Partitioning the components of soil respiration: a research challenge

Little is known about the respiratory components of CO₂ emitted from soils and attaining a reliable quantification of the contribution of root respiration remains one of the major challenges facing ecosystem research. Resolving this would provide major advances in our ability to predict ecosystem responses to climate change. The merits and technical and theoretical difficulties associated with different approaches adopted for partitioning respiration components are discussed here. The way forward is suggested to be the development of non-invasive regression analysis validated by stable isotope approaches to increase the sensitivity of model functions to include components of rhizosphere microbial activity, changing root biomass and the dynamics of a wide range of soil C pools. Section Editor: A. Hodge     

Partitioning of soil respiration components and evaluating the mycorrhizal contribution to soil respiration in a semiarid grassland

土壤呼吸组分的区分对于理解地下碳循环过程非常重要。而菌根真菌在地下碳循环过程中扮演着重要的角色, 但是有关菌根呼吸在草原生态系统中的研究相对较少。该研究在内蒙古半干旱草原应用深浅环网孔法, 结合浅环、深环(排除根系)和一个带有40 μm孔径窗口的土壤环(排除根系但是有菌根菌丝体)将根和菌丝物理分离, 来区分不同的呼吸组分。结果表明: 异养呼吸对总呼吸的贡献比例为51%, 根呼吸的贡献比例为26%, 菌根呼吸的贡献比例为23%, 菌根呼吸的比例3年变化范围为21%-26%。与国内外研究相比, 此方法提供了一个相对稳定的菌根呼吸测量精度范围, 在草原生态系统中切实可行。对菌根呼吸的准确定量将有助于预测草原生态系统土壤碳释放过程对未来气候变化的响应。     

半干旱草原土壤呼吸组分区分与菌根呼吸的贡献

土壤呼吸组分的区分对于理解地下碳循环过程非常重要。而菌根真菌在地下碳循环过程中扮演着重要的角色, 但是有关菌根呼吸在草原生态系统中的研究相对较少。该研究在内蒙古半干旱草原应用深浅环网孔法, 结合浅环、深环(排除根系)和一个带有40 μm孔径窗口的土壤环(排除根系但是有菌根菌丝体)将根和菌丝物理分离, 来区分不同的呼吸组分。结果表明: 异养呼吸对总呼吸的贡献比例为51%, 根呼吸的贡献比例为26%, 菌根呼吸的贡献比例为23%, 菌根呼吸的比例3年变化范围为21%-26%。与国内外研究相比, 此方法提供了一个相对稳定的菌根呼吸测量精度范围, 在草原生态系统中切实可行。对菌根呼吸的准确定量将有助于预测草原生态系统土壤碳释放过程对未来气候变化的响应。     

Organic carbon cycling in Taylor Valley, Antarctica: quantifying soil reservoirs and soil respiration

Organic carbon reservoirs and respiration rates in soils have been calculated for most major biomes on Earth revealing patterns related to temperature, precipitation, and location. Yet data from one of the Earth's coldest, driest, and most southerly soil ecosystems, that of the McMurdo Dry Valleys of Antarctica, are currently not a part of this global database. In this paper, we present the first regional calculations of the soil organic carbon reservoirs in a dry valley ecosystem (Taylor Valley) and report measurements of CO₂ efflux from Antarctic soils. Our analyses indicate that, despite the absence of visible accumulations of organic matter in most of Taylor Valley's arid soils, this soil environment contained a significant percentage (up to 72%) of the seasonally unfrozen organic carbon reservoir in the terrestrial ecosystem. Field measurements of soil CO₂‐efflux in Taylor Valley soils were used to evaluate biotic respiration and averaged 0.10 ± 0.08 μmol CO₂ m^?2 s^?1. Laboratory soil microcosms suggested that this respiration rate was sensitive to increases in temperature, moisture, and carbon addition. Finally, a steady‐state calculation of the mean residence time for organic carbon in Taylor Valley soils was 23 years. Because this value contradicts all that is currently known about carbon cycling rates in the dry valleys, we suggest that the dry valley soil carbon dynamics is not steady state. Instead, we suggest that the dynamic is complex, with at least two (short‐ and long‐term) organic carbon reservoirs. We also suggest that organic carbon in the dry valley soil environment may be more important, and play a more active role in long‐term ecosystem processes, than previously believed.     

