1、BSI Standards PublicationPD CEN ISO/TS 29843-2:2014Soil quality Determinationof soil microbial diversityPart 2: Method by phospholipid fattyacid analysis (PLFA) using the simple PLFAextraction method (ISO/TS 29843-2:2011)PD CEN ISO/TS 29843-2:2014Incorporating corrigendum November 2014PD CEN ISO/TS
2、29843-2:2014 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of CEN ISO/TS 29843-2:2014. It is identical to ISO/TS 29843-2:2011. It supersedes DD ISO/TS 29843-2:2011 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee EH
3、/4, Soil quality.A list of organizations represented on this committee can 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. Publi
4、shed by BSI Standards Limited 2014ISBN 978 0 580 88685 0ICS 13.080.30Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 October 2011.Amendments/corrigenda issued
5、 since publicationDate Text affected31 October 2014 This corrigendum renumbers DD ISO/TS 29843-2:2011 as PD CEN ISO/TS 29843-2:201430 November 2014 Implementation of CEN Correction Notice 5 November 2014: ISO corrected text 1 February 2012 inserted, equation (1) added to subclause 6.1TECHNICAL SPECI
6、FICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 29843-2 October 2014 ICS 13.080.30 English Version Soil quality - Determination of soil microbial diversity - Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method (ISO/TS 29843-2:2011, Co
7、rrected version 2012-02-01) Qualit du sol - Dtermination de la diversit microbienne du sol - Partie 2 : Mthode par analyse des acides gras phospholipidiques (PLFA) en utilisant la mthode simple dextraction des PLFA (ISO/TS 29843-2:2011) Bodenbeschaffenheit - Bestimmung der Diversitt von Bodenmikroor
8、ganismen - Teil 2: Verfahren mittels Phospholipidfettsure(PLFA)-Analyse unter Verwendung des einfachen PLFA-Extraktionsverfahrens (ISO/TS 29843-2:2011) This Technical Specification (CEN/TS) was approved by CEN on 11 August 2014 for provisional application. The period of validity of this CEN/TS is li
9、mited 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 CEN/TS in the same way as for an EN and
10、 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 reached. CEN members are the national stan
11、dards 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, Poland, Portugal, Romania, Slovakia, Sl
12、ovenia, 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 exploitation in any form and by any means reserv
13、ed worldwide for CEN national Members. Ref. No. CEN ISO/TS 29843-2:2014 ECEN ISO/TS 29843-2:2014 (E) 3 Foreword The text of ISO/TS 29843-2:2011, Corrected version 2012-02-01 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO)
14、 and has been taken over as CEN ISO/TS 29843-2:2014 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 be the subject of patent rights. CEN and/or CENELEC shall no
15、t 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: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Den
16、mark, 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, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement n
17、otice The text of ISO/TS 29843-2:2011, Corrected version 2012-02-01 has been approved by CEN as CEN ISO/TS 29843-2:2014 without any modification. PD CEN ISO/TS 29843-2:2014CEN ISO/TS 29843-2:2014 (E)ISO/TS 29843-2:2011(E) ISO 2011 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope
18、12 Normative references. 13 Symbols and abbreviated terms (except chemical products and reagents). 14 Principle. 15 Test materials . 25.1 Soil. 25.2 Reagents 25.3 Apparatus 46 Procedures 56.1 Lipid extraction (Bligh-Dyer extraction) 56.2 Separation of lipids by SI column. 56.3 Derivatization Transme
19、thylation Clean-up. 56.4 PLFA analysis. 6Bibliography . 7PD CEN ISO/TS 29843-2:2014ISO/TS 29843-2:2011(E)ISO/TS 29843-2:2011(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Internatio
20、nal 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-governmental, in liaison with ISO
21、, 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 2.The main task of technical commi
22、ttees 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 vote.In other circumstances, parti
23、cularly 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 and is accepted for publication if i
24、t 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 members of the committee casting a vote.
