Stable isotopes of C and H in methane fermentation of agriculture substrates at different temperature conditions

1 Introduction

Anaerobic digestion (AD) is a microbiological decomposition of high molecular organic compounds yielding, among others, methane and carbon dioxide [1, 2]. A specific community of hydrolysing and acetogenic microorganisms as well as acetoclastic methanogens (Archaea), is responsible for the methane fermentation process [3]. Such processes are observed in many environments, eg. in marshes, wetlands, river sediments, in many coal basins, on landfills, as well as in the digestive tract of ruminants [16, 47].

AD depends on a number of physical and chemical factors, including chemical composition of the substrate, nutrient concentration, fermentation temperature, pH, volatile fatty acid composition, total nitrogen content, toxic substance content, hydration, salinity and dissolved oxygen level. These factors may stimulate or inhibit the speed and efficiency of the biological conversion of organic matter [4, 5, 6, 7].

Research in the area of isotope geochemistry, including isotopic fingerprints of carbon and hydrogen, are commonly used to distinguish gas origin (thermogenic, biogenic), and to distinguish the main methane generation pathways [45, 46]. In natural environment methane production usually follows two main paths (apart from others) [8]. It is either a product of a biological decomposition of acetic acid by heterotrophic microorganisms [9] or

reduction of carbon dioxide by autotrophic microorganisms [10]. According to Fey et al. [11], in natural environment, methane production is most often observed via acetoclastic methanogenesis, and then via hydrogenotrophic methanogenesis. Whiticar et al. [12] noticed that the isotopic composition of carbon in the methane generated during decomposition of acetic acid ranged from −60‰ to −33‰, while for the methane produced from carbon dioxide the value of δ¹³C varied from −110‰ to −60‰. Stable isotope analysis of carbon and hydrogen in raw materials and fermentation products might be useful to identify metabolic pathways of the fermentation [13, 14]. It is also a tool used to monitoring and describing the AD process state [15]. The isotopic analysis is important in studies on methane fermentation as it improves our understanding of complex biological processes. It allows for determining the efficiency of the methanogenesis and enables preliminary identification of carbon sources preferred by the methane-producing microorganisms [16].

Research on the interactions between bacterial methanogenesis and methanotropy are still in the area of interest among geochemists and microbiologists [16, 35, 45, 46]. This article outlines a way to identify methanogenic pathways in a precise manner using techniques related to stable isotope mass spectrometry. Moreover, it is possible to track changes in biogas production and the isotopic composition of substrates and products depending on the temperature used during methane fermentation. In these experiments agricultural raw materials such as maize silage and cattle manure served as substrates for experimental methane fermentation in bioreactors under controlled laboratory conditions. Bioreactors contained identical proportions of substrates, but were run under different temperature (20, 30 and 40ºC).

2 Materials and methods

3 Results

3.2 Biogas production

In each experiment pH ranged from about 5.1 to 7.9, depending on the methane fermentation stage. In the first days, acidification usually occurred and then the pH oscillated around neutral. As the experiment progressed, we observed a downward trend in oxygen content in the fermentation solution.

The yield of biogas production was 3.44 in FA variant, 4.14in FB variant, and in FC was 5.42 mM of gas per gram of organic carbon (see Fig. 1). At higher experimental temperatures, the biogas production increased (FA=84.5, FB=101.8 and FC=133.3dm³/kg VS), and the maximum of its production occurred sooner, than at the lowest temperature (FA=43 days, FB=36 days, FC=34 days).

Figure 1

Biogas production over time in the FA agriculture substrate incubation experiment.

Figure 2

Biogas production over time in the FB agriculture substrate incubation experiment.

Figure 3

Biogas production over time in the FC agriculture substrate incubation experiment.

Gas chromatography first detected methane presence on the third day of FA experiment and it was 0.02 [mM]/C_org[g]. Methane concentration was the highest on 42^nd day of FC experiment, when it reached 1.64 [mM]/C_org[g]. In all experiments, methane concentration increased around 14^th day of the incubation and grew until the last day of the observation. Methane concentration in all experiments was in the range from 0.01 to 1.64 [mM]/C_org[g] (see Tab. 2). An average concentration of methane in all headspace gas samples from the experiments presented in [mM]/C_org[g] and it was 0.39 for FA variant, 0.35 for FB, and 0.84 for FC. Carbon dioxide concentration fluctuated from 0.01 to 0.31 [mM]/C_org[g] in all experiments (see Tab. 2). An average concentration of CO2 in all headspace gas samples from the experiments presented in [mM]/C_org[g] and it was 0.17 for FA variant, 0.11 for FB, and 0.16 for FC.

