Published online 2 February 2006
Published in J Environ Qual 35:516-521 (2006)
DOI: 10.2134/jeq2004.0443
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS
Waste Management
A Method for Measuring Low-Weight Carboxylic Acids from Biosolid Compost
Marina Himanena,*,
Kyösti Latva-Kalab,
Merja Itävaarab and
Kari Hänninena
Department of Biological and Environmental Sciences, University of Jyväskylä, Survontie 9, 40500 Finland
VTT Biotechnology, VTT Technical Research Centre of Finland, Tietotie 2, Espoo, Finland
* Corresponding author (marina.himanen{at}bytl.jyu.fi)
Received for publication November 19, 2004.
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ABSTRACT
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Concentration of low-weight carboxylic acids (LWCA) is one of the important parameters that should be taken into consideration when compost is applied as soil improver for plant cultivation, because high amounts of LWCA can be toxic to plants. The present work describes a method for analysis of LWCA in compost as a useful tool for monitoring compost quality and safety. The method was tested on compost samples of two different ages: 3 (immature) and 6 (mature) months old. Acids from compost samples were extracted at high pH, filtered, and freeze-dried. The dried sodium salts were derivatized with a sulfuric acid–methanol mixture and concentrations of 11 low-weight fatty acids (C1–C10) were analyzed using headspace gas chromatography. The material was analyzed with two analytical techniques: the external calibration method (tested on 11 LWCA) and the standard addition method (tested only on formic, acetic, propionic, butyric, and iso-butyric acids). The two techniques were compared for efficiency of acids quantification. The method allowed good separation and quantification of a wide range of individual acids with high sensitivity at low concentrations. Detection limit for propionic, butyric, caproic, caprylic, and capric acids was 1 mg kg–1 compost; for formic, acetic, valeric, enanthoic and pelargonic acids it was 5 mg kg–1 compost; and for iso-butyric acid it was 10 mg kg–1 compost. Recovery rates of LWCA were higher in 3-mo-old compost (57–99%) than in 6-mo-old compost (29–45%). In comparison with the external calibration technique the standard addition technique proved to be three to four times more precise for older compost and two times for younger compost. Disadvantages of the standard addition technique are that it is more time demanding and laborious.
Abbreviations: LWCA, low-weight carboxylic acids
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INTRODUCTION
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AMOUNT OF LOW-WEIGHT CARBOXYLIC ACIDS (LWCA) in compost mass is an important parameter for the composting process as these acids can have a toxic effect to plants (Epstein, 1997). Additionally, LWCA are contributors to odor nuisance during the composting process. Therefore, choosing a proper method for LWCA analysis is a significant task for monitoring composting processes and avoiding toxicity to plants when compost is applied for plant growth. The purpose of this paper is to attract attention of researchers to the importance of choosing an analytical method for analysis of LWCA in composts of different age.
In many published studies focusing on LWCA dynamics in compost, methods used for acids analysis are slightly modified from the methods applied to different matrixes, for example, wastewater (Brinton, 1998), waste material (Schuman and McCalla, 1976), and soil (Kirchmann and Widén, 1994; García et al., 1991). In these references and many others it is not mentioned whether suitability of the method for the compost matrix was tested. All the methods mentioned above are based on distillation of acids at low pH and their analysis using liquid or gas chromatography. This procedure leads to losses of volatile substances as in acidic conditions LWCA are in the most volatile form. One preferable option for acids extraction from compost is using solution with basic pH, which leads to formation of acid salts and prevents losses of substance due to volatilization. A number of articles contain examples of this type of extraction method (Elliott and Travis, 1975; Brinton, 1998; Baziramakenga and Simard, 1998). This method also allows storage of extracted salts for a rather long time before analysis is conducted, which makes it more convenient.