东北东部森林生态系统土壤呼吸组分的分离量化

对森林生态系统的土壤呼吸组分进行分离和量化,确定不同组分CO2释放速率的控制因子,是估测局域和区域森林生态系统碳平衡研究中必不可少的内容。采用挖壕法和红外气体分析法测定无根和有根样地的土壤表面CO2通量(RS),确定东北东部6种典型森林生态系统RS中异养呼吸(RH)和根系自养呼吸(RA)的贡献量及其影响因子。具体研究目标包括:(1)量化各种生态系统的RH及其与主要环境影响因子的关系;(2)量化各种生态系统RS中根系呼吸贡献率(RC)的季节动态;(3)比较6种森林生态系统RH和RA的年通量。土壤温度、土壤含水量及其交互作用显著地影响森林生态系统的RH(R2=0.465~0.788),但其影响程度因森林生态系统类型而异。硬阔叶林和落叶松人工林的RH主要受土壤温度控制,其他生态系统RH受土壤温度和含水量的联合影响。各个森林生态系统类型的RC变化范围依次为:硬阔叶林32.40%~51.44%;杨桦林39.72%~46.65%;杂木林17.94%~47.74%;蒙古栎林34.31%~37.36%;红松人工林33.78%~37.02%;落叶松人工林14.39%~35.75%。每个生态系统类型RH年通量都显著高于RA年通量,其变化范围分别为337~540 gC.m-2.a-1和88~331 gC.m-2.a-1。不同生态系统间的RH和RA也存在着显著性差异。     

Partitioning soil respiration of temperate forest ecosystems in northeastern China

Quantifying soil respiration components and their relations to environmental controls are essential to estimate both local and regional carbon (C) budgets of forest ecosystems. In this study, we used the trenching-plot and infrared gas exchange analyzer approaches to determine heterotrophic (R_H) and autotrophic respiration (R_A) in the soil surface CO₂ flux for six major temperate forest ecosystems in northeastern China. The ecosystems were: Mongolian oak forest (dominated by Quercus mongolica), aspen-birch forest (dominated by Populous davidiana and Betula platyphylla), mixed wood forest (composed of P. davidiana, B. platyphylla, Fraxinus mandshurica, Tilia amurensis, Acer amono, etc.), hardwood forest (dominated by F. mandshurica, Juglans mandshurica, and Phellodendron amurense), Korean pine (Pinus koraiensis), and Dahurian larch (Larix gmelinii) plantations, representing the typical secondary forest ecosystems in this region. Our specific objectives were to: (1) quantify R_H and its relationship with the environmental factors of the forest ecosystems, (2) characterize seasonal dynamics in the contribution of root respiration to total soil surface CO₂ flux (RC), and (3) compare annual CO₂ fluxes from R_H and R_A among the six forest ecosystems. Soil temperature, water content, and their interactions significantly affected R_H in the ecosystems and accounted for 46.5%–78.8% variations in R_H. However, the environmental controlling factors of R_H varied with ecosystem types: soil temperature in hardwood and Dahurian larch forest ecosystems, soil temperature, and water content in the others. The RC for hardwood, poplar-birch, mixed wood, Mongolian oak, Korean pine, and Dahurian larch forest ecosystems varied between 32.40%–51.44%, 39.72%–46.65%, 17.94%–47.74%, 34.31%–37.36%, 33.78%–37.02%, and 14.39%–35.75%, respectively. The annual CO₂ fluxes from R_H were significantly greater than those from R_A for all the ecosystems, ranging from 337–540 g Cm^-2a^-1 and 88‐331 gCm^-2a^-1 for R_H and R_A, respectively. The annual CO₂ fluxes from R_H and R_A differed significantly among the six forest ecosystems.     

