CEN ISO/TS 29843-2:2021

Soil quality - Determination of soil microbial diversity - Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method (ISO/TS 29843-2:2021)

This document specifies a simple method for the extraction of only phospholipid fatty acids (PLFA) from soils.

Bodenbeschaffenheit - Bestimmung der Diversität von Bodenmikroorganismen - Teil 2: Verfahren mittels Phospholipidfettsäure(PLFA)-Analyse unter Verwendung des einfachen PLFA-Extraktionsverfahrens (ISO/TS 29843-2:2021)

Dieses Dokument legt ein einfaches Verfahren ausschließlich zur Extraktion von Phospholipidfett-säuren (PLFA) aus Böden fest.

Qualité du sol - Détermination de la diversité microbienne du sol - Partie 2: Méthode par analyse des acides gras phospholipidiques (PLFA) en utilisant la méthode simple d'extraction des PLFA (ISO/TS 29843-2:2021)

Le présent document spécifie une méthode simple permettant d'extraire uniquement les acides gras phospholipidiques (PLFA) du sol.

Kakovost tal - Določevanje mikrobne raznolikosti tal - 2. del: Analiza fosfolipidnih maščobnih kislin (PLFA) z enostavno ekstrakcijsko metodo PLFA (ISO/TS 29843-2:2021)

General Information

• Status: Published
• Publication Date: 27-Jul-2021
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 28-Jul-2021
• Completion Date: 28-Jul-2021
• Ref Project: SIST-TS CEN ISO/TS 29843-2:2021 - Soil quality - Determination of soil microbial diversity - Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method (ISO/TS 29843-2:2021)

Relations

• Replaces: CEN ISO/TS 29843-2:2014 - 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, Corrected version 2012-02-01)
• Effective Date: 04-Aug-2021

Overview

CEN ISO/TS 29843-2:2021 - "Soil quality - Determination of soil microbial diversity - Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method" - specifies a straightforward, widely accessible procedure for extracting and analysing phospholipid fatty acids (PLFA) from soils. Published as a CEN-endorsed technical specification in 2021, it updates and supersedes the 2014 edition and aligns with ISO/TC 190 guidance on soil quality.

Key topics and requirements

• Scope: Extraction of only PLFAs from soil samples to provide quantitative and qualitative indicators of viable microbial biomass and broad microbial community structure.
• Principle: Extraction of membrane phospholipids followed by conversion to fatty acid methyl/alkyl esters for chromatographic analysis.
• Core procedures (document structure):
  • Lipid extraction (Bligh–Dyer type extraction)
  • Lipid class separation (silica column or commercial cartridges)
  • Derivatization / transmethylation and clean‑up to produce FAME/BAME
  • Analysis by GC or GC–MS
• Analytical options: The 2021 edition adds specification for qualitative and quantitative PLFA analysis, use of BAME (qualitative) or FAME (quantitative) standards, and explicit allowance for GC–MS instrumentation.
• Performance notes: The simple PLFA method is designed for accessibility and speed. Compared to the extended PLFA method, it provides:
  • Good ability to differentiate communities and estimate biomass
  • Lower resolution (fewer detectable fatty acids - typically <50 vs 200–400 for extended methods)
  • Reduced capacity to detect low–concentration or unusual fatty acids and to fully assign individual taxa

Applications and users

• Primary uses: Rapid assessment of soil microbial biomass, monitoring shifts in microbial community structure, phenotypic diversity screening, and detecting stress‑related physiological changes (e.g., changes in cyclopropyl fatty acids).
• Who uses it: Soil scientists, microbial ecologists, environmental testing laboratories, agronomy and land‑management researchers, remediation practitioners, and regulatory bodies concerned with soil quality, biodiversity monitoring, or impact assessment.
• Advantages: Accessible to most research and analytical labs, adaptable to a wide range of soil types, and useful for time‑series or disturbance studies where rapid detection of community shifts is needed.

Related standards

• ISO/TS 29843‑1 (extended PLFA extraction method) - for higher‑resolution fingerprinting and linkage of fatty acids to lipid classes.
• ISO 18400‑206 - sampling, handling and storage for microbiological assessment (referenced normative document).

