1、BSI Standards PublicationDD ISO/TS 29843-1:2010Soil quality Determinationof soil microbial diversityPart 1: Method by phospholipid fatty acidanalysis (PLFA) and phospholipid ether lipids(PLEL) analysisPD CEN ISO/TS 29843-1:2014PD CEN ISO/TS 29843-1:2014 PUBLISHED DOCUMENTNational forewordThis Publis
2、hed Document is the UK implementation of CEN ISO/TS 29843-1:2014. It is identical to ISO/TS 29843-1:2010. It supersedes DD ISO/TS 29843-1:2010 which is withdrawn.This publication is not to be regarded as a British Standard.It is being issued in the Published Document series of publications and is of
3、 a provisional nature. It should be applied on this provisional basis, so that information and experience of its practical application can be obtained.Comments arising from the use of this Published Document are requested so that UK experience can be reported to the international organization respon
4、sible for its conversion to an international standard. A review of this publication will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the start of the review period will be made in an announ
5、cement in the appropriate issue of Update Standards.According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into an international Standard, to extend the life of the Technical Specification or to withdraw it. Comm
6、ents should be sent to the Secretary of the responsible BSI Technical Committee at British Standards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to Technical Committee EH/4, Soil quality.A list of organizations represented on this committee can
7、be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014ISBN 978 0 580 85503 0ICS 13.080.30Compli
8、ance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 October 2010.Amendments/corrigenda issued since publicationDate Text affected30 November 2014 This corrigendum ren
9、umbers DD ISO/TS 29843-1:2010 as PD CEN ISO/TS 29843-1:2014TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 29843-1 October 2014 ICS 13.080.30 English Version Soil quality - Determination of soil microbial diversity - Part 1: Method by phospholipid fatty acid analys
10、is (PLFA) and phospholipid ether lipids (PLEL) analysis (ISO/TS 29843-1:2010)Qualit du sol - Dtermination de la diversit microbienne du sol - Partie 1: Mthode par analyse des acides gras phospholipidiques (PLFA) et par analyse des lipides ther phospholipidiques (PLEL) (ISO/TS 29843-1:2010) Bodenbesc
11、haffenheit - Bestimmung der Diversitt von Bodenmikroorganismen - Teil 1: Verfahren mittels Phospholipidfettsure(PLFA)-Analyse und Phospholipidetherlipid(PLEL)-Analyse (ISO/TS 29843-1:2010) This Technical Specification (CEN/TS) was approved by CEN on 11 August 2014 for provisional application. The pe
12、riod of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CE
13、N/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reach
14、ed. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Po
15、land, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitatio
16、n in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN ISO/TS 29843-1:2014 EPD CEN ISO/TS 29843-1:2014CEN ISO/TS 29843-1:2014 (E)CEN ISO/TS 29843-1:2014 (E) 3 Foreword The text of ISO/TS 29843-1:2010 has been prepared by Technical Committee ISO/TC 190 “Soil quality“
17、 of the International Organization for Standardization (ISO) and has been taken over as CEN ISO/TS 29843-1:2014 by Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN. Attention is drawn to the possibility that some of the elements of this document may
18、be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this Technical Specification: Aust
19、ria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Swede
20、n, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO/TS 29843-1:2010 has been approved by CEN as CEN ISO/TS 29843-1:2014 without any modification. DD ISO/TS 29843-1:2010ISO/TS 29843-1:2010(E) ISO 2010 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scop
21、e1 2 Normative references1 3 Abbreviated terms .1 4 Principle2 5 Reagents and materials 3 5.1 Soil 3 5.2 Reagents.3 5.3 Buffers and standards 4 5.4 Apparatus.4 6 Procedures.5 6.1 Lipid extraction (Bligh-Dyer-extraction)5 6.2 Separation of lipids by sI-column5 6.3 PLFA analysis 5 6.3.1 Mild alkaline
22、hydrolysis 5 6.3.2 NH2column: Separation of FAME from OH-substituted FAME (= PLOH) and unsaponifiable lipids.5 6.3.3 SCX column: Separation of unsubstituted ester-linked PLFA (EL-PLFA).6 6.3.4 Acidic methylation of unsaponifiable lipids and separation into UNOH and UNSFA 6 6.3.5 TMSI derivatization
23、of PLOH and UNOH (see 5.2.22).6 6.3.6 DMDS derivatization of MUFA (see 5.2.8)6 6.4 PLEL analysis 7 6.4.1 General .7 6.4.2 Acidic methylation.7 6.4.3 Cleavage of etherbonds with hydroiodic acid (HI).7 6.4.4 Reductive dehalogenization with zinc.7 6.5 Measurement of PLFA/PLEL fractions 7 7 Identificati
24、on and calculation.8 Bibliography9 PD CEN ISO/TS 29843-1:2014ISO/TS 29843-1:2010(E)DD ISO/TS 29843-1:2010ISO/TS 29843-1:2010(E) iv ISO 2010 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies).