Constraining estimates of global soil respiration by quantifying sources of variability

Quantifying global soil respiration (R_SG) and its response to temperature change are critical for predicting the turnover of terrestrial carbon stocks and their feedbacks to climate change. Currently, estimates of R_SG range from 68 to 98 Pg C year^?1, causing considerable uncertainty in the global carbon budget. We argue the source of this variability lies in the upscaling assumptions regarding the model format, data timescales, and precipitation component. To quantify the variability and constrain R_SG, we developed R_SG models using Random Forest and exponential models, and used different timescales (daily, monthly, and annual) of soil respiration (R_S) and climate data to predict R_SG. From the resulting R_SG estimates (range = 66.62–100.72 Pg), we calculated variability associated with each assumption. Among model formats, using monthly R_S data rather than annual data decreased R_SG by 7.43–9.46 Pg; however, R_SG calculated from daily R_S data was only 1.83 Pg lower than the R_SG from monthly data. Using mean annual precipitation and temperature data instead of monthly data caused +4.84 and ?4.36 Pg C differences, respectively. If the timescale of R_S data is constant, R_SG estimated by the first‐order exponential (93.2 Pg) was greater than the Random Forest (78.76 Pg) or second‐order exponential (76.18 Pg) estimates. These results highlight the importance of variation at subannual timescales for upscaling to R_SG. The results indicated R_SG is lower than in recent papers and the current benchmark for land models (98 Pg C year^?1), and thus may change the predicted rates of terrestrial carbon turnover and the carbon to climate feedback as global temperatures rise.     

Autotrophic and heterotrophic soil respiration in a Norway spruce forest: estimating the root decomposition and soil moisture effects in a trenching experiment

The two components of soil respiration, autotrophic respiration (from roots, mycorrhizal hyphae and associated microbes) and heterotrophic respiration (from decomposers), was separated in a root trenching experiment in a Norway spruce forest. In June 2003, cylinders (29.7 cm diameter) were inserted to 50 cm soil depth and respiration was measured both outside (control) and inside the trenched areas. The potential problems associated with the trenching treatment, increased decomposition of roots and ectomycorrhizal mycelia and changed soil moisture conditions, were handled by empirical modelling. The model was calibrated with respiration, moisture and temperature data of 2004 from the trenched plots as a training set. We estimate that over the first 5 months after the trenching, 45% of respiration from the trenched plots was an artefact of the treatment. Of this, 29% was a water difference effect and 16% resulted from root and mycelia decomposition. Autotrophic and heterotrophic respiration contributed to about 50% each of total soil respiration in the control plots averaged over the two growing seasons. We show that the potential problems with the trenching, decomposing roots and mycelia and soil moisture effects, can be handled by a modelling approach, which is an alternative to the sequential root harvesting technique.     

Partitioning sources of soil respiration in boreal black spruce forest using radiocarbon

Separating ecosystem and soil respiration into autotrophic and heterotrophic component sources is necessary for understanding how the net ecosystem exchange of carbon (C) will respond to current and future changes in climate and vegetation. Here, we use an isotope mass balance method based on radiocarbon to partition respiration sources in three mature black spruce forest stands in Alaska. Radiocarbon (Δ¹⁴C) signatures of respired C reflect the age of substrate C and can be used to differentiate source pools within ecosystems. Recently‐fixed C that fuels plant or microbial metabolism has Δ¹⁴C values close to that of current atmospheric CO₂, while C respired from litter and soil organic matter decomposition will reflect the longer residence time of C in plant and soil C pools. Contrary to our expectations, the Δ¹⁴C of C respired by recently excised black spruce roots averaged 14‰ greater than expected for recently fixed photosynthetic products, indicating that some portion of the C fueling root metabolism was derived from C storage pools with turnover times of at least several years. The Δ¹⁴C values of C respired by heterotrophs in laboratory incubations of soil organic matter averaged 60‰ higher than the contemporary atmosphere Δ¹⁴CO₂, indicating that the major contributors to decomposition are derived from a combination of sources consistent with a mean residence time of up to a decade. Comparing autotrophic and heterotrophic Δ¹⁴C end members with measurements of the Δ¹⁴C of total soil respiration, we calculated that 47–63% of soil CO₂ emissions were derived from heterotrophic respiration across all three sites. Our limited temporal sampling also observed no significant differences in the partitioning of soil respiration in the early season compared with the late season. Future work is needed to address the reasons for high Δ¹⁴C values in root respiration and issues of whether this method fully captures the contribution of rhizosphere respiration.     