In all the references mentioned above LWCA were analyzed using so-called external calibration methods, when acids in the unknown sample are analyzed and concentration of compounds calculated on the basis of a calibration graph obtained from the analysis of standard solution. Although the external calibration method is widely applied, the differences in pH, ionic strength, temperature, impurities, and sample matrix structure, which are very typical for compost mass, may interfere or change the analytical signal produced by the chromatograph and cause erroneous results (Bader, 1980). The standard addition method overcomes this problem. In this case the acids are directly added to the sample, which increases the peak area in the chromatogram. By making a series of additions, the original unknown concentration of component can be calculated by extrapolating the straight line built from a series of points until it crosses the abscissa, which represents the absolute value of the original concentration (Robards et al., 1997), or in other words, the amount of compound both extracted and trapped in the matrix.
Another aspect is that methods differ in a range of acids that can be analyzed with them. In many articles variety of acids mentioned is rather small. For example, García et al. (1991) report data on acetic, propionic, iso-butyric, and butyric acids studied in water extracts of sewage sludge compost; Chanyasak et al. (1982) present results of two groups of compounds in water extracts of composted garbage (acetic and other low fatty acids); and DeVleeschauwer et al. (1982) discuss dynamics of acetic, propionic, iso-butyric, butyric, and iso-valeric acids in refuse compost. Very few articles contain information on dynamics of formic acid, which is an interesting compound due to the structure, small size, and activity of the acid molecule. Usually restrictions for a variety of acids that can be analyzed are defined by analytical instruments and columns that are used in chromatographic analysis.
The method described in this work has been designed with the intention that special features of compost, like maturation, absorption of compounds to compost matrix, volatilization of LWCA, and microbial degradation, could be minimized. The method has been developed at the Technical Research Center of Finland and has been previously applied for other materials (e.g., bread and diary products). The objectives of the present research were to (i) measure the amounts of a wide range of low-weight fatty acids (C1–C10) in compost by applying basic extraction–derivatization and headspace sample injection, (ii) compare the amount of low-weight fatty acids (C1–C4) analyzed by the external calibration and standard addition methods, and (iii) calculate and compare recovery rates of small quantities of LWCA from compost samples of two different ages.
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MATERIALS AND METHODS
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Sampling and Preparation of Compost Material
Samples of biosolid compost were collected at the full-scale composting plant of the Helsinki Metropolitan Area Council, Finland. At the plant, source-separated biowaste mixed with wood chips is processed in composting tunnels for 2 wk and after that transferred outdoors and piled in windrows for maturation. Samples for experiments were collected from two piles of different age: 3 and 6 mo old. Compost of two different ages was chosen to study applicability of the method for analysis of mature and immature composts, which have different chemical and microbial structures. The samples were packed in polyethylene bags (200-L volume) and transported to the laboratory. Upon arrival samples were sieved through a 5-mm screen, thoroughly mixed, divided into approximately 1-L subsamples, and stored frozen at –20°C in the sealed plastic bags. Subsamples were used for LWCA analysis when needed.
Dry matter, ash content, pH, and conductivity of the samples were analyzed according to standard methods CEN 13039, CEN 13037, and CEN 13038, respectively (European Committee for Standardization, 1999a, 1999b, 1999c). Concentrations of dissolved ammonium, nitrate, and nitrite ions in compost water extract (1:5) were detected using test strips (Aquamerk for NH4+ and Merckoquant for NO2– and NO3–; Merck, Darmstadt, Germany). Phytotoxicity of compost samples was tested with a plant bioassay. The tested mixture was compost and peat-based growth medium (1:3). The control was peat-based growth medium; the tested plant was gardencress pepperweed (Lepidium sativum L.), 100 seeds per pot, with four replicates. Plants were grown in a phytotron. The temperature was 25°C and relative humidity 60%, with a day–night regime of 16 and 8 h, respectively. Cultivation lasted for 8 d, after which germination percent and dry weight of seedling shoots were measured. Results are presented as percent germination or weight to control.
Analysis of Low-Weight Carboxylic Acids
Calculation of LWCA concentrations in compost mass and their recovery rates was done in two stages. First, amounts of LWCA (background levels) were analyzed and calculated using the external calibration method and then the standard addition method.
External Calibration Method
For the external calibration analysis, 5 g of subsample, unfrozen at 4°C, was weighted into a glass beaker (500-mL volume), 50 mL of 0.1 M NaOH was added to the sample, and the mixture was homogenized with a blender (Bamix, Mettlen, Switzerland) for 2 min (pH >12). The slurry was filtered through a prerinsed filter paper (Number 4, 
15 cm; Whatman, Maidstone, UK) into a 100-mL glass volumetric flask. The beaker was rinsed twice with 15 mL of 0.1 M NaOH and washings were filtered through the same filter paper. After all the solvent was drained, the flask was filled to the mark with 0.1 M NaOH and shaken.
One- and ten-milliliter aliquots were pipetted into 22-mL headspace vials and the vials were covered with foil and frozen at –20°C. Each aliquot had two parallels. Frozen aliquot was dried in a vacuum drier and stored at room temperature covered with aluminum foil. Before analysis freeze-dried aliquot was derivatized with 2 mL of methylating reagent (163 mL 50% sulfuric acid + 138 mL methylalcohol), sealed immediately with a Teflon-faced rubber septa and aluminum seal, and temperated at 80°C in a water bath for 10 min. Methylated samples were cooled down to room temperature and used for LWCA chromatographic analysis.
The acid methyl esters were analyzed with a gas chromatograph (HP5890 Series II; Hewlett-Packard, Palo Alto, CA), which was interfaced to a static headspace autosampler (Tekmar 7000; Teledyne Tekmar, Mason, OH). The samples were equilibrated at 80°C for 30 min. Pressurization time was 0.5 min, pressure equilibrate time 0.25 min, loop fill time 0.15 min, and loop equilibrate time 0.05 min. Both sample loop and transfer line temperatures were 180°C. The gas chromatograph was equipped with a 60-m x 0.53-mm RTX-1701 column (SGE, Austin, TX) and the flame ionization detector operated at 250°C. The injector temperature was 220°C. The oven temperature program was the following: from –20 to 130°C temperature rise rate was 5°C min–1, from 130 to 240°C rise rate was 20°C min–1, and time at 240°C was 6.5 min. Helium was used as a carrier gas with a velocity rate of 25 cm s–1. The split ratio was 1:30. Injection volume was 1 mL. Concentration of each acid was calculated based on the peak area of acids in the sample and standard solution of acids. Number of replicates analyzed by this technique was 14.
The following acids were identified with the described technique: formic (methanoic), acetic (ethanoic), propionic (propanoic), butyric (n-butanoic), iso-butyric (iso-butanoic), valeric (n-pentanoic), caproic (n-hexanoic), enanthic (n-heptanoic), caprylic (n-octanoic), pelargonic (n-nonanoic), and capric (n-decanoic).
Standard Addition Method
For conducting analysis of LWCA with the standard addition method five acids were added (formic, acetic, propionic, butyric, and iso-butyric) to each compost sample at increasing concentration in proportion to the background concentration measured with the external calibration method. The amounts of acids added are presented in Table 1.
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Table 1. The amount of individual low-weight carboxylic acids (LWCA) added to 3- and 6-mo-old compost samples for conducting the standard addition method of LWCA analysis.
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A corresponding amount of each acid was added to a 5-g compost sample and weighted into a glass vial (38-mL volume). The vial was closed immediately with a screw lid, thoroughly shaken, and allowed to stand for 2 h at room temperature so that the compost could absorb the acids but the microbes existing in compost would decompose the acids as little as possible. Twenty milliliters of 0.1 M NaOH was added in to the vial. After mixing the compost mass was allowed to stand for another 30 min. Content of the vial was transferred into a glass beaker (500-mL volume), the vial rinsed twice with 15 mL of 0.1 M NaOH, and washings combined to the same beaker. The sample was homogenized for 2 min and filtered through a prerinsed filter paper (Number 4, 15 cm; Whatman) into a 100-mL glass volumetric flask. After all solvent was drained, the bottle was filled to the mark with 0.1 M NaOH and shaken. For methylation and analysis procedures, see the External Calibration Method section (above). Each vial had four replicates.
Results of the standard addition method were used for recalculation of acid concentrations in 3- and 6-mo-old composts. For each acid it was done based on five concentration points. For a detailed equation refer to Harris (1999).
Recovery rate of each acid was calculated based on results of the standard addition method. Calculations were done according to the formula:
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where Asample is peak area of acid determined in the sample after addition of acids; Abackground is average peak area of acid in the original compost sample; and Astandard is peak area of acid determined in standard solution.
Statistical Analysis
Concentrations were analyzed by two different analytical methods. Significance of differences between the mean concentrations measured with two methods was tested with a t test using SPSS 12.01 (SPSS, 2003). To minimize effect of difference in variability and in number of replicates (14 for the external addition and 4 for the standard addition tests) concentrations to be compared were log transformed before statistical analysis. Differences in variability between the two treatments were tested with Levene's test using SPSS 12.01.
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RESULTS AND DISCUSSION
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Results of the basic analysis and plant bioassay of compost samples are presented in Table 2.
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Table 2. Basic characteristics of 3- and 6-mo-old composts and results of plant growth bioassay on gardencress pepperweed (Lepidium sativum L.).
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Application of Basic Pretreatment and Headspace Gas Chromatography for Low-Weight Carboxylic Acids Analysis
Extraction of LWCA from compost samples with basic solution and further derivatization with methanol at low pH was an effective pretreatment method that allowed successful identification and quantification of acids with headspace gas chromatography. In spite of a high number of "noise" peaks produced by compounds originated from compost and solvent, peaks of 11 methyl esters of low-weight fatty acids (from formic to capric) could be clearly identified using standard solution. Figure 1 exhibits an example of a chromatography profile of 3-mo-old compost sample to illustrate peak shape. Although peaks of some methyl esters are small due to low concentrations, they are sharp and narrow and good for integration. The pretreatment method allowed us to prevent evaporation of volatile substances and store treated samples for up to 2 wk before analysis of acids without affecting the final result. This increases convenience of method application especially when a large number of samples should be analyzed. Therefore, the method gives an opportunity to study dynamics of these acids during the composting process in the future and investigate the role of LWCA in generating noxious odors during the composting process as well as toxicity to plants. In this research we have concentrated only on 11 carboxylic acids with the smallest molecular size, but this method has potential for analysis of a broader range of carboxylic acids, although this might need some tuning of chromatograph parameters.
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Fig. 1. Typical gas chromatography profile of 3-mo-old compost sample extracted at basic pH and derivatized with methanol at low pH. Peaks of methyl esters of formic (1), acetic (2), propionic (3), iso-butyric (4), butyric (5), valeric (6), caproic (7), enanthoic (8), caprylic (9), pelargonic (10), and capric (11) acids are sharp, symmetric, and quantifiable.
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Although 1- and 10-mL aliquots were used for LWCA analysis, in further discussion results of 10-mL aliquots only will be used because amounts of acids in 1-mL aliquots were close to detection limits of the method. Detection limits of individual acids with both methods varied considerably. For propionic, butyric, caproic, caprylic, and capric acids it was 1 mg kg–1 compost; for formic, acetic, valeric, enanthoic, and pelargonic acids it was 5 mg kg–1 compost; and for iso-butyric acid it was 10 mg kg–1 compost.
As it was described in Materials and Methods, the standard addition method was tested on five fatty acids only: formic, acetic, propionic, iso-butyric, and butyric. Linear response of standard solutions of these acids is presented in Fig. 2. Among all acids iso-butyric acid was the most sensitive and easy to identify, the most difficult to measure was acetic acid. Similar results were obtained by Paul and Beauchamp (1989) claiming that among three acids (acetic, propionic, butyric), acetic acid was the most difficult to measure especially at low concentrations.
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Fig. 2. Linear response of low-weight carboxylic acids (LWCA) standard solutions to increasing acid concentrations. The most sensitive and easiest for identification is iso-butyric acid, while the most difficult is acetic acid.
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Percent recovery of added fatty acids is presented in Table 3. In samples of 3-mo-old compost average recovery rates were higher than in 6-mo-old composts. For 3-mo-old samples the percentage was in a range of 57 to 99% and for 6-mo-old compost it was 29 to 45%. In older samples recovery rates of formic and acetic acids were higher than propionic, butyric, and iso-butyric acids; in younger samples no similar trend could be observed. These percentages were lower than reported by Paul and Beauchamp (1989), who tested the volatile fatty acid standard addition method on silt loam soil. They obtained recovery rates of acetic, propionic, and butyric acids ranging from 94 to 114% in a 3:1 water to soil extract. Lower recovery rate in this study especially in older sample can be explained by the structure of compost matrix. As compost is getting more mature and amount of complex organic molecules (e.g., humic substances) increases, they bind LWCA more tightly and make compounds more difficult for extraction. Therefore, the matrix effect plays a very significant role in analysis of LWCA. This assumption may bring an important aspect in selecting the proper analytical method when studying dynamics of LWCA during maturation of compost.
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Table 3. Average recovery rates of low-weight carboxylic acids (LWCA) in 3- and 6-mo-old biowaste compost samples pretreated with a basic solution and analyzed with headspace gas chromatography.
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Standard Addition versus External Calibration
Concentrations of LWCA in compost samples were analyzed using both the external calibration and standard addition methods. Comparison of acid concentrations analyzed by the two methods is presented in Fig. 3 and 4. Results of both methods showed that formic and acetic acids were dominating in composts of both ages; their concentrations several hundreds times exceeded concentrations of propionic, butyric, and iso-butyric acids. Baziramakenga and Simard (1998) in their work have obtained similar data stating that both acetic and formic acids were dominating in composted manures. It is a very interesting fact that these acids were of similar concentration levels, because a lot of authors claim that on a large range of LWCA only acetic acid plays major role in producing phytotoxic effects (DeVleeschauwer et al., 1982; Lynch, 1977). Such a conclusion is made in those studies where concentration of formic acid was not measured. Results of a bioassay test (see Table 2) showed that compost of both ages significantly suppressed plant growth of cress salad. Therefore, it might be assumed that high concentrations of both formic and acetic acids could be the reason for it. But a toxicological study of acids is needed for proving this hypothesis.
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Fig. 3. Concentrations of formic and acetic acids analyzed by the external calibration and standard addition methods in 3- and 6-mo-old compost samples (mean ± SD; for external calibration n = 14, for standard addition n = 4). Amounts of acids analyzed by the standard addition method are significantly higher than by the external calibration method (** indicates significance at the 0.01 probability level), especially in 6-mo-old compost, where the difference is three to four times.
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Fig. 4. Concentrations of propionic, butyric, and iso-butyric acids analyzed by the external calibration and standard addition methods in 3- and 6-mo-old compost samples (mean ± SD; for external calibration n = 14, for standard addition n = 4). Amounts of acids analyzed by the standard addition method are significantly higher than by the external calibration method (* indicates significance at the 0.05 probability level, ** significance at the 0.01 probability level). Only for butyric acid is the difference not significant.
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Statistical analysis of results showed that the standard addition method had higher variability than the external calibration method, which could be due to the extrapolation procedure of several points of the sample. But even in spite of high variability the standard addition method allowed us to analyze significantly higher concentrations of all acids except butyric. An interesting fact is that for more mature compost the difference between means of the methods was three to four times, while for younger compost the difference was about two times. Again this proves the idea that choosing an improper analytical method can give a substantially erroneous result especially in analyzing LWCA in older composts.
An advantage of the standard addition technique is that it allows us to obtain more precise data on concentrations of LWCA in compost samples in comparison with the external calibration technique, as it takes into consideration the effect of acid entrapment to the compost matrix. Disadvantages of this technique are that it is laborious, time consuming, and costly.
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ACKNOWLEDGMENTS
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Acknowledgments are made to the National Technology Agency of Finland (TEKES) (Project no. 40689) and the Maj and Tor Nessling Foundation (Project no. 2003232) for financial support of the work and to Anna-Liisa Ruskeepää for technical assistance in analyzing LWCA with gas chromatography.
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REFERENCES
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ABSTRACT
INTRODUCTION