25、 The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-
26、governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part
27、2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a
28、 vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group
29、and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the memb
30、ers of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further th
31、ree years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights
32、. ISO/TS 29843-1 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. ISO/TS 29843 consists of the following parts, under the general title Soil quality Determination of soil microbial diversity: Part 1: Method by phospholipid fatty acid analysis (PLFA
33、) and phospholipid ether lipids (PLEL) analysis Part 2: Method by phospholipid fatty acid analysis (PLFA) using the “simple PLFA extraction method” PD CEN ISO/TS 29843-1:2014ISO/TS 29843-1:2010(E)DD ISO/TS 29843-1:2010ISO/TS 29843-1:2010(E) ISO 2010 All rights reserved vIntroduction Phospholipids ar
34、e essential components of membranes of all living cells, and their fatty acid (PLFA: phospholipid fatty acids) or ether-linked isoprenoid side chains (PLEL: phospholipid ether lipid) allow for taxonomic differentiation within complex microbial communities (References 5 and 7). This approach is now w
35、ell established in soil ecology and serves as a phenotypic and thus complementary tool to genotypic (molecular genetic) approaches for determining microbial diversity. Different methodologies for determination of soil fatty acids are available. These methodologies present different levels of complex
36、ity when applied and provide different levels of resolution in the description of soil microbial communities. The determination of total PLFA and PLEL provides a quantitative measure of the viable biomass of soil: microorganisms of all three domains of the biosphere (bacteria, fungi and archaebacter
37、ia). Viable microbes have an intact membrane, which contains phospholipids. Cellular enzymes hydrolyze and release the phosphate group within minutes or hours following cell death (Reference 6). Apart from taxonomic descriptions, the PLFA technique enables the determination of physiological changes
38、within microbial consortia. For instance, the monoenic PLFA 16:17c and 18:1 7c are increasingly converted to the cyclopropyl fatty acids cy17:0 and cy19:0 in Gram-negative bacteria in response to environmental stress (Reference 2). Besides the method described in this part of ISO/TS 29843, other met
39、hods for the determination of PLFA are available (References 3 and 6). With these methods, only bacterial and fungal PLFA can be estimated; the determination of hydroxy-substituted fatty acids (PLOH), non-ester-linked (NEL) fatty acids and PLEL is not possible. PD CEN ISO/TS 29843-1:2014ISO/TS 29843
40、-1:2010(E)DD ISO/TS 29843-1:2010PD CEN ISO/TS 29843-1:2014DD ISO/TS 29843-1:2010TECHNICAL SPECIFICATION ISO/TS 29843-1:2010(E) ISO 2010 All rights reserved 1Soil quality Determination of soil microbial diversity Part 1: Method by phospholipid fatty acid analysis (PLFA) and phospholipid ether lipids
41、(PLEL) analysis 1 Scope This part of ISO/TS 29843 specifies an extended method for the extraction and determination of both phospholipid fatty acids (PLFA) and phospholipid ether lipids (PLEL) from soils. ISO/TS 29843-2 specifies a simple method for the extraction of only PLFA from soils. 2 Normativ
42、e references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 10381-6, Soil quality Sampling P
43、art 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory 3 Abbreviated terms FAME fatty acid methyl ester(s) (EL-)PLFA (ester-linked) phospholipid fatty acid(s) PLEL phospholipid
44、ether lipid(s) SATFA saturated fatty acid(s) MUFA mono-unsaturated fatty acid(s) PUFA poly-unsaturated fatty acid(s) PLOH hydroxy-substituted fatty acid(s) NEL-PLFA non-ester-linked phospholipid fatty acid(s) UNSFA unsubstituted fatty acid(s) UNOH hydroxy-substituted fatty acid(s) GC/MS gas chromato
45、graphy/mass spectrometry PD CEN ISO/TS 29843-1:2014DD ISO/TS 29843-1:2010ISO/TS 29843-1:2010(E) 2 ISO 2010 All rights reservedSCX strong cation exchange HPLC high-performance liquid chromatography 4 Principle Lipids are extracted using the Bligh and Dyer9extraction procedure. Lipid extracts are sepa
46、rated by liquid chromatography using a silica column (si-column). Phospholipids are transformed into fatty acid methyl esters (FAME) by mild alkaline hydrolysis and into phospholipid ether lipids (PLEL) by acid hydrolysis and methylation. Separation of FAME into saturated (SATFA), mono-unsaturated (
47、MUFA), poly-unsaturated (PUFA), hydroxy-substituted (PLOH), non-ester-linked unsubstituted (NEL-UNSFA) and non-ester-linked hydroxy-substituted (NEL-UNOH) fatty acids is achieved on solid-phase extraction columns. The different FAME are measured using gas chromatography/mass spectrometry (GC/MS). A
48、schematic overview of the procedures is given in Figure 1. Figure 1 Schematic overview of PLFA and PLEL analysis PD CEN ISO/TS 29843-1:2014ISO/TS 29843-1:2010(E)DD ISO/TS 29843-1:2010ISO/TS 29843-1:2010(E) ISO 2010 All rights reserved 35 Reagents and materials 5.1 Soil Take soil samples and prepare
49、them as specified in ISO 10381-6. If samples which have been sieved in the fresh state are not analysed immediately, they may be kept at 20 C or stored in chloroform after lipid extraction (see 6.1). 5.2 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade. 5.2.1 Acetone, C3H6O, residue analysis. 5.2.2 Acetonitrile, CH3CN, for high-performance liquid chromatography (HPLC). 5.2.3 Bis(trimethylsilyl)trifluoroacetamide (BSTFA). 5.2.4 Celite 5451), particle size 0,02 m