南方型杨树人工林土壤呼吸及其组分分析

采用开沟隔离法,利用LI-8100型土壤呼吸测定系统,对15年生的南方型杨树(Populus deltoides)人工林土壤呼吸进行了研究,并试图区分根系呼吸和土壤微生物呼吸。结果表明,开沟隔离处理后的10个月内,由于土壤中被截断根系具有自养呼吸和分解作用,土壤呼吸中的根系呼吸与微生物呼吸尚难以区分。尽管如此,研究表明15年生杨树人工林的土壤总呼吸通量为9.74 tC.hm-.2a-1,其中,枯枝落叶等土壤表层凋落物分解所释放的碳通量是2.63 tC.hm-.2a-1,占总量的27.0%;林木根系呼吸与土壤微生物呼吸通量的和为7.11 tC.hm-.2a-1,占总量的73.0%。土壤各组分呼吸速率与10 cm深处的土壤温度之间存在着显著的指数函数关系。不同直径的杨树根系被截断后的活力变化有所不同,根系越粗,存活时间越长。     

Examination of the method for measuring soil respiration in cultivated land: Effect of carbon dioxide concentration on soil respiration

An acceleration of soil respiration with decreasing CO₂ concentration was suggested in the field measurements. The result supporrs that obtained in laboratory experiments in our previous study. The CO₂ concentrations in a chamber of the alkali absorption method (the AA-method) were about 150–250 parts/10⁶ lower than that in the atmosphere (about 350 parts/10⁶), while those observed in the open-flow IRGA method (the OF-method) were nearly equal to the soil surface CO₂ levels. The AA-method at such low CO₂ levels in the chamber appears to overestimate the soil respiration. Our results showed that the rates obtained by the AA-method were about twice as large as those by the OF-method in field and laboratory measurements. This finding has important consequences with respect to the validity of the existing data obtained by the AA-method and the estimation of changes in the terrestrial carbon flow with elevated CO₂     

Partitioning of respiration from soil,litter and plants in a mixed-grassland ecosystem

Summary Carbon dioxide effluxes from plants, litter and soil were measured in two mixed-grassland sites in Saskatchewan, Canada. Ecosystems at both locations were dominated by Agropyron dasystachyum (Hook.) Scribn. Respiration rates of intact and experimentally-modified systems were measured in field chambers using alkali-absorption. Removal of green leaves, dead leaves, and litter from a wet sward reduced respiration to as low as 58% of the rate in an intact system. In a dry sward green shoots were the only significant above-ground source of CO₂.Carbon dioxide effluxes from different parts of A. dasystachyum plants, and from soil samples were measured in laboratory vessels at 20° using alkali-absorption. Respiration of green leaves (1.46 mg CO₂ g^-1 h^-1) was significantly higher than microbial respiration in moist, dead leaf samples (0.79 mg CO₂ g^-1 h^-1) or litter (0.75 mg CO₂ g^-1 h^-1). Microbial respiration in air-dried, dead plant material was very low. Average repiration rates of roots separated from soil cores (0.24 mg CO₂ g^-1 h^-1) were lower than many values reported in the literature, probably because the root population sampled included inactive, suberized and senescent roots. Root respiration was estimated to be 17–26% of total CO₂ efflux from intact cores.Laboratory data and field measurements of environmental conditions and plant biomass were combined in order to reconstruct the CO₂ efflux from the shoot-root-soil system. Reconstructed rates were 1.3 to 2.3 times as large as field measured rates, apparently because of stimulation to respiration caused by the experimental manipulations. The standing dead and litter fractions contributed 26% and 23% of the total CO₂ efflux in a wet sward. Both field-measured and reconstructed repiration values suggest that in situ decomposition of standing dead material under moist conditions can be a significant part of carbon balance in mixed grassland.     

中国南亚热带森林不同演替阶段土壤呼吸的分离量化

量化森林土壤呼吸(R_S)及其组分对准确地评估森林土壤碳吸存极其重要。该文以鼎湖山南亚热带季风常绿阔叶林及其演替系列针阔叶混交林和马尾松(Pinus massoniana)林为研究对象, 采用挖壕沟法结合静态气室CO₂测定法对这3种林分类型的R_S进行分离量化。结果表明: 鼎湖山3种森林演替系列上的森林R_S及其组分(自养呼吸R_A、异养呼吸R_H)均呈现出明显的季节动态, 表现为夏季最高、冬季最低的格局。在呼吸总量上, 季风常绿阔叶林显著高于针阔叶混交林和马尾松林, 但混交林与马尾松林之间差异不显著; R_A除季风常绿阔叶林显著大于针阔叶混交林外, 其余林分之间差异不显著; 对于R_H来说, 3个林分之间均无显著差异。随着森林正向演替的进行, 由马尾松林至针阔叶混交林至季风常绿阔叶林, R_A对土壤总呼吸的年平均贡献率分别为(39.48 ± 15.49)%、(33.29 ± 17.19)%和(44.52 ± 10.67)%, 3个林分之间差异不显著。方差分析结果表明, 土壤温度是影响R_S及其组分的主要环境因子, 温度与R_S及其组分呈显著的指数关系; 土壤含水量对R_S的影响不显著, 甚至表现为轻微的抑制现象, 但未达到显著性水平。对温度敏感性指标Q₁₀值的分析表明, 3个林分均为R_A的温度敏感性最大, R_H的温度敏感性最小。     