Keywords: soil quality, soil microbial diversity, PLFA, phospholipid fatty acid analysis, ISO/TS 29843-2:2021, Bligh-Dyer extraction, GC-MS, FAME, biomass estimation.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2021
Nadomešča:
SIST-TS CEN ISO/TS 29843-2:2014
Kakovost tal - Določevanje mikrobne raznolikosti tal - 2. del: Analiza fosfolipidnih
maščobnih kislin (PLFA) z enostavno ekstrakcijsko metodo PLFA (ISO/TS 29843-
2:2021)
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-
2:2021)
Bodenbeschaffenheit - Bestimmung der Diversität von Bodenmikroorganismen - Teil 2:
Verfahren mittels Phospholipidfettsäure(PLFA)-Analyse unter Verwendung des einfachen
PLFA-Extraktionsverfahrens (ISO/TS 29843-2:2021)
Qualité du sol - Détermination de la diversité microbienne du sol - Partie 2: Méthode par
analyse des acides gras phospholipidiques (PLFA) en utilisant la méthode simple
d'extraction des PLFA (ISO/TS 29843-2:2021)
Ta slovenski standard je istoveten z: CEN ISO/TS 29843-2:2021
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN ISO/TS 29843-2
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
July 2021
TECHNISCHE SPEZIFIKATION
ICS 13.080.30 Supersedes CEN ISO/TS 29843-2:2014
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-
2:2021)
Qualité du sol - Détermination de la diversité Bodenbeschaffenheit - Bestimmung der Diversität von
microbienne du sol - Partie 2: Méthode par analyse des Bodenmikroorganismen - Teil 2: Verfahren mittels
acides gras phospholipidiques (PLFA) en utilisant la Phospholipidfettsäure(PLFA)-Analyse unter
méthode simple d'extraction des PLFA (ISO/TS 29843- Verwendung des einfachen PLFA-
2:2021) Extraktionsverfahrens (ISO/TS 29843-2:2021)
This Technical Specification (CEN/TS) was approved by CEN on 28 June 2021 for provisional application.

The period 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 CEN/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 reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 29843-2:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (CEN ISO/TS 29843-2:2021) has been prepared by Technical Committee ISO/TC 190
"Soil quality" in collaboration with Technical Committee CEN/TC 444 “Environmental characterization
of solid matrices” 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 shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN ISO/TS 29843-2:2014.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN websites.
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, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO/TS 29843-2:2021 has been approved by CEN as CEN ISO/TS 29843-2:2021 without any
modification.
TECHNICAL ISO/TS
SPECIFICATION 29843-2
Second edition
2021-07
Soil quality — Determination of soil
microbial diversity —
Part 2:
Method by phospholipid fatty acid
analysis (PLFA) using the simple PLFA
extraction method
Qualité du sol — Détermination de la diversité microbienne du sol —
Partie 2: Méthode par analyse des acides gras phospholipidiques
(PLFA) en utilisant la méthode simple d'extraction des PLFA
Reference number
ISO/TS 29843-2:2021(E)
©
ISO 2021
ISO/TS 29843-2:2021(E)
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved

ISO/TS 29843-2:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms (except chemical products and reagents) .1
5 Principle . 2
6 Test materials . 2
6.1 Soil . 2
6.2 Reagents. 2
6.3 Apparatus . 4
7 Procedures . 5
7.1 Lipid extraction (Bligh-Dyer extraction) . 5
7.2 Separation of lipids by SI column . 5
7.3 Derivatization — Transmethylation — Clean-up . 6
7.4 PLFA analysis . 6
Bibliography . 7
ISO/TS 29843-2:2021(E)
Foreword
ISO (the International 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 has 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 standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with
the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO/TS 29843-2:2011), which has been
technically revised.
The main changes compared to the previous edition are as follows:
— addition of specification for qualitative and quantitative analysis of PLFAs;
— use of BAME (qualitative) or FAME (quantitative) standards;
— use of GC-MS apparatus;
— precisions in 7.2 and 7.3;
— possibility to use commercial cartridges in addition to, or replacement of, home-made cartridges;
— update of bibliographic references.
A list of all the parts in the ISO/TS 29843 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved

ISO/TS 29843-2:2021(E)
Introduction
Phospholipids are essential components of membranes of all living cells. Extracted from soil samples
in fatty acid form (phospholipid fatty acids, PLFA) or ether-linked isoprenoid side chains (phospholipid
ether lipid, PLEL), they provide quantitative and qualitative insights into the soil's viable/active
microbial biomass. Cellular enzymes hydrolyse and release the phosphate group within minutes to
[1]
hours following cell death . The determination of total PLFA and PLEL provides a quantitative measure
of the viable biomass of soil, i.e. microorganisms of all three primary domains of the biosphere (bacteria,
archaea and microeukaryota). PLFA and PLEL can also allow for rough taxonomic differentiation within
[2],[3]
complex microbial communities . Each microbial species contains several fatty acids, with a total
[4]
composition in PLFA subject to the environmental conditions . The approach is performed to evaluate
[5]
biomass and shifts in microbial community composition , in what regards dominance of main groups
[6] 13 14
of organisms . Furthermore, combined with the use of isotope ( C or C) labelled substrates, the
lipid methods can also be used to identify the metabolically active part of the microbial community.
This approach is well established in soil ecology and serves as a phenotypic, and thus complementary,
tool to genotyping approaches for determining microbial diversity. Apart from taxonomic descriptions,
the PLFA technique enables the determination of physiological changes within microbial consortia. For
instance, the monoenic PLFA 16:1ω7c and 18:1ω7c are increasingly converted to the cyclopropyl fatty
[7]
acids cy17:0 and cy19:0 in Gram-negative bacteria in response to environmental stress .
Different methodologies are available for the determination of soil fatty acids. These methodologies
present different 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 document deals with the generally called “simple PLFA extraction
[8],[9]
method” .
This document 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 soil
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 microbial community
structure. It can also be used to give a rough description of microbial groups present in soil samples
[6]
(e.g. Gram-positive bacteria, actinomycetes, fungi ). Table 1 (adapted from Table 1 in Reference [8])
...