applied sciences Article Theoretical Methane Emission Estimation from Volatile Fatty Acids in Bovine Rumen Fluid Sang-Ryong Lee * , Yunseo Cho, Hyuck K. Ju and Eunjeong Kim Department of Biological Environmental Science, College of Life Science and Biotechnology, Dongguk University, Seoul 04620, Korea; [email protected] (Y.C.); [email protected] (H.K.J.); [email protected] (E.K.) * Correspondence: [email protected]; Tel.: +82-31-961-5723 Abstract: Methane production from livestock farming is recognized as an important contributor to global GHGs. Volatile fatty acids (VFAs) found in bovine rumen may be utilized as a substrate for methanogens to form CH4, and thus improvement of quantitative VFA measurements can help facilitate greater understanding and mitigation of CH4 production. This study aims to contribute to the development of more accurate methods for the quantification and specification of VFAs in bovine rumen. The VFAs were analyzed using the conventional method and an alternative catalytic esterification reaction (CER) method. Substantial differences in the detected concentrations of the C3+ VFAs (chain length ≥ 3) were observed between both methods, especially for butyric acid. Evaluation of the sensitivity of both methods to detecting the VFA concentrations in standard solutions confirmed that the values resulting from the CER method were closer to the known concentrations of the standard solution than those from the conventional method. The results of this study provide the first quantitative proof to show the improved accuracy of the measurements of C3+ VFAs when using the CER method compared with the conventional method. Therefore, the CER Citation: Lee, S.-R.; Cho, Y.; Ju, H.K.; method can be recommended to analyze the VFAs found in rumen, especially butyric acid and other Kim, E. Theoretical Methane C3+ VFAs. Emission Estimation from Volatile Fatty Acids in Bovine Rumen Fluid. Keywords: rumen; odor; volatile fatty acids; methane; catalytic esterification Appl. Sci. 2021, 11, 7730. https:// doi.org/10.3390/app11167730 Academic Editor: Leonarda 1. Introduction Francesca Liotta Food and Agriculture Organization (FAO) statistics show that the worldwide sup- ply of animal protein has risen from 34 to 43 kg per capita per year between 1993 and Received: 19 July 2021 2013 [1]. Large inequalities in protein consumption between countries mean that the an- Accepted: 18 August 2021 Published: 22 August 2021 nual consumption of animal proteins in many wealthier nations far exceeds this amount. For example, in 2013, the annual per capita animal protein supply in North America, the Publisher’s Note: MDPI stays neutral European Union, and Australia and New Zealand stood at 113 kg, 81 kg, and 114 kg, respec- with regard to jurisdictional claims in tively [1]. To sate our massive demand for meat and dairy products, concentrated animal published maps and institutional affil- feeding operations (CAFOs) have inevitably served the long-term viability of the livestock iations. industry over the last three decades [2–4]. Despite their economic benefits and production efficiencies, CAFOs have triggered unwanted environmental problems due to the large production of manure waste, far exceeding the capacity of land to assimilate the loadings of organic carbon and nutrients [5,6]. One of the recent urgent issues associated with CAFOs is the loss of gaseous species to the ambient air stream, which has contributed to climate Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. change by means of emitting potent greenhouse gasses such as CH4 and N2O[7,8]. For This article is an open access article instance, animal agriculture contributes 9% of anthropogenic CO2 emissions, 37% of CH4 distributed under the terms and emissions, and 65% of N2O emissions, and the combined emissions expressed as a CO2 conditions of the Creative Commons equivalent amounts to about 18% of anthropogenic greenhouse gas (GHG) emissions [7]. Attribution (CC BY) license (https:// Despite the well-defined guidelines to estimate GHG emissions from the Intergovernmental creativecommons.org/licenses/by/ Panel on Climate Change (IPCC), the whole rationale for estimating GHG emissions from 4.0/). the livestock sector is only sensible with the support of robust and accurate data sets [9,10]. Appl. Sci. 2021, 11, 7730. https://doi.org/10.3390/app11167730 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 7730 2 of 9 To this end, it is highly desirable to establish technical advancements to provide robust and accurate data sets for estimating GHG emissions from the livestock sector. Among the various GHGs from the livestock industry, CH4 is a major ubiquitous GHG during the normal digestive process in ruminant animals, and its global warming