the subject of much discussion and controversy. On one side, proponents of cloning argue that it has the potential to bring about significant advancements in fields such as medicine, agriculture, and scientific research. They contend that the ability to clone individuals could lead to the developmentof new treatments for diseases, the production of more resilient and nutritious crops, and the preservation of endangered species.However, opponents of cloning raise a number of ethical and practical concerns. They argue that the process of cloning raises issues of bodily autonomy, personal identity, and the potential for exploitation. There are also concerns about the long-term consequences of cloning, such as the impact on genetic diversity and the possibility of unintended consequences.Ultimately, the decision to allow or ban cloning in high schools is a complex one that requires carefulconsideration of the potential benefits and risks. Proponents of cloning argue that it is a valuable tool that should be explored and utilized, while opponents argue that the ethical and practical concerns outweigh the potential benefits.Chinese Response:高中克隆与不克隆的辩论是一个复杂多面的问题,一直是讨论和争议的焦点。