Manual on measurement of methane and nitrous oxide emissions from agricultureand nitrous oxide emissions from agriculture
A joint undertaking by the Food and Agriculture Organization of the United Nations
and the International Atomic Energy Agency
INTERNATIONAL ATOMIC ENERGY AGENCY
FROM AGRICULTURE IAEA, VIENNA, 1992 IAEA-TECDOC-674 ISSN 1011-4289
Printed by the IAEA in Austria November 1992
PLEASE BE AWARE THAT ALL OF THE MISSING PAGES IN THIS DOCUMENT
WERE ORIGINALLY BLANK
FOREWORD
The Inter-Governmental Panel on Climate Change (IPCC) was established by the ( înited Nations Environmental Programme and the World Meteorological Organi/ation to identity the causes and effects of climate change as well as strategies to limit and adapt to such changes. In order to reduce uncertainties and thereby predict climate and climate change on a global and regional basis and to design appropriate response strategics, the Ministerial Declaration of «he 2nd World Climate Conference held in Geneva in 1990 stressed the need to sustain and strengthen national and regional research activities directed to the areas of uncertainty identified by the IPCC. This Declaration furthermore cmphasi/ed that to achieve sustainable development, measures to meet the climate challenge must minimi/c the causes and adverse consequences of environmental degradation.
While there is some degree of uncertainty as to the rate and therefore potential impact of climate change, one of the basic causes, i.e. accumulation of "greenhouse gases" such as carbon dioxide (CO:), methane (CH4) and nitrous oxide (N;,O) is already well documented. Emissions of greenhouse gases arise from a variety of human activities, and these have been comprehensively documented through a report prepared in 199! by the Organisation for Economic Co-operation and Development (OECD) lor the IPCC. Amongst the activities highlighted in this report and in an earlier report produced for the IPCC by the United States Environmental Protection Agency, is Agriculture. Agricultural activities contribute directly to the "greenhouse effect" primarily through emissions of CH4 arising from rearing ruminant livestock, storing animal manure and growing rice, and of N:O as a result of using nitrogen fcrtili/.ers. Other activities related to agriculture which result in greenhouse gas emissions (e.g. land clearing and burning of biomass) are also covered in the OECD report.
Against this background, the IPCC identified two priority tasks: compilation of national greenhouse gas emissions inventories and assessment of opportunities for reducing these emissions, and urged all relevant national, regional and international organi/ations to co-ordinate their efforts towards attaining these objectives. Since then, a substantial amount of data has been collected and published on the different sources and sinks of greenhouse gases associated with agriculture, on their relative contributions to overall global warming, and on strategics for reducing emissions. In addition. FAO has developed an overall programme strategy which encompasses a special action field and other activities and which specifically addresses the question of agricultural sustainability and the environment.
Notwithstanding these encouraging developments, one of the major constraints to developing reliable greenhouse gas emissions estimates and hence assessing options for control which are consistent with sustained but environmentally-sound agricultural production, is that most developing countries lack information about the methodology for measuring greenhouse gas emissions and fluxes and for studying underlying mechanisms. To fulfill this need and hence assist the IPCC and FAO in support of their policy of sustainable and environmentally-sound agricultural development, the FAO decided to convene a meeting of specialists to consider the present methodologies for measuring CH4 and N:O emissions from Agriculture and to prepare detailed procedures and guidelines for their use. This publication arises from a meeting of such specialists, held at the Vienna International Center, Aus ria. in April 1992 under the auspices of the Joint FAO/1AEA Division of Nuclear Techniques in Food and Agriculture. It contains recommended methodologies for measuring CH., and N,O emissions (including requirements for equipment, sampling procedures, quality control) from different agricultural practices with the focus on livestock and rice production. Isotopic and non-isotopic methodologies are also described which can be used within the framework of research to measure pools and the dynamics of gas production and hence assess emission-reduction approaches. U is hoped that by encouraging the generation of data on greenhouse gas emissions from agricultural sources and the use of uniform and standardi/ed methodologies, this publication will assist governments in their efforts to develop environmentally-friendly and sustainable agricultural practices.
FAO and IAEA wish to record their sincere appreciation of the skill and dedication of the consultants who prepared this publication, i.e. Drs D. Beever, O. Van Cleemput. J.W. C/crkawski, M. Gibbs, K. Johnson, R. Leng, A. Mosier. W.H. Patrick, Jr.. J. Rowe, K. Smith, J. Wallace and R. Wassmann.
EDITORIAL NOTE
In preparing this material for the press, staff of the International Atomic Energy Agency have mounted and paginated the original manuscripts and given some attention to presentation.
The views expressed do not necessarily reflect those of the governments of the Member States or organizations under whose auspices the manuscripts were produced.
The use in this book of particular designations of countries or territories does not imply any judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and institutions or of the delimitation of their boundaries.
The mention of specific companies or of their products or brand names does not imply any endorsement or recommendation on the part of the IAEA.
CHAPTER 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CHAPTER 2. METHANE PRODUCTION IN RUMINANT ANIMALS . . . . . . . . . . . . . . 13
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1. INDIVIDUAL ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.1.1. Enclosure techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.1.1. Total enclosure of animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.1.1.2. Head box and mask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.1.2. Tracer methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2. 2.
.2.1. Isotopic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
.2.2. Non-isotopic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.1.3. Indirect methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2. .3.1. As VFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2. .3.2. From feed characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2. .3.3. In vitro incubations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2. GROUPS OF ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.2.1. Enclosure of animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.2.1.1. Continuous air flow approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.2.1.2. Restricted air flow approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.2. From feed characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2.3. Micrometeorological estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.3. GAS HANDLING. STORAGE AND ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.3.1. Sampling and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.3.2. Measuring CH4 concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.3.2.1. Flame ionization detector (FID) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 2.3.2.2. Thermal conductivity detector (TCD) . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.3.2.3. Infrared detector (IRD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.3.3. Measurement of SF6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2.3.4. Standards and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.4. METHANE FROM ANIMAL WASTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 General reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
CHAPTER 3. METHANE AND NITROUS OXIDE FLUX MEASUREMENTS FROM SOIL AND PLANT SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.1. SAMPLING TECHNIQUES AND SAMPLE HANDLING . . . . . . . . . . . . . . . . . . . . . . 45 3.1.1. Chamber (cover-box) methods - manual sampling . . . . . . . . . . . . . . . . . . . . . . . . 46
3. 3. 3. 3. 3.
,1.1. Chamber size and numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 . 1.2. Closed (static) chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 .1.3. Chambers for flooded rice systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 .1.4. Operating procedure and example of calculation . . . . . . . . . . . . . . . . . . . 50
1.5. Sampling from remote locations - Sample handling . . . . . . . . . . . . . . . . . 52
3.1.1.6. Comments on the chamber method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.I.Î.7. Open (dynamic) chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.2. Chamber method - automatic sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.1.2.1. General system description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.1.2.2. Suggestions for alternatives to equipment . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1.3. Micrometeorological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 3.1.3.1. Flux-gradient method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.1.3.2. Aerodynamic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.1.3.3. Bowcn ratio (energy balance method) . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.1.3.4. Eddy correlation approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.1.3.5. Alternative micrometeorological methods . . . . . . . . . . . . . . . . . . . . . . . . 63
3.1.4. Ultra-large chambers with long-path IR spectrometers . . . . . . . . . . . . . . . . . . . . . 64 3.1.5. Examples of flux measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1.5.1. Methane flux measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 3.1.5.2. Nitrous oxide flux measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 3.1.5.3. Factors affecting fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.2. METHODS FOR MECHANISTIC STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 3.2.1. Isotope techniques for identifying greenhouse gas production mechanisms and
quantifying emission rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 3.2.2. The acetylene inhibition method: Core and closed chamber protocols . . . . . . . . . . 72
3.2.2.1. Static core protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3.2.2.2. Closed chamber protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.3. ANALYTICAL ASPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.3.1. Methane analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.3.2. Nitrous oxide analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.3.3. Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 General reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5
Chapter 1 INTRODUCTION
The greenhouse effect
Carbon dioxide, water vapour, chlorofluorocarbons (CFCs), methane, nitrous oxide and ozone in the atmosphere arc the gases mainly responsible for atmospheric warming, or the "greenhouse effect". These gases are nearly transparent to the visible and near-infrared wavelengths in sunlight, and they absorb and re-emit downward a large fraction of the longer infrared radiation emitted by Earth. As a result of this heat-trapping, the atmosphere radiates large amounts of long-wavelength energy downward to the earth's surface. Consequently, long-wavelength radiant energy received on Earth is nearly double that received directly from the sun. Although the magnitude and timing of climate changes are uncertain, it is known that atmospheric concentrations of radiatively active gases are increasing.
The increased levels of greenhouse gases in the atmosphere are caused primarily by industrial activity. Increasing levels of CO,, the most important greenhouse gas, are mostly the result of fossil fuel combustion. Similarly, CFC emissions, which are being phased out under international agreement due to their o/.one-depleting characteristics, arc only produced by industrial processes and products. Nevertheless, as a major source of CH4 and N2O emissions and as a secondary contributor to CO-, emissions, increasing levels of agricultural activities are contributing to the buildup of greenhouse gases in the atmosphere (Tables 1.1 and 1.2).
The total annual "injection" of CH4 into the atmosphere is estimated at 400 to 600 Tg and emissions exceed oxidation by about 50 Tg/year1. So the abundance of CH4 in the atmosphere (currently about 1.7 ppm (v/v)), is increasing at about 1% per year. Even though the amount of CH4 in the atmosphere is increasing at the rate of only l/100th that of CO2, its high infrared absorption properties mean that it accounts for about 15 to 20% of the increased greenhouse effect.
Nitrous oxide is present in the Earth's atmosphere at very low concentrations, about 310 ppb (v/v). This low concentration is important to atmospheric warming because each molecule of N2O emitted into the atmosphere has an average life time of about 150 years and a net greenhouse effect about 300 times greater than a molecule of CO,. So despite its low and slowly increasing concentration (0.25%/year), N2O is becoming increasingly important in the overall global warming picture.
Contributions of agricultural practices
Methane
Globally, rice and ruminant animal production arc the two largest sources of anthropogenic CH4. While the actual magnitude of the emissions from each source remains uncertain, it has been estimated that these two sources (including emissions from animal waste management facilities) account for about 40 % of global CH4 emissions (Table 1.1). Methane is also produced in anaerobic environments such as marine and aquatic sediments and the hindgut of many animals and insects, but the rumen produces CH4 at a considerably higher rate per unit of biomass than these other systems.
Methane is also produced in soil during microbial decomposition of organic materials and carbon dioxide reduction under strictly anaerobic conditions, such as in natural freshwater wetlands and in flooded rice. Methane emissions from a particular ecosystem are basically controlled by two different microbial processes : CH4 production and CHa oxidation. Only thai part of CH4 which is not oxidized will enter the atmosphere. While CH4-producing bacteria (methanogens) require anoxic conditions, the
'Tg = teragram = H)12 gram.
SOURCES
Anthropogenic Sources
SINKS AND ACCUMULATION
Reaction with hydroxyl (OH) Removal by soils Removal in the Stratosphere Atmospheric Accumulation
Total Sources = Total Sinks + Accumulation
Estimate
100
420 30 10 37
70-120
34-40
420-600
Scientific Assessment of Climate Change prepared for the IPCC by Working Group I, published by the World Meteorological Organization and the United Nations Environment Programme, 1992 NR - Not reported
CH4-oxidizing bacteria (methanotrophs) require oxygen for metabolism (Conrad, 1989). When soils in these terrestrial systems dry out, they may oxidize methane from the atmosphere and become net CH4 sinks. Although these sinks have been known for some time, their importance and the factors affecting them are only now being investigated (Mosier and Schimel, 1991). In addition to soil methanotrophic bacteria, bacteria which oxidize ammonium to nitrite can oxidize CH4 to CO2. Exchange of CH4 with the stratosphere where it is oxidized is another small CH4 sink.
Other important agricultural sources of CH4 are the rumen of animals and animal waste. In the rumen, CH4 production is a specialized biochemical function which is generally considered to occur in archaebacteria rather than eubacteria. Some eubacteria produce small amounts of CH4, but only methanogenic archaebacteria derive energy from methanogenesis (Jones et al., 1987). The archaebacteria include methanogens, sulphate reducers, thermophiles and halophiles The melhanogens are very different to the vast majority of rumen microorganisms (which are eubacteria mostly fermenting a variety of fibres, sugars, peptides, aniino acids and other small molecules) because of
8
SOURCES
Temperate Soils - Forests — Grasslands
SINKS AND ACCUMULATION
Total Sources = Total Sinks + Accumulation
Range
0.05 - 2.0 ?
0.03 - 3.0 0.2 - 1.0 0.1 - 0.3 0.2 - 0.6 0.4 - 0.6 0.1 - 0.3
7 7- 13 3-4.5
10 - 17.5
Scientific Assessment of Climate Change prepared for the IPCC by Working Group I, published by the World Meteorological Organization and the United Nations Environment Programme, 1992
their specialized energy metabolism. Again, CH4 is an electron sink product in environments which are strictly anaerobic. In the rumen the primary population of fermentative bacteria form fermentation products such as volatile fatty acids (VFA), formate and H2. The methanogens then use the formate and H2 -f CO2 to form CH4. Methane production from manure, on the other hand, involves mainly the reduction of acetate generated by primary fermentation, a process which is of minor importance in the rumen because of its high turnover rate. Between 5 and 15% of the digestible energy consumed by animals is lost from the gut as CH4, although this depends on the efficiency of microbial growth. The fermentation stoichiometry of rumen fermentation is diet-dependent. Different populations of microorganisms give rise to different end-products of fermentation, so while CH4 production is not a constant proportion of the food consumed, in general it is related to the amount consumed. The amount
öl CH4 produced per unit amount of milk or meat is particularly high under dietary conditions where growth of rumen microorganisms is limited by nitrogen, phosphorus or sulfur availability rather than by energy.
Nitrous oxide
Terrestrial systems are the principal source of N2O (Table 1.2). Nitrous oxide is pioduccd not only during denitrification (reduction of nitrate to nitrous oxide and then to dinitrogen), but also during nitrification (oxidation of ammonium to nitrate). Both of these microbial processes are influenced by soil mineral N content, and therefore are influenced by N-fertiii/ation.
A large body of information concerning N2O emissions from agricultural systems and the influence of N-fertilization was accumulated during the late 1970s and early 1980s. Of the approximately 100 Tg of N fertilizer consumed annually worldwide, about 2-3 Tg N2O-N are evolved from the soil where the N was applied. The amount of N2O directly emitted from in-field biologically fixed N is not known. Mineral N applications along with organic matter additions generally increase total denitrification and N2O production.
Agricultural technologies are needed that maintain or enhance animal and rice production while decreasing or limiting increases in production of CH4 and N2O. Clearly, for those billions of people who are dependent upon rice production for survival, the question of CH4 and N2O production and the global greenhouse effect is only of secondary importance and thereto«« any new technologies developed to decrease emissions must increase animal and crop productivity. Nitrogen fertilizer use will obviously increase to meet the growing demands for food required by a rapidly expanding world population, and while fertilization is not the largest source of N2O, it is susceptible to management for reduced impact. Increased N2O production and decreased CH4 uptake caused by fertilization can be mitigated by agricultural management without decreasing production. While the application of nitrification inhibitors clearly decreases N2O production, this reduction is at the expense of decreased soil methane oxidation, although the latter consequence is less important. The role of fertilization on N2O production in tropical agriculture is particularly uncertain and the intensification of tropical agriculture highlights the importance of research into these effects.
Purpose of this manual
While adequate data exist to conclude that agricultural activities are an important source of CH4 and N2O emissions, measurements are 'acking in many areas. In particular, measurements are required to evaluate the variations in emission? from the diversity of current crop and animal management systems and the effects of emission-reduction options. This manual describes techniques to evaluate both current emissions from diverse animal and crop production practices and suggests methods for decreasing emissions of CH4 and N2O from agricultural systems.
In particular, it is suggested that the measurement techniques described are used to obtain data for the emissions sources that arc most uncertain and the options for decreasing emissions that are most likely to succeed. For example, CH4 and N2O emissions from the diverse flooded rice conditions found throughout Asia require study. Considerable research is needed to show which rice cultivars, improved nutrient management, water management and cultural practices may help reduce CH4 emissions while maintaining productivity and the generally low N2O emission rates.
Similarly, improved estimates of N2O emissions and CH4 emissions and sinks from other cropping systems are needed. Evaluation of emission-decreasing options should be encouraged, including: timing N application to periods when plant uptake is highest; applying fertilizers in a form so that N is released at a controlled, optimum rate (e.g. through the use of coatings); or applying nitrification inhibitors.
10
In the case of CH4 emissions from animals, attention should be focused on those animal management systems for which data are currently lacking, such as large ruminants subsisting on poor quality forages with and without supplementation. Opportunities for decreasing methane emissions from animals require considerable study, including : supplementation to improve fermentative digestion efficiency and/or to enhance productivity; manipulation of the fermentative digestion system (e.g. using feed additives): and feed treatment to improve feed quality.
With improvi/ation, the techniques described in this manual can be used under field conditions to obtain the desired data on CH4 and N2O emissions from agricultural sources. These techniques may also find value as they can be adapted to examine emissions from other N2O and CH4 sources such as landfills and waste treatment lagoons.