potential is 25 times that of CO2 [11]. For instance, ruminal methanogens use the methanogenesis pathway to maintain low H2 partial pressure and to facilitate fiber digestion in the rumen by converting H2 to CH4 [12]. There are two strategies to reduce CH4 emissions from the livestock sector. One strategy is dietary manipulation [11], and another strategy is to improve the efficiency of ruminal function and to mitigate methane release [12]. However, quantification of CH4 emissions from the livestock industry is challenging because a limited number of individual animals are monitored in any study, and correction factors are required to calculate actual CH4 emission values [10]. Moreover, direct quantification of individual animal CH4 emissions in open-circuit respiration chambers or using the sulfur hexafluoride (SF6) tracer technique requires considerable investment in infrastructure and technical support and may impact animal feeding behavior [10,12]. VAF quantification methods through a catalytic esterification reaction (CER) requires a biochemical methane potential (BMP) test that allows alternative measurements compared with the conventional methane estimation methods such as respiration chamber techniques or direct stomach porthole treatment through the livestock. It is urgent that a quantitative methodology for estimating CH4 emissions from the livestock sector should be developed. Better accuracy in CER VFA measurements will provide compensation to methane emission inventory, especially from livestock. This case study aims to contribute to the develoment of more accurate methods for the quantification and specification of volatile fatty acids (VFAs) in bovine rumen. Improved quantitative measurements of VFAs could be correlated with the theoretical production of CH4 due to the likelihood that VFAs can be utilized as a substrate for methanogens to form CH4 [13,14]. Methanogens live in a variety of environments, including in the human and animal gut, and in ruminants that are responsible for emitting abundant amounts of methane during the digestion of food [15,16]. To ensure the accuracy of quantitative measurements of VFAs in bovine rumen and excreta, a reliable analytical technique should be developed. In addition, for quantification of VOCs using gas chromatography (GC), quantifying VFAs by their corresponding methyl esters via derivatization is favorable over their direct analysis as VFAs themselves, because the hydrogen bonds derived from their carboxyl group cause low resolution and tailing peaks [17]. However, conventional derivation methods contain several difficulties. First, a homogenous acid catalyst is needed, which means a washing process is required to remove salt [18–20]. Second, the yield of methyl ester is readily affected by impurities such as contaminants and moisture in the sample, thereby resulting in a low conversion efficiency [21–23]. Third, hazardous and potentially explosive solvents such as diazomethane are needed for the esterification reaction [24–26]. Moreover, the conventional analytical methods require extraction and isolation steps prior to analysis [27]. To overcome these technical challenges, quantification of VFAs was conducted via catalytic esterification [21,23–25,27] without the pretreatment of the bovine rumen and excreta samples. To evaluate the efficacy of this new approach, quantification of VFAs by the CER method was compared with that of the conventional method. 2. Materials and Methods 2.1. Chemical Reagents and Materials Rumen fluids were obtained from the National Institute of Animal Science (NIAS) in Korea, and the samples were stored in a freezer at −37 ◦C. The VFA standard mixtures were prepared by using the pure VFAs and deionized water. All pure VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) and all pure VFA methyl ester standard solutions (acetic acid methyl ester, propionic acid methyl ester, butyric acid methyl ester, isobutyric acid methyl ester, valeric acid methyl ester, and isovaleric acid methyl ester) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Appl. Sci. 2021, 11, 7730 3 of 9 Methanol and silica with pore size of 60 Å were purchased from Sigma-Aldrich (St. Louis, MO, USA) as well. 2.2. Conventional Method Five milliliters of rumen fluid was mixed with 1 mL of a 25% metaphosphoric acid solution and 0.05 mL of saturated mercuric chloride solution in a 15 mL PTFE tube. The mixture was centrifuged at 4000 rpm for 20 min (20 ◦C). The sets of 1 mL of the super- natants were moved into 1.5-mL centrifuge tubes and then centrifuged at 12,000 rpm for 10 min (20 ◦C). After centrifugation, the supernatants were filtered using a 0.2-µm syringe filter and extracted to a 20-mL GC vial. The prepared samples were injected