Estimation of autotrophic and heterotrophic components of soil respiration by trenching is sensitive to corrections for root decomposition and changes in soil water content

This study aims to assess the effects of corrections for disturbances such as an increased amount of dead roots and an increase in volumetric soil water content on the calculation of soil CO₂ efflux partitioning. Soil CO₂ efflux, soil temperature and superficial soil water content were monitored in two young beech sites (H1 and H2) during a trenching experiment. Trenching induced a significant input of dead root mass that participated in soil CO₂ efflux and reduced the soil dissolved organic carbon content, while it increased superficial soil water content within the trenched plot. Annual soil CO₂ efflux in control plots was 528 g C m⁻² year⁻¹ at H1 and 527 g C m⁻² year⁻¹ at H2. The annual soil CO₂ efflux in trenched plots was 353 g C m⁻² year⁻¹ at H1 and 425 g C m⁻² year⁻¹ at H2. By taking into account annual CO₂ efflux from decaying trenched roots, the autotrophic contribution to total soil CO₂ efflux reached 69% at H1 and 54% at H2. The partitioning calculation was highly sensitive to the initial root mass estimated within the trenched plots. Uncertainties in the remaining root mass, the fraction of root C that is incorporated into soil organic matter during root decomposition, and the root decomposition rate constant had a limited impact on the partitioning calculation. Corrections for differences in superficial soil water content had a significant impact on annual respired CO₂ despite a limited effect on partitioning.     

Separation of root and heterotrophic respiration within soil respiration by trenching, root biomass regression, and root excising methods in a cool-temperate deciduous forest in Japan

Trenching (Tr), root biomass regression (RR), and root excising (RE) methods were used to estimate the contribution of root (RR) and heterotrophic (HR) respiration to soil respiration (SR) in a cool-temperate deciduous forest in central Japan. The contribution ratios of RR to SR were 23 % (?16 to 46 %), 11 % (?19 to 61 %), and 115 % (20 to 393 %), as estimated by the Tr, RR, and RE methods, respectively. The contribution ratio showed clear seasonal variation with high values in summer for the Tr method, while they were undetectable for the RR and RE methods because of some methodological problems. These results suggest the Tr method is the best of the three methods used to estimate the contribution ratio of RR and HR to SR in the forest. Annual SR, RR, and HR rates, estimated by the Tr method, were 479, 369, 110 gC m^?2 year^?1, respectively. The seasonal variation of SR was mainly influenced by HR (77 %) throughout the year, while the influence of RR on SR was strongest in summer (46 %). This effect occurred because RR (Q ₁₀ = 7.5) is more sensitive to temperature than HR (Q ₁₀ = 3.2). Also, the contribution of fine RR to total RR was higher than that of coarse RR because of high respiratory activity (Q ₁₀ and R ₁₀) as well as the large biomass of fine roots. These results suggest that each component of SR responds differently to the same environmental factors and their relative influence on SR changes across the seasons.     

Partitioning the effect of composition and diversity of tree communities on leaf litter decomposition and soil respiration

The decomposition of plant material is an important ecosystem process influencing both carbon cycling and soil nutrient availability. Quantifying how plant diversity affects decomposition is thus crucial for predicting the effect of the global decline in plant diversity on ecosystem functioning. Plant diversity could affect the decomposition process both directly through the diversity of the litter, and/or indirectly through the diversity of the host plant community and its affect on the decomposition environment. Using a biodiversity experiment with trees in which both functional and taxonomic diversity were explicitly manipulated independently, we tested the effects of the functional diversity and identity of the living trees separately and in combination with the functional diversity and identity of the decomposing litter on rates of litter decomposition and soil respiration. Plant traits, predominantly leaf chemical and physical traits, were correlated with both litter decomposition and soil respiration rates. Surface litter decomposition, quantified by mass loss in litterbags, was best explained by abundance‐weighted mean trait values of tree species from which the litter was assembled (functional identity). In contrast, soil respiration, which includes decomposition of dissolved organic carbon and root respiration, was best explained by the variance in trait values of the host trees (functional diversity). This research provides insight into the effect of loss of tree diversity in forests on soil processes. Such understanding is essential to predicting changes in the global carbon budget brought on by biodiversity loss.     

A novel method of quantifying root exudation in the presence of soil microflora

A microcosm is described in which root exudation may be estimated in the presence of microorganisms. Ryegrass seedlings are grown in microcosms in which roots were spatially separated from a microbial inoculant by a Millipore membrane. Seedlings grown in the microcosms were labelled with [¹⁴C]-CO₂, and the fate of the label within the plant and rhizosphere was determined. Inoculation of the microcosms with Cladosporium resinae increased net fixation of the [¹⁴C] label compared to plants grown under sterile conditions. Inoculation also increased root exudation. The use of the microcosm was illustrated and its applications discussed.     

Measurement of soil respiration using closed chamber method: An IRGA technique

A closed chamber method (CC-method) using an infra-red gas analyzer (IRGA) for measuring soil respiration was examined. Two major factors which potentially cause errors: (i) volume of air sampled from the chamber; and (ii) measuring period of time, were examined in laboratory experiments. Field measurements were also conducted with both the CC-method and the open-flow IRGA method (OF-method) throughout a year. The results of laboratory experiments showed that (i) sampling volume of air should be less than 0.2% of the volume of the chamber; and (ii) the air within the chamber should be sampled several times within 20 min. Field measurements showed that soil respiration rates measured by the CC-method were not significantly different from those by the OF-method. The results of this study indicate that the CC-method is as effective for the measurement of the soil respiration rates as the OF-method.     

Thawing permafrost increases old soil and autotrophic respiration in tundra: Partitioning ecosystem respiration using δ13C and ∆14C

Ecosystem respiration (R_eco) is one of the largest terrestrial carbon (C) fluxes. The effect of climate change on R_eco depends on the responses of its autotrophic and heterotrophic components. How autotrophic and heterotrophic respiration sources respond to climate change is especially important in ecosystems underlain by permafrost. Permafrost ecosystems contain vast stores of soil C (1672 Pg) and are located in northern latitudes where climate change is accelerated. Warming will cause a positive feedback to climate change if heterotrophic respiration increases without corresponding increases in primary production. We quantified the response of autotrophic and heterotrophic respiration to permafrost thaw across the 2008 and 2009 growing seasons. We partitioned R_eco using Δ¹⁴C and δ¹³C into four sources–two autotrophic (above – and belowground plant structures) and two heterotrophic (young and old soil). We sampled the Δ¹⁴C and δ¹³C of sources using incubations and the Δ¹⁴C and δ¹³C of R_eco using field measurements. We then used a Bayesian mixing model to solve for the most likely contributions of each source to R_eco. Autotrophic respiration ranged from 40 to 70% of R_eco and was greatest at the height of the growing season. Old soil heterotrophic respiration ranged from 6 to 18% of R_eco and was greatest where permafrost thaw was deepest. Overall, growing season fluxes of autotrophic and old soil heterotrophic respiration increased as permafrost thaw deepened. Areas with greater thaw also had the greatest primary production. Warming in permafrost ecosystems therefore leads to increased plant and old soil respiration that is initially compensated by increased net primary productivity. However, barring large shifts in plant community composition, future increases in old soil respiration will likely outpace productivity, resulting in a positive feedback to climate change.     

Microsampling of rhizosphere soil and laboratory artifacts

A parallel analysis of the macrosamples of surrounding soil and microsamples of rhizosphere soil did not reveal the so-called rhizospheric effect. The data obtained showed that dilution significantly influences the results of determination of the number of soil microorganisms. The actual number of microorganisms revealed in soil samples greatly differed from the theoretically predicted values. The enumeration of microorganisms in soil microsamples by direct count and, especially, by the plating method with the use of conversion coefficients based on the degree of sample dilution gave erroneous results.