REFERENCES
Conrad, R. (1989). Control of methane production in terrestrial ecosystems. In : Andreae, M.O. and Schimel, D.S. (Eds) Exchange of trace gases between terrestrial ecosystems and the atmosphere. J. Wiley and Sons, Chichester, 39-58. Jones, W.J., Nagle, D.P. and Whitman, W.B. (1987). Methanogens and the diversity of archaebacteria. Microbiol. Rev. 51 135-177. Mosier, A.R. and Schimel. D.S. (1991). Influence of agricultural nitrogen on atmospheric methane and nitrous oxide. Chemistry and Industry, 2 December 1991, 874-877.
GENERAL READING
Bouwman, A.F. (Ed.) (1990). Soils and the greenhouse effect. J. Wiley and Sons, Chichester. Braatz, B.V. and Hogan, K.B. (Eds.) (1991). Sustainable rice productivity and methane reduction. Research Plan. U.S. Environmental Protection Agency, Office of Air and Radiation, Washington, D.C. Crutzen, P.J., Aselmann I., and Seiler, W. (1986). Methane production by domestic animals, wild ruminants, other herbivorous fauna and humans. Tellus 38 B 271-284. Houghton. J.T. Jenkin,G.J. and Ephraums, J.J. (Eds) (1990). Climate change: the IPCC scientific assessment. Cambridge University Press. Cambridge. Kimball, B.A. (Ed.) (1990). Impact of carbon dioxide, trace gases, and climate change on global agriculture. ASA Special Publication Number 53. Amer. Soc. Agron., Crop Sei. Soc. Amer, Soil Sei. Soc. Amer, Madison. Rogers, J.E. and Whitman, W.B. (Eds) (1991). Microbial production and consumption of greenhouse gases: methane, nitrogen oxides, and halomethanes. Amer. Soc. Microbiology. Washington, D.C. Scharpenseel, H.W., Schomaker, M. and Ayoub, A. (Eds) (1990). Soils on a warmer earth. Developments in Soil Science 20. Elsevier, Amsterdam.
11
INTRODUCTION
Many methods are available for measuring CH4 production in ruminant animals. They vary from simple short-term incubations of rumen contents (indirect method) to more elaborate systems such as respiration calorimetry. In all cases, the methods have strengths and weaknesses and need to be selected with care for a particular purpose.
The choice of method will depend on whether the measurements are to be made in individual animals or in groups of animals, whether the animals can be confined to a chamber or whether they should move freely during the measurements. If it is impossible to use any of the direct methods, then a much less accurate indirect method might have to be used, and examples of some of these methods are also given here.
2.1. INDIVIDUAL ANIMALS
2.1.1. Enclosure techniques
2.1.1.1. Total enclosure of animal (see Figure 2.1)
The technique of open circuit indirect respiration calorimetry has been routinely used with all animal species to determine the partition of dietary energy. This comprises the quantitative estimation of O2 consumption and CO2 production, but in ruminant livestock, the procedures have been extended to determine total CH4 production, arising largely from rumen fermentation, in order to provide more precise estimates of relationships between dietary metabolizable energy content and feed intake.
Estimation of total CH4 production by open circuit calorimetry requires a chamber in which the animal can be restrained, and in which a sustained slightly negative pressure with respect to atmospheric pressure can be achieved through continuous removal of air (Cammell et al., 1980). Devices to estimate the outflow rate of dry air at Standard Temperature and Pressure (STP), are also required, along with analytical equipment to measure the CH4 concentration of air removed from the chamber in either continuous or batch mode.
The chamber design does not need to be elaborate. Laboratories in developed countries tend to use galvanized or stainless steel constructions, but it is also possible to use cheaper and perhaps more readily available materials such as softwood and laminated wood panels. During construction however, it is crucial that all joints between panels, doors, windows, etc. are made to a high standard, for whilst these do not need to be perfectly air-tight, minimization of air-leaks will certainly improve the operator's ability to maintain satisfactory environmental conditions within the chamber. The use of an inert seal in this respect is recommended.
In most chambers, animal access is provided through doors normally situated at the rear of the chamber. In addition, it is advisable to construct a smaller door at the front of the chamber to permit feeding of the animal, whilst the provision of transparent window space at the animal's head-end of the chamber is desirable so that the animal does not become claustrophobic.
• ~ / 13
Animal access
pump
Chamber
) Small p. d. pump
IR gas analyser
FIG. 2.1. Schematic diagram of open circuit calorimeter to measure CH4 production in animals; the chamber can be replaced by a head box or mask {see Section 2.1.1.2).
Recommended dimensions (m) of chambers for different livestock classes are as follows:
Dairy cows: Growing beef cattle: Sheep and goats: 1.6 1.0
Length
(m)
3.6
3.0
2.0
,0.75Note. The minimum space is 150 l/(kg body weight)'
It is necessary to have air inlet and outlet parts in the chamber. These can be readily constructed from PVC pipe fittings, but may need some simple in-line valve devices to regulate air flows. It is essential that the air (inlet) supply is provided from an outside source for if this supply is taken from within the animal house itself, this may give rise to substantial and often highly variable background CH4 concentrations. It should also be free from possible blockage.
The chamber should be sufficiently rigid to tolerate normal animal behaviour and if possible a metabolism stall should be provided within the chamber to restrain the animal. Provision should be made for collection and regular (3 x per day) removal of faeces and urine along with suitable devices for feeding, watering and (if applicable), milking of the animal.
Within the chamber it may be necessary to control the environmental conditions with respect to temperature and relative humidity. In most systems this is achieved by constructing an air conditioning plant which can be placed above or adjacent to the chamber. The plant comprises principally of a fan to move the air, and a cooling coil of sufficient capacity to maintain relative humidity within the chamber at around those levels prevailing in the normal atmosphere at that time.
14
The amount of water vapour produced by the animal will depend upon its body weight, feed intake and metabolic rate. It is also necessary to provide a suitable drainage point to ensure that the condensed water is removed from the plant, thus minimizing corrosion of any surrounding metal.
Within the air conditioning plant, it is advisable to pass the air through a simple filter. Depending upon the local environment, this procedure will limit dust/debris levels in the air, which if not controlled can adversely affect both gas flow meters and analyzer function. Prior to reentry of the air into the chamber it may under certain conditions be necessary to reheat the air; this can be achieved using an in-line heating coil with thermostatic control. The desired rate of air movement through the air conditioner will depend on the volume of the chamber, and the metabolic activity of the subject animal. In this respect, a variable speed fan is recommended but not essential. Furthermore, in order to facilitate good gas mixing within the chamber, reentry of conditioned air can be at more than one site; this can be achieved by suitable arrangement of the air ducting.
It is necessary to remove chamber air continually for subsequent determination of CH4 concentration, and ultimately CH4 production. Under normal circumstances, sufficient air can be removed by an inexpensive vacuum pump, situated up to 10 m or so from the chamber, with the rate of air removal adjusted to easurc that a pressure slightly lower than atmospheric is maintained within the chamber at all times. A simple U-tubc manometer on the outside of the chamber with an air tube leading into the chamber is sufficient for this purpose.
List of items:
1. Air conditioning part comprising cooling coil and associated refrigerator is essential in hot regions, air fan, air filter and coil heater (optional).
2. Air ducting - preferably PVC ( 2" - 4" diam) for air inlet, recirculation and removal. 3. Manometer - easily constructed. 4. Metabolism stall with suitable feeding, watering and as applicable milking facilities. 5. Vacuum pump with suitable valve to adjust air removal rate.
The use of closed circuit calorimetry is not recommended for measuring CH4 production particularly in developing countries. Undoubtedly, such cisterns can provide reliable estimates of gaseous exchange in animals, but the need for complete air tightness, coupled with elaborate gas flushing and/or absorption systems make such options untenable in many countries.
Measurement of air volume
A number of methods are available to determine the volume of air passing through the chamber. The simplest device (which requires no electrical supply) is a commercial dry gas meter which gives a direct measure of total gas flow. Supersonic flow nozzles are also used by some laboratories to control the gas flow rates precisely but these require calibration with other methods that are often complex and therefore not recommended. Other alternatives include electronic turbine flowmeters. These give instantaneous rates of gas flow, and are highly reliable. However, the need to integrate ihe individual air flow rates and the relative sophistication of such apparatus precludes their use in all laboratories except those which have adequate and reliable electrical supply and computer support.
It is necessary to correct the total flow of wet gas to dry gas volume at Standard Temperature Pressure (STP). This requires regular determination of atmospheric pressure changes (approx. 4 x per day) and estimation of the relative humidity of the outflowing gas, which can be determined with a simple laboratory hygrometer.
Measurement of CH4 from chamber exhaust
After the exhaust air leaves the gas meter for measurement of total volume leaving the chamber, it needs to be analyzed for CH4 concentration. Several possibilities exist for CH4 measurement. A
15
small aliquot of the exhaust gas may be collected over the entire run and stored in a butyl rubber bag or other gas collection flask. It is important that the percentage of the aliquot is constant, thereby providing an integrated representation of the entire collection. Small flow meters installed in the gas line after the gas volume measurement device can be used to ensure that a constant aliquot is removed to the collection device. After the run has been completed, CH4 concentration may be measured by the methods outlined in Section 2.3.2.
At all parts in the system up to and including the air outflow flowmetcrs, it is essential that the pressure is maintained below that of the atmosphere, so that any air leakage is inwards. Thereafter, once the volume has been measured, it is necessary to achieve a slight positive pressure compared with the atmosphere. This ensures that any leaks will be outwards so that CH4 concentration remains unchanged.
Alcohol recoveries
A method that can be used to validate the gas collection and aliquoting systems as well as the chambers themselves is to bum a known quantity of absolute alcohol (1 mol). Absolute alcohol when burned uses 3 mol O2 and produces 2 mol CO2 and 3 mol H2O. The procedure is as follows:
(1) Set all flow rates for the chambers). (2) Fill alcohol lamps, set the wicks up and weigh the lamps accurately on a reliable balance. (3) Place the lamps inside the chamber and light them. (4) Seal the chamber and begin collection of gases. (5) Allow the alcohol to bum out and continue to collect gas for at least as long as the lamps
burned. (6) Collect a constant percentage of the total air flow (5-20%) over the entire gas collection period
in a collection apparatus (bag or jar). (7) At the end of the run, reweigh the lamps and calculate the expected amount of CO2 using the
following equation:
CH3CH2OH + 3O2 = 2CO2 + 3H2O
(8) Weigh out an amount of barium hydroxide appropriate for the amount of alcohol burned (i.e. a 2:1 molar ratio).
(9) Flush the collected gas through the Ba(OH)2 which will trap the CO2. Dry and weigh the resultant precipitate and calculate the actual CO2 recovery.
(10) The relationship between the actual and the expected CO2 gives the recovery percentage.
Actual CO, x 100 Recovery percentage = ——————:————
Predicted CO2
(11) Recoveries should be between 97-103%. If they are not the chambers should be examined for leaks.
2.1.12. Head box and mask
The use of face masks and head stalls for estimating CH4 production provides a robust and direct measurement. The principle is straightforward and there are a number of options which can be applied to almost all situations. The method relies on collecting a representative sample of gas from the head of the animal (eructated and expired gas) and then estimating concentration. These two aspects are considered later. In considering the methods it is most important to understand the precautions required in handling and storing gas samples prior to analysis in order to obtain accurate data. Analytical methods and details of gas measuring equipment are considered in more detail in Section 2.3.
16
Principles
Without the use of a tracer that moves with CH4 it is essential to collect all of the expired and eructated gas. This can be done by drawing a stream of air past the muz/le of the animal using a face mask or by enclosing the animal's head in a hood. Both methods require considerable training of the animals. A face mask can be more difficult to use than a hood as the animal can break the mask against solid objects and/or damage or obstruct the collection tube. On the other hand, a hood can be sufficiently large to prevent the animal from damaging it with its head. A further advantage of the hood is that the animal can be fed and have access to water during the collection of gas samples. In both methods, the principle is to have a sufficient outflow of gas to ensure there is lower gas pressure in the hood and gas lines and that all of the "leaks" are inward. This ensures that none of the expired or eructated gas is lost before subsampling for analysis. With the accuracy of available analytical equipment, the dilution of CH4 by air drawn past the animal's head does not present a problem.
Training of animals
Stockmen skilled in training animals for show presentation and for leading by the halter have the talent and ability to prepare animals for intensive handling and experimental work where this is needed. It is often worth employing a person with these skills in order to prepare a group of animals for a set of experiments or, alternatively send animals away for training. This preparation pays dividends in the long run in that animals behave "normally" during experiments and repeated sampling is easily accomplished. Without proper training, equipment can be damaged, animals may refuse to eat and there can also be physical danger for operators. Compared to the costs associated with carrying out experiments and analysing the data the time and cost of preparing the animals represents an extremely good investment. In addition to general training, the animals must be familiar with the equipment to be used and the surroundings of the animal house and/or yards. The use of a hood has advantages in training animals in that feeding the animal every time they are restrained provides positive reinforcement. On the other hand a face mask prevents the animal from eating and drinking, and some animals find masks uncomfortable.
Design of hoods
The hood should be designed to provide sufficient feeding space and enough room for the animal to move its head in an unrestricted way. A wide variety of materials may be used to build a box which is reasonably air tight. The most common materials used arc wood and metal. While they can be custom built, it is also possible to use plastic or metal drums or pre-constructed packing crates. It is a major advantage to have a clear removable panel to provide access for feeding and for checking the animal. This clear panel helps to maintain normal animal behaviour, particularly if other animals are visible to the experimental animal during the period it is in the hood. The animal needs to be restrained with its head in the hood and the design of the hood depends on the facility available for restraint. For example, if animals arc held in metabolism cages where they cannot turn around a canvas sleeve can be fitted around the neck and connected to the hood as shown in Figure 2.2. This allows the animal to stand, eat and lie down during the measurement period . U may also be necessary to restrain the animal within the hood by means of a halter or collar. Hoods may also be built around yokes or even head bales at the end of a working race.
It is desirable to minimize the amount of air leakage around the neck and head and a sleeve is an effective means of achieving this. This can be tied around the neck using a draw string. The sleeve can be constructed from any material but canvass or heavy cotton are most suitable. The length of the sleeve should be sufficient to allow the animal to stand up, lie down and have unrestricted access to feed and water.
Example for sheep (arrangement similar to that shown in Figure 2.2). The box made of 9 mm plywood has solid sides 0.9 m x 0.4 m top and bottom (0.4 m x 0.6 m). The front and back panel
17
Sampling port
Pump (A)
bag
FIG. 2.2. Gas samp//«ff from hood or over 24 h for subsequent analysis.
(0.9 m x 0.6 m) have windows 0.25 m x 0.25 m for the animal's head and 0.5 m x 0.5 m for feeding and observation. A removable clear plastic or perspex panel 0.6 m x 0.6 m is required for the feeding/observation window. The dimensions of the box can be varied to accommodate standard feeders, a water trough and the layout of the animal housing. The length of the sleeve should be around 0.35 m and tapers from a diameter of 0.25 m attached to the hood to 0.15 m to fit over the animal 's head and to be secured around the neck.
The dimensions of the hood should be increased for cattle (approximately 3 times larger than for sheep). This may vary considerably according to the size of the cattle and the type of diet. The basic principle for the hood is that it should be sufficiently large for the animal's comfort and it should have an appropriate facility for feeding concentrate and roughage diets. Attention should also be given to minimis the places where gas can leak from the system.
Masks
The mask provides a means of drawing a volume of air past the animal's muzzle without the need for a hood. An example of a fibreglass mask is shown in Figure 2.2. Masks can be made from a variety of materials and there is considerable scope for improvization. They should be light but sufficiently sturdy to attach the sampling tube and to secure it to the animal's head. The inlet and gas sampling vents need to be at least 50 mm for both sheep and cattle to allow for a free flow of gas. Elastic straps are needed and can be secured behind the ears or horns. Fibreglass is ideal as it can be moulded for the shape and size of the animal's muzzle. A strip of foam rubber can be glued around the edge of the mask to make a better seal and provide a comfortable fitting for the animal.
Gas sampling
The methods for collecting the gas sample including gas lines, pumps and meters are similar irrespective of whether hoods or masks are used.
18
Sampling and airflow: An airflow of 50 to 70 1/min is suggested for measuring CH4 production in sheep. This gives concentrations of between 100 and 500 ppm (v/v) of methane in the airflow from the hood and accurate analysis is possible in this concentration range (sec Section 2.3.2). For cattle a flow rate of around 5001/min is recommended. This flow rate is suitable for withdrawing gas from a hood as well as drawing air past the animal's muzzle when using a mask. These levels of gas flow will also be sufficient to provide the animal wilh fresh air and maintain CO2 levels below 1%. Gas How rates arc easily controlled using a by-pass needle valve and a variable aperture flow meter. A single pump can be used to sample a number of animals providing each sampling line has independent control of flow rate and a separate airflow meter. A schematic diagram is shown in Figure 2.2.
Sampling Unes: The gas sampling lines can be secured overhead. The diameter of the tubing should be between 15 and 20 mm for sheep and 50 mm for cattle and can be constructed of materials such as copper, PVC or flexible rubber or plastic hoses. Although CH4 will leak from plastic or PVC tubing, the use of these materials in a sampling line is acceptable since there is negative pressure and leaks arc inward. When using a mask, a flexible tube of around 3 m length (50 mm diameter) is sufficient to allow movement of the animal. The tube should be suspended above the animal using elastic ties.
Filter: It is advisable to have a simple filter system in the main sampling line in order to remove insects and feed particles which may enter the line. A jar containing plastic scourers or glass wool is adequate.
Gas flow measurement: The total volume of gas drawn past the animal must be measured accurately using a commercial dry gas meter.
Analysis and sub-sampling systems
Continuous gas analysis: It is possible to analyse the CH4 content of the gas from the animal by incorporating an infrared analyser in the main line or in a stream of gas taken from the main line (see Figure 2.3). With this system the output from the meter needs to be recorded continually and
Filter
Pump
Chart Infrared recorder methane
FIG. 2.3. Gas sampling and continuous analysis ofsubsampled air stream.
19
integrated over time. Calibration of this equipment requires relatively large quantities of gas and this can present a potential source of error and an ongoing cost. In addition, it requires relatively sophisticated recording and integration equipment or a datalogger. The infrared analyser in the main line is not recommended unless there is a specific need to measure changes over time.
Sub-samples: A subsample of gas can be taken from the main line and analysed separately. There are two options. A series of separate samples can be taken over time and analysed individually. This requires relatively large numbers of samples but provides flexibility if time-dependent measurements are important. The second option is to continuously withdraw a subsample over an extended period of time. When 24-hour measurements of CH4 production are required this provides a robust and simple system. With any system involving sub-sampling care must be taken to ensure that no CH4 is lost between taking and analysing the sample (sec Section 2.3).
Continuous sub-sampling for subsequent analysis: A subsample taken at the rate of around 2 ml/min (approx 3 1/24 h) gives a manageable volume for storage prior to analysis. The subsample may be taken into a butyl rubber bag suspended in a 10 1 glass jar filled with water (sec Fig 2.2). It is advisable not to use other plastic or rubber bags as these are permeable to CH4 and are not suitable. The subsample of gas can be withdrawn from the main sampling line by pumping or controlled syphoning of water from the sealed jar. The butyl bag submerged in water retains CH4 over periods of 2 to 3 days. Samples of gas can be withdrawn from the bag into syringes (see Section 2.3.1) and analysed in the laboratory. This method is very useful for obtaining a representative sample of gas over a 24 hour period which is essential for accurate measurement of daily CH4 production.
Time scale for measuring CH4 production: The extrapolation of short-term measurements of CH4 production using a mask by only subsampling over part of the day can be very misleading. There is considerable variation in the rate of CH4 production during the day and it can change considerably after feeding. Unless the measurement is for specific screening purposes, and the time of sampling is standardized with respect to feeding and other animal husbandry practices, measuring for periods of less than 24 hours is not recommended. However, when animals are fed continuously or at hourly intervals, accurate measurement of CH4 production may be achieved using shorter periods of collection.
Calculation: The production of CH4 is calculated simply by multiplying the concentration of methane in the subsample by the total volume of gas passing through the hood or the mask. For example, a daily airflow of 86 (XX) litres past the head of a sheep and a CH4 concentration of 200 ppm (v/v) gives a daily production of 17.2 1/d (0.77 mol/d). In cattle, corresponding values might be a daily airflow past the animal of 720 000 litres and a concentration of 250 ppm (v/v). In this instance, CH4 production would be 180 1/d (8.04 mol/d).
2.1.2. Tracer methods
2.1.2.1. Isotop k method
Methane production by ruminants can be estimated by isotope dilution using either 3H or 14C- labelled CH4. As with any technique of this type, its accuracy depends on the efficient mixing of the labelled gas with the pool which is to be measured, in this case CH4 in the rumen. The solubility of CH4 is low and its diffusibility is high, so these properties cause difficulties not encountered with solid solutes. However, with care these problems can be minimized, and the continuous infusion of CH4 into the rumen can yield valuable measurements of production. The technique can be used to estimate production in the rumen by sampling the gas phase in the dorsal rumen, or alternatively total tract CH4 production (less a small correction lor methane lost from the anus) if total expired gases are collected.
20
Experimental animals
Cattle or sheep cannulated in the rumen are the most convenient animals to use.The animals should have leak-proof cannulas and metal infusion lines to deliver the 14C -methane solution into the ventral rumen. A stainless steel tube covered with a nylon gauze should be located in the gas phase in the dorsal rumen for the withdrawal of gas samples. Continuous feeding is recommended to minimis variations in production with time.
Preparation of infusion solution
The main problem with CH4 is its very low solubility and the possibility of losses during handling due to diffusion and leakage. Thus at ail stages during the preparation of a solution suitable for infusion, strict precautions must be taken to avoid loss of gas. Achieving the correct solution for infusion is the most difficult part of the whole procedure, and where most errors arise.
The apparatus for prepaiing 14C-methane solution consists entirely of glass and metal, with butyl rubber bungs and seals (Figure 2.4) except for a flexible plastic bottle whose purpose is to enable the system to be flushed with water and made gas-tight. Distilled water is de-gassed by boiling for 20-30 min and introduced into the conical flask and flexible plastic bottle. The vial breaker with a 1 ml vial of 14C-methane (37 MBq or 1.0 mCi) is then introduced into the flask, the flask is closed and all pockets of air are expelled by squeezing the collapsable container. The effluent line is then closed and the whole assembly is allowed to cool. The plastic bottle collapses as the volume contracts.
The glass vial containing the stock 14C-methane gas is then crushed by turning the screw, and the methane is allowed to dissolve for 3-6 h. Cooling improves the rate of dissolution. This l4C-mcthane solution can be used as prepared, or alternatively can be transferred to glass air-tight bottles and stored in a cold room.
Methane production by sheep
FIG. 2.4. Apparatus used to transfer radioactive methane gas to the infusion solution. A: vial breaker; B: sealed 2 I flask; C: outlet tube; D: collapsible plastic bottle; E: outlet tube.
21
Experimental procedure
An infusion rate of 18 to 54 kBq/min (or 0.5-1.5 uCi/min) is required, delivered at a rate of 1.2 ml/min. In the original method published by Murray et al. (1975), the solution was delivered by a Palmer slow injection pump. Modern piston pumps would also be suitable, but a peristaltic pump will lead to the loss of gas by diffusion through the rubber tubing. Samples are removed from the gas phase of the rumen at 4 to 12 h and up to 24 h using a gas-tight syringe. A 20 ml plastic syringe, provided with a 3-way tap or a needle stuck into a rubber bung and kept under water, will keep the gas for up to 24 h without any serious loss. Where available, glass syringes are preferred (see Section 2.3.1).
Determination of specific radioactivity
Various methods can be used for the determination of the specific radioactivity. Devices are available for the simultaneous measurement of CH4 concentration and radioactivity following gas chromatography from which the specific radioactivity can be calculated. Alternatively, the CH4 can be burned to CO2 and the radioactivity of the resultant CO2 can be subsequently determined in apparatus similar to that shown in Figure 2.5. The CO2 of the gas sample is first removed by adding 3 ml of l M NaOH to 20 ml of the gas sample using a pair of gas-tight syringes (Figure 2.6) and mixing thoroughly. The NaOH solution is then removed and the remaining gas injected into the oxidation apparatus via a 3-way tap. The gas passes through copper oxide or a similar catalyst at 780- 790°C where the CH4 is oxidized to CO2. The CO2 is then stripped from the gas mixture in a mixing coil containing l M NaOH. This solution can be counted directly using a scintillation cocktail, but it is recommended that CO2 losses be minimized by converting the CO2 to barium carbonate.
Gas sample (3)
N2<2) Tube furnace (4) Mixing coil (6)
Auloanalyser pumps
Absorbent (1) Collection tube (7)
FIG. 2.5. Apparatus used for oxidation ofCH4 to CO2 in determination of specific radioactivity ofCH4 in rumen gas samples 1. Absorbent: IN CO2-free NaOH is stored in a reservoir equipped with a carbasorb C02 trap. The flow
rate is 2 ml/min. 2. Carrier gas: Nitrogen is stored in an impermeable butyl rubber bag and pumped through the system at a
rate of 9 mlfmin. 3. Sample entry: The sample is injected from a syringe via a rubber septum into the carrier gas stream and
into the column or a 3-way tap. 4. Oven: The column is heated by an element to 780-790°C. 5. Combustion A quartz glass tube containing a catalyst of copper oxide, ISO g {wire form 0.6 mm BDH
chamber: Chemicals or Merck). Alternative catalysts include nickel oxide or cobalt oxide.
6. Mixing coil: An autoanalyser type coil (l m in length) is used to mix gas and absorbent streams and the latter is collected.
7. Collection A quick-fit test tube is used to collect the sample and the BaCO, is precipitated tube: with 2.0 ml 5% NHjCl and 0.8 ml 20% BaCl2.
22
Connector
=C
NaOH
as
E> F/G. 2.6. A simple apparatus for removal of CO2 from samples of gas.
Ammonium chloride (1 ml of 5% w/v) and 0.4 ml of 20% (w/v) BaQ2 are added to 5 ml of the alkaline solution. The BaCO3 precipitate is centrifuged and washed twice with distilled water, and then dried with acetone. The precipitate is ground and added to a weighed scintillation vial, the vial is re- weighed and the scintillation cocktail is added, and then the vial is counted. The specific radioactivity of the BaCO3 is the same as that of CH4.
Calculation
The rate of CH4 production is calculated from the specific radioactivity using the equation:
Infusion rateCH4 production = Mean CHt specific radioactivity
where methane production is in g C/min, specific radioactivity is in kBq/g C and the infusion rate is in kBq/min.
23
Mean SR
Time (h)
FIG. 2.7. Changes in the specific radioactivity rf methane during infusion of'4CH4 into the rumen.
The calculation can be made by averaging specific radioactivity determinations of a number of samples taken over a 24 h period, or by bulking the gas samples, storing and performing duplicate analyses.
For animals fed once or twice daily, the rate of CH4 production will fluctuate (Figure 2.7). The analysis of hourly samples then gives a more reliable picture of the overall pattern of production.
Usefiilness of method
Once established, the method is relatively straightforward and calculation is simple. An accurate determination of CH4 production is obtained. The main problem is the initial preparation of the labelled CH4 solution. A possible solution to this difficulty is to replace labelled CH4 with l4C-choline, which is easier to handle. However, up to 20% of the label may be incorporated into protozoa rather than methane, and this would have to be minimized or taken into consideration.
'4C-labelled sodium formate is an alternative label that might be used, but no information is available regarding its suitability (for example what proportion of formate is absorbed in the rumen).
2.1.2.2. Non-isotopic method
Use of SF6 bolus
This method involves inserting a permeation tube that emits the inert gas sulfur hexafluoride (SF6) at a low but steady rate into the rumen (see Figure 2.8). This tracer gas behaves similarly to CH4 in the rumen, and is released with CH4 during the normal respiration and eructation processes. Because the tracer is released at a known rate, the ratio of the measured concentrations of tracer gas and CH4 can be used to calculate the rate of CH4 emission . This can be done without measuring breathing or air exchange rates in the rumen because both CH4 and SF6 will be diluted by air to the same degree. This method eliminates the need to sample directly from the animal's throat, because changes in dilution associated with head movement or ambient movements of air are accounted for directly.
24
FIG. 2.8. Structure of the slow permeation bolus for releasing control amounts ofSF6.
FIG. 2.9. Position of the evacuated vessels on the animal.
25
AH experimental animals need to become accustomed to wearing a halter and a neck rope with suspended collection can. Failure to allow acclimation could result in destruction of the apparatus and loss of samples.
The permeation tubes consist of a closed stainless steel tube capped at one end with a Teflon disk held in place with a standard Swagelok1"1 nut. The tube is filled with liquid SF6 while being cooled with liquid N2 and then allowed to equilibrate to a fixed temperature in a water bath. Permeation rates are determined in two ways: gravimetrically through repeated weighing on a precision balance over a period of several weeks; and analytically by measuring the concentration of SF6 in dilution air passed over the tube at a carefully measured rate. Typical permeation rates are of the order of 500 ng/min and under controlled temper?iurcs the rates are extremely stable.
Permeation tubes should be inserted into the rumen a minimum of three days prior to the first scheduled collection to allow steady state conditions to be reached. Insertion can be accomplished with a balling/drenching gun or other similar methods for inserting boluses into the rumen.
A leather pad attached to the nose band of a halter serves as an anchor point for the sample line near the animal's nose and mouth. A small piece of plastic tubing is attached to a filter and oriented such that it is placed over one of the nostrils (see Figure 2.9). A filter (10 urn) connected to the upstream end of the sample line protects the flow restrictor from becoming plugged. Fastening the tube to the sides of the halter helps to protect the capillary tubing and reduces animal irritation. The capillary tube-collection flask connection should be via a Quick-connect fitting in order to simplify flask exchange. A soft rope fastened around the neck with a clasp that can be attached to the collection flask helps stabilize the flask and takes pressure off the capillary tube.
The collection flask should be large enough to accommodate the size of sample desired, should be able to withstand a vacuum, and should have a valve for sealing the flask. Immediately prior to use, all air should be removed from the flask and the valve closed. After fastening the flask to the supporting neck rope and attaching the capillary tube, the time of day should be noted and the valve opened. When the sampling time is complete, the flask is removed for analysis. If repeated collections are desired, another flask should be added after the first one is filled. It is recommended that many measurements are made on each animal and that total 24 h emissions are reported. The diameter and length of the capillary tube needed depend on the rate at which sampling is desired. The size of the capillary tube bore should be such that the evacuated sample flask fills to about IA atmospheric pressure over the desired sampling time. The flask pressure should he measured and the flask then filled with N2 to bring it to positive pressure (around 1.5 atmospheres) Both pressures need to be recorded to enable the extent of dilution by N2 to be calculated.
The flask samples can be quickly and accurately analysed for the tracer using an electron capture gas Chromatograph. Methane can be analysed using a gas Chromatograph with an FID detector (see Section 2.3.2.1).
The emission rate of CH4 (1/h) is calculated from:
ßC7/4 = QSF{ [CH,4
6
where QSF6 is the release rate of SF6 in l/h, [CHJ is the concentration of CH4 in the gas sample and (SFJ is the concentration of SF6 in ppm (v/v).
2.13. Indirect methods
The procedures described below are not really methods for measuring CH4 production per se in ruminant animals. Therefore they should be used only if more appropriate measurements cannot be
26
made and there is a need to fill gaps in much larger surveys, or in some preliminary comparative work to obtain data before embarking on more extensive studies using the proper direct techniques.
2.1 J.I. As VFA
When the main substrate of rumen microorganism, i.e. the carbohydrate, is converted to volatile fatty acids (VFA) under anaerobic conditions, there is a net accumulation of H2 when acetic and butyric acids are produced and a net uptake of H2 when propionic acid is the end-product. Since the proportions of acetic and butyric acids are normally greater than that of propionic acid, there is net accummulation of metabolic H2 which is converted to CH4 by methanogenic bacteria of the rumen. A small proportion of metabolic H2 is used in the synthesis of microbial cells (about 10-20%).
The ratio of CH4 to VFA produced will depend on the composition of the VFA and may range (in an in vitro system) from 0.23 with roughage diets to 0.28 with concentrate diets (mol/mol). However in work with animals, the ratio CHJVFA can range from 0.20 to 0.35. With mixed balanced diets when the proportions of acetic: propionic: butyric are 60:20:10, the CH4 production may be about 0.25 mol/mol VFA produced.
If the measured VFA production in an animal is R mol/d, then 0.25 R mol/d will give a reasonable estimate of CH4 production. For example, a sheep digesting 1 kg mixed balanced food per day may produced 6.2 mol VFA and would be expected to produce 1.6 mol CH4 or about 36 1 CH^d.
A better estimate of CH4 production would be R.f(c) (mol/d), where R is the rate of production of VFA (mol/d) and f(c) is a function of molar proportion of VFA which can be obtained from stoichiometric equations (Czerkawski, 1986). However, for comparative work the value of 0.25 mol
per mol VFA would be quite sufficient.
Estimation of VFA production
If a ruminant is fed continuously or at very frequent intervals (i.e. hourly), ruminai fermentation and hence VFA production can be regarded as occurring at a relatively constant rate. With non-steady state conditions, no technique can be recommended for meaningful measurement, and even with steady state there are problems in assuming that the molar proportions of VFA represent the molar proportions in which they are produced. This is acceptable at high rumen pH (about 7.0) when absorption rates of the individual VFA are similar, but at low rumen pH the relative proportion of acetic acid in the rumen is higher and those of propionic and butyric acids are lower than their relative rates of production.
The production rate (more correctly, the irreversible loss rate) of the acid can be calculated from the plateau specific radioactivity and the rate of administration of radioactivity as follows:
Production rate (mol/d) = Rate of is0t°Pe «*"***"*"» plateau specific radioactivity (MBq/mol)
The tracer, which is normally a 14C-labelled volatile acid, is administered by continuous infusion via a rumen fistula. The samples of rumen liquor are also removed through the fistula for estimation of specific activity.
Total ruminai VFA consists of six to seven acids of which acetic, propionic and butyric acids predominate and therefore the isotope dilution method demands that separate tracers be used for each acid. Using the assumption that production rate is proportional to concentration, a single tracer (usually I4C-acetate) may be infused and an estimate of total VFA production rate obtained. However, considerable exchange of carbon occurs in the rumen between acetate and butyrate, and to a lesser
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extent between acetate and propionate, and therefore determining total VFA production rate using a single tracer is an approximation which will give net production rate. Measurement of total production rate and estimation of exchange requires infusion of all three major acids.
Animals and feeding: The animals are housed in metabolism cages during measurements and should be prepared with rumen fistulac of sufficient size to enable samples of rumen liquor to be removed without interfering with the infusion of tracer. A suitable period (at least 10 days) is necessary to allow the animal to adapt to the dietary regime. To achieve conditions of steady rate, feeding must be as frequent as possible. As water intake may not be constant when dry feeds are offered, fluctuations in the rumen due to drinking may be reduced by infusing the tracer in a relatively large volume of water (at least 1 1/d for sheep).
Choice of tracers: Either all or one of the carbon atoms within a VFA molecule can be labelled. The 14C-labelling position of acetate and butyrate tracers appears to have little if any effect upon the estimates of production rates obtain while the labelling position of propionate tracer is very important. The use of l-14C-propionate gives a much higher estimate of propionate production rate than does 2- l4C-propionate. For normal production rate estimates 2-l4C-propionate should be used.
Infusion technique: Tracers should be infused as the sodium salts after being diluted with carrier VFA salts and dissolved in sterile water (e.g. 1-2 umol carrier per kBq; 2.96-3.7 MBq/d (80-100 uCi/d) for sheep). A simple way of achieving constant infusion rates is to use a multichannel peristaltic pump (one channel per animal). The infusion solution is carried to each animal by flexible tubing and introduced into the rumen through a rigid tube (stainless steel or polyethylene) passing through a rubber bung fitted in the rumen fistula. One infusion should last at least 16 hours so that plateau specific radioactivity values can be obtained for all VFAs.
Sampling of rumen liquor: It is necessary to obtain rumen liquor samples that are representative of the rumen contents as a whole, but which are not contaminated with highly radioactive material close to the site of infusion. Immediately after removing the sample, a preservative (e.g. concentrated H2SO4) must be added to prevent further microbial action. Samples can then be stored in a deep-freeze. Normally 5-9 samples should be taken hourly through to the end of each infusion.
There are essentially three options for making these measurements. The first is to infuse one VFA and determine the total activity in the steam distillate of the VFA. This option gives the net production of total VFA. Rumen liquor samples are steam-distilled and the total VFA content determined by titration of the distillate, retaining an aliquot for radioactivity estimation. The relative concentrations of individual VFA are determined by gas-liquid chromatography. The second option is to infuse one VFA but ui^n to separate the individual VFAs to determine radioactivity and extent of interconversion. Finally, it is possible to inject labelled acetate, propionate and butyrate on different occasions and then to separate by column chromatography the individual VFAs (International Atomic Energy Agency, 1985). The quantitative analysis of interconversions can then be assessed by calculations using a three- compartment model.
2.1.3.2. From feed characteristics
Undoubtedly, most CH4 produced by ruminants arises from anaerobic fermentation in the rumen, with the caecum unlikely to contribute more than 15% of total yield. Within the rumen, fermentation of hexose for the essential production of ATP for microbial growth is associated with the production of VFAs and CH4, but the yield of CH4 per mole of hexose passing through the pathways to VFA will be influenced by the microbial population in the rumen. Two examples of this are given below for contrasting high roughage and high concentrate diets (all in mois):
Roughage [hexose] -» 1.34 acetate + 0.45 propionate + 0.1 butyrate + 0.61 methane. Concentrate (hexose] —> 0.9 acetate + 0.7 propionate + 0.2 butyrate + 0.38 methane.
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These indicate the inverse relation and competitive roles of propionate and CH4 production in the assimilation of metabolic H2.
The situation is further complicated when it is recognized that only a proportion of available hexose in the rumen is fermented to VFA and CH4. Hexose is extensively utilized by microorganisms in the synthesis of microbial protein, nucleic acids and some lipid, and the established propensity of some microbes to synthesize substantial quantities of microbial polysaccharide provides another route by which hexose may be disposed. Consequently, depending on the extent to which hexose is partitioned between fermentation and synthesis, the ultimate yield of CH4 per mole of total ruminally digested carbohydrate can be highly variable. This makes prediction of CH4 production from feed characteristics rather difficult. Against this background, several potential prediction equations have been proposed. Some of the earlier ones were based solely on the total intake of dry matter by the animal, but given current knowledge regarding CH4 production in the rumen, such equations have no universally acceptable applicability and can give wrong predictions.
From a series of CH4 measurements obtained largely with mature sheep fed a range of diets (from poor quality hay to sugar beet pulp). Blaxter and Clapperton (1965) proposed the following equation:
CH4(kcal/100 kcal GE) = 1.30 + 0.112D + L(2.37 - 0.05D)
where
D = digestibility of gross energy, and L = level of intake relative to maintenance.
When this equation was applied in other experiments to compare predicted versus observed yields, overestimations of between 10% and 30% were reported for a diet of lucerne fed at 200-1000 g DM/d (Murray et al., 1975). There are now further instances where this equation appears to be unreliable in predicting methane output.
Subsequently, other equations were proposed by Maynard and Loosli (1976) and by Church (1979) for sheep and cattle, but the reasons for the apparent differences between sheep and cattle are not given.
Methane production in sheep (g) = 2.41 x digested carbohydrates (100 g) + 9.80 Methane production in cattle (g) = 4.01 x digested carbohydrates (100 g) + 17.68
More recently, Moe and Tyrrell (1979) proposed the following equation:
CH4(MJ/d) = 3.406 + 0.510 soluble residue (kg fed) + 1.736 hcmiccllulose (kg fed) + 2.648 cellulose (kg fed)
This may represent some improvement over the Blaxter and Clapperton (1965) equation since it relates methanogenesis to specific feed characteristics rather than simply to the feed intake and digestibility.
The equation proposed by Moe and Tyrell (1979) based on high quality dairy rations has not been extensively evaluated. Its use to predict CH4 production needs to be qualified with respect to the overall objectives of the study. If the need is to revise current national and international inventories, it is possible that this equation is an improvement on the present situation where estimates are based largely on the relationship given by Blaxter and Clapperton (1965). If the requirements are more specific (e.g. to examine the effect of deliberate nutritional or microbial manipulations of the rumen ecosystem on CH4 generation), it is unlikely that any simple equation based on the macrocharacteristics of feed will be sufficient.
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Through the improvement of feed characterization, and in particular estimation of the likely extent of carbohydrate fermentation in the rumen, together with some recognition of the likely pathways of VFA production, it should be possible to improve the accuracy of empirical equations to predict CH4 production. Such procedures are not yet available, but their development should be encouraged. Also empirical equations or models are purely descriptions of the input data and contain no detail regarding the underlying mechanisms; they are therefore strictly applicable only within the range of the database used in their development.
Alternatively, it may be possible in the long term to predict CH4 production by mechanistic mathematical modelling. Several such models have been proposed and recently Baldwin et al. (1987) examined a revised rumen model for the specific purpose of predicting CH4 production. To date, however, no suitable models are available, largely because considerable debate still exists over the definitive factors which regulate the various pathways of VFA production, and hence the relative contributions of these factors to total carbohydrate fermentation. This is demonstrated in part in Table 2.1, which attempts to describe the theoretical disposal of 1 mole of carbohydrate when supplied from forage, concentrate or a molasses-containing diet, and assuming different patterns of VFA production.
In order to be applicable to systems that range from grazing dry tropical pastures and feeding crop residues and by-products, to concentrate diets and high protein pastures, all equations must be able to predict the carbon balance of the rumen. This is not yet possible, and therefore estimations of CH4 production from feed characteristics should at best be considered as preliminary calculations before the start of proper measurements.
TABLE 2.1. FERMENTATION OF CONTRASTING CARBOHYDRATE SOURCES; PYRUVATE FORMATION AND METABOLISM*
Diets
Pyruvate utilized
134 0.45 0.22 4.62*
0.90 0.70 0.40 438*
0.94 0.40 0.60 4.54*
* 1 mol carbohydrate •» 2 mol pyruvate + 2mol H2 + 2 mol ATP. ** Includes H? produced iii above »e act ion. * Includes ATP produced in above reaction and 1 mol per mol methane.
Note. The calculations assume that no hydrogen is used for cell synthesis but this can be allowed for (see for example Czerkawski, 1986).
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2J.3J. In vitro incubations
There are two main forms of artificial rumen, one in which rumen contents freshly removed from a donor animal are incubated in vitro in batch culture, and the other in which a continuous culture is established. The former type of culture is valid for a period of hours while the latter which represents an anaerobic system not necessarily identical to the rumen, can be sustained for days or weeks. A procedure for batch-type culture is described below, while the rumen simulation technique (Rusitec; C/.erkawski and Breckenridge, 1977) is a proven system used widely throughout the world.
Neither type of in vitro system is ideal for predicting methane production. The batch culture system generally lacks pH control and the stoichiometry of product formation cannot be guaranteed to be the same as that occurring in the donor animal. For example, lactic acid is often detected in short-term in vitro incubât ioas but not in the animal. In the Rusitec, the pattern of volatile fatty acids produced is usually similar, but not identical, to the in vivo situation.
Nevertheless, in vitro systems are extremely valuable for comparative purposes, e.g. for measuring the effects of additives on factors that control fermentation. The continuous culture is more valuable than the batch system, because it can take into account any adaptation the microbial population makes in response to the additive. This is particularly important for additives that affect CH4 production. Some types of material, such as chlorinated hydrocarbons, are highly effective in the short term, but the rumen microbial population adapts eventually to become insensitive to the inhibitor. On the other hand, some types of additive such as microbial additives require adaptation of the population to detect an effect that does not occur in the batch system.
Batch systems
A variety of types of apparatus can be used. These differ in their cost and ease of use and range from a simple conical flask to a pH and Eh controlled fermenter. The following criteria are essential to all of the systems:
rumen contents must be removed from the rumen just before use, anaerobic conditions must be maintained during transfer of the sample to the incubation vessel and during the incubation, the fermentation liquid must be incubated at 39°C and agitated sufficiently to maintain some of the ciliate protozoa in suspension, the donor animals should be fed the feed to be used in the incubations.
Incubation of rumen contents in glass syringes: a simple artificial rumen
The simplest type of short-term artificial rumen consists of test tubes or flasks with rumen contents incubated at 39°C. Anaerobic conditions are maintained by bubbling CO2 during incubation or by providing the vessels with Bunsen valves. The latter arrangement is difficult to manipulate (addition of substances, withdrawal of samples), an important component of fermentation products (gas) cannot be quantified, and the free-venting of ga? can result in contamination when radioisotopes are used. The procedure described, which was developed by Czerkawski and Breckenridge (1977) is simple, the apparatus is inexpensive, it does not suffer from the disadvantages discussed above, and can be readily adapted to different requirements.
Procedure
(1) Connect a 3-way tap to a 50 ml glass syringe as shown in Figure 2.10. When the stopcock is turned to any particular opening, that opening is closed.
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GAS
REACTION MIXTURE
FIG. 2.10. Simple apparatus for incubating small samples of rumen contents in a water bath.
(2) Fill the syringe with water and empty it. This is to wet the plunger and the barrel. Turn the tap to C and inject 20 ml rumen contents through the opening B, using a 20 ml syringe. Turn the tap to A and remove the 20 ml syringe.
(3) Fill a 10 ml syringe with an inert gas (e.g. N2), connect it to B and press the plunger, allowing the gas to escape through C until exactly 5 ml of gas is left in the syringe. Turn the tap to C and transfer the 5 ml of gas to the bigger syringe. Turn the tap to A and remove the small syringe. The syringe now contains 20 ml rumen contents and 5 ml of gas to make it possible to agitate the liquid when the syringe is lying on its side.
(4) Place the syringe in a water bath and incubate. If a shaking water bath is available, fix the syringes to a frame with clips. If no such bath is available, allow the syringes to float (they will be 70-80% submerged) and take them out and agitate the contents by inversion at regular intervals.
(5) Take the syringe out at regular intervals, keep it vertical as shown in Figure 2.10 (making sure that the plunger is free to move) and read the volume of gas on the scale. The difference between this reading and the amount of gas added gives the amount of gas produced. This is not a very accurate parameter, but it is extremely useful. With care, a curve relating gas production to time can be drawn. The shape of this curve can be very informative about the extent and magnitude of reaction in the incubated sample.
(6) At the end of incubation take the syringe out of the bath, connect an empty syringe (20 ml) to the opening B, turn the tap to C and transfer the gas to this syringe. If the volume of gas produced is small, use a 10 ml syringe; the measurement of gas produced will be more accurate. Turn the tap to B and disconnect the 3-way tap (plus the small syringe) from the big syringe at A. Read the volume of gas and set the sealed gas sample aside to further analysis. The proportion of CH4 can be estimated by recovering the CO2 using the apparatus shown in Figures 2.5 and 2.6.
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(7) Small amounts of liquid (e.g. substrates, labelled compounds, inhibitors) can be introduced into the reaction mixture in the same way as the gas (sec step (3) above), but it is best to use a small ( 1 ml) plastic syringe. Similarly, samples of liquid or gas can be taken (the former, with the syringe upside down).
Continuons fermenters
Three main types of continuous system arc used. The first is actually a sequential culture, where a small amount of solid feed is added to sample of rumen fluid which is subcultured daily (Merry et al., 1987). Whether methane production is sustained and mimics accurately the real situation is not clear. The second involves the continuous flow of both solid and liquid phases (Hoover et al., 1976), while the third type is the Rusitcc, which receives solids once daily but liquid phase by continuous flow. Rusitcc requires a considerable input of time and expertise and would not be recommended unless the specific objective is particularly suited to the apparatus. Typically, Rusitec would be used only where small quantities of the potential modifying agents are available, but the small scale and between-vcsscl reproducibility offers a major advantage over animal experiments.
2.2. GROUPS OF ANIMALS
2.2.1. Enclosure of animals
It is possible to estimate CH4 emissions from groups of animals that are housed in a bam or similar structure. To be suitable, it is best that the building be a simple, one-room structure and that the air flow through the building can be controlled to some degree. This approach is useful for measuring emissions from a group of animals of various sixes and types, and can be used as a complement to the individual animal measurements discussed earlier. Additionally, if there are emissions from animal wastes within a building, this approach can be used to measure the emissions from the animals and the waste, or the waste alone when the animals are removed from the bam.
The technique relics on the release of a known quantity of an inert tracer gas and measuring the concentration of CH4 and the inert tracer in a mixed sample of the air in the enclosure. The inert tracer provides a basis for evaluating the air flow and dispersion of CH4 in the enclosure, so that the flux rate of CH4 can be computed using the known release rate of the tracer and the measured tracer and CH4 concentrations. This technique has been applied successfully in a variety of situations, including animal management facilities (see Section 2.1.2.2).
On-site immediate gas analysis is required so that portable analytical capabilities arc necessary. A portable CH4 analyser, such as a portable gas Chromatograph with an integrator or a portable FID capable of measuring between 2 and 100 ppm (v/v) of CH4 in air with a precision of at least ±0.5 ppm (v/v) or better is required. The precision of the measurement is improved when the measurement error of the analyser is small compared to the concentration of CH4 detected (see Section 2.3.2.).
An inert tracer gas is required that can be released at a known and constant rate and can be detected with a portable analyser and integrator. It is essential that there is a very low background concentration of the tracer gas and that it can be detected with a precision of at least ±10%. Also, a tracer gas should be selected that disperses in a manner similar to CH4. Sulfur hexafluoride (SF6) has been used successfully in conjunction with an electron capture detector, and is consequently recommended, but other possibilities, such as for example propane, should be considered. In any case, gas standards are also required.
Gas handling equipment including flow regulators, flow meters, tubing, and sampling containers are required. Large fans are useful for promoting mixing of the air within the building and a precise mass balance is needed to confirm the flow rate of the tracer gas. It is essential that the equipment
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used with the inert tracer (e.g., the gas canister, flow regulator, flow meter, tubing, and mass balance) be kept physically separate from all analytical equipment and sampling equipment to prevent contamination.
Because on-sile immediate analysis is needed to use this method, a reliable power source is needed for the analysers and fans.
Personnel
At least 3 scientists trained in gas handling, sampling, and analysis are required. One person works with the inert tracer gas, including regulating and measuring its flow. Two technicians are required to take air samples and analyse them for methane and the inert tracer. In no case should the individual regulating the inert tracer release be involved in taking or analysing samples (contamination).
Experiments using this method can be conducted using continuous or restricted air flow. The two approaches are very similar, and the preferred method will depend on the ability to control the ventilation of the structure.
2.2.1.1. Continuous airflow approach
The air flow through the structure must be controllable. A typical situation is shown in Figure 2.11 in which the windows or shutters of the building are closed so that most of the air enters the building at Point A and exits at Point B. Although Figure 2.11 shows Points A and B being doors, they can be windows, vents, or similar structures.
Ventilation fans turned off
<8> animai stalls Porta
® - Air flow
FIG. 2.11. Enclosure for measuring the emission of methane in groups of animals.
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Once the air flow through the building is established reliably, the following are recommended:
( 1 ) Fans should be