25、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 three years, at which time it must eith
26、er 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.ISO/TS 29843-2 was prepared by Techni
27、cal 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) and phospholipid ether lipids (PLEL) a
28、nalysis Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction methodIn this corrected version, the missing Equation (1) in 6.1 has been added.iv ISO 2011 All rights reservedPD CEN ISO/TS 29843-2:2014ISO/TS 29843-2:2011(E)ISO/TS 29843-2:2011(E)ForewordISO (the Int
29、ernational Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). 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 ha
30、s been established has the right to be represented on that committee. International organizations, governmental and non-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
31、standardization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 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
32、for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a 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
33、Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group 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 agreem
34、ent between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members 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
35、International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three 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 m
36、ay be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights.ISO/TS 29843-2 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
37、 Soil quality Determination of soil microbial diversity: Part 1: Method by phospholipid fatty acid analysis (PLFA) and phospholipid ether lipids (PLEL) analysis Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction methodIn this corrected version, the missing Equ
38、ation (1) in 6.1 has been added.iv ISO 2011 All rights reservedISO/TS 29843-2:2011(E)IntroductionPhospholipids are essential components of membranes of all living cells. Extracted from soil samples in fatty acid form (PLFA: phospholipid fatty acids) or ether-linked isoprenoid side chains (PLEL: phos
39、pholipid ether lipid), they provide quantitative and qualitative insights into the soils viable/active microbial biomass. Cellular enzymes hydrolyse and release the phosphate group within minutes to hours following cell death (Reference 1). The determination of total PLFA and PLEL provides a quantit
40、ative measure of the viable biomass of soil, i.e. microorganisms from all three domains of the biosphere (bacteria, fungi and archaebacteria). PLFA and PLEL can also allow for taxonomic differentiation within complex microbial communities (References 2 and 3). This approach is now well established i
41、n soil ecology and serves as a phenotypic, and thus complementary, tool to genotypic (molecular genetic) approaches for determining microbial diversity. Apart from taxonomic descriptions, the PLFA technique enables the determination of physiological changes within microbial consortia. For instance,
42、the monoenic PLFA 16:17c and 18:17c are increasingly converted to the cyclopropyl fatty acids cy17:0 and cy19:0 in Gram-negative bacteria in response to environmental stress (Reference 4).Different methodologies are available for the determination of soil fatty acids. These methodologies present dif
43、ferent levels of complexity when applied and provide different levels of resolution in the description of soil microbial communities. ISO/TS 29843-1 deals with the generally called “extended PLFA extraction method” while this part of ISO/TS 29843 deals with the generally called “simple PLFA extracti
44、on method” (References 5 and6).This part of ISO/TS 29843, which deals with the simple PLFA extraction method, is accessible to most research and analytical laboratories involved in soil sciences. This methodology can be used for a wide range of soils. It provides a broad diversity measurement of a s
45、oil microbial community at the phenotypic level. It can be applied to biomass estimation and can be used to differentiate microbial communities among different soil samples (with the aid of an adapted statistical method). This method is especially adapted for detecting rapid changes in the soil micr
46、obial community structure. It can also be used to give a rough description of microbial groups present in soil samples (e.g. Gram-positive bacteria, actinomycetes, fungi). Table 1 (adapted from Table 1 in Reference 5), presents a comparison of the sensitivity of the “extended PLFA” versus “simple PL
47、FA” techniques.Table 1 Comparison of the sensitivity of the “simple” and “extended” PLFA techniques in characterizing shifts in the composition of microbial communitiesProperty PLFA (simple) PLFA (extended)Ability to differentiate between two communities (with the aid of multivariate statistical met
48、hods)Yes YesApplicability for biomass estimation Yes YesAbility to register all single components of an entire community structure (“fingerprint”)No YesAbility to register FAs other than EL-FAs No YesEstimation of number of FAs in soil samples 50 200 to 400Capacity to determine the linkage of the FA
49、s to lipids in the moleculeYes, EL Yes, EL, NELCapacity to detect defined FAs in lower concentrations in the soil extractNo YesCapacity to detect unusual FAs in the soil extract No YesNumber of available signatures of FAs for defined organismsFew High numbersRelationships of FAs widespread in the profile High NaturaAbility to identify the organisms causing the shift in microbial communityNo Basically yesThis method has been derived from the one first proposed in Reference 7 and later modified in Reference 1. This revised met