Table 2

Characteristics of gaseous products from incubation experiments of agriculture substrates at different temperatures.

	FA 20°C	FB 30°C	FC 40°C
Biogas yield [mM]/Corg[g]	3.44	4.14	5.42
CH4 [mM]/Corg[g]	0.02 – 1.13	0.01 – 1.22	0.14 – 1.64
CO2 [mM]/Corg[g]	0.07 – 0.26	0.01 – 0.22	0.04 – 0.31
δ13C(CH4) [h]	−30.0 to −21.1	−41.3 to −19.9	−43.7 to −26.8
δ2H(CH4) [h]	−370.3 to −253.6	−379.4 to −294.5	−410.4 to −380.0
δ13C(CO2) [h]	+4.9 to +12.3	−10.0 to +13.9	−8.3 to +2.5

3.3 Isotopic analysis of headspace gases

Isotopic composition of methane and carbon dioxide in the collected biogas changed over time. In the FA variant (see Fig. 4), at 20^°C, δ¹³C(CH₄) ranged from −20.1‰at the beginning of the study to −30.0‰at the end of the study. Negative trend and decrease in value by nearly 10‰ from 3^th day of incubation to 34^th day with one episode of enrichment in heavy carbon isotopes on 28^th day of incubation has been observed. The value of δ²H(CH₄) varied from −370.3 to −253.6‰. Negative trend and decrease in value

Figure 4

Time-dependent values of δ¹³C(CH₄), δ¹³C(CO₂), δ²H(CH₄) and pH in FA experiment.

by nearly 117‰ from 14 day of incubation to 28 day has been recorded. Contrary to that, δ¹³C(CO₂) values ranged from +4.9 to +12.3‰ and also a negative trend and a decrease in the value of 7.4‰ from the 3^rd day of incubation to the 34^th day has been observed.

In FB variant (see Fig. 5), where the incubation was carried out at 30^°C, δ¹³C(CH₄) values varied from −41.3 to −19.9‰, δ²H(CH₄) values ranged from −379.4 to −294.5‰, and δ¹³C(CO₂) values varied from −10.0 to +13.9‰. In the case of δ¹³C(CH₄), a negative trend and a decrease in value by almost 15‰ from the 8^th to 28^th day of incubation has been observed, followed by a change in the trend and enrichment of the values in heavy isotopes by over 20‰to 42^nd of incubation. Regarding changes in the δ²H(CH₄), a negative trend has been observed from 8^th to 28^th day of incubation and a decrease in value by 46‰, followed by a change in the trend and enrichment the values in heavy isotopes of carbon by 9.5‰ up to the 42^nd day of incubation. In the case of δ¹³C(CO₂), also a negative trend and a decrease in value by nearly 24‰ from 4 to 20 days of incubation has been observed, followed by a change in the trend to positive and enrichment the values in heavy carbon isotopes by 6.5‰.

Figure 5

Time-dependent values of δ¹³C(CH₄), δ¹³C(CO₂), δ²H(CH₄) and pH in FB experiment.

The FC variant (see Fig. 6), in which methane fermentation proceeded at the highest temperature of 40^°C, was characterized by δ¹³C(CH₄) values varying from−43.7 to −26.8‰. δ²H(CH₄) values ranged from −410.4 to −380.0‰, and δ¹³C(CO₂) from −8.3 to +2.5‰. In this experiment, in the case of δ¹³C(CH₄) and δ¹³C(CO₂), a clear negative trend and a decrease in the value of nearly 12‰ and 11‰ respectively from the 14^th to 28^th day of incubation

Figure 6

Time-dependent values of δ¹³C(CH₄), δ¹³C(CO₂), δ²H(CH₄) and pH in FC experiment.

has been observed, and then change the trend to positive and enrichment the value in heavy isotopes by nearly 17‰ and 10.5‰, respectively, until the 42^nd day. Values of δ²H(CH₄), changed in a similar way, from the 14^th to the 20^th day of incubation, a negative trend and a drop in value by almost 12‰ has been observed, and then the trend changed to positive and enrichment in heavy isotopes by over 30‰, up to 34^th day of incubation has been observed.

4 Discussion

Maintaining kinetic equilibrium at individual fermentation stages is important for proper course of anaerobic digestion. For this reason, it is crucial to ensure optimal starting conditions for fermentation e.g. by providing the right composition of fermentation solutions with an optimal amount of nutrients for microorganisms [18]. In our experiment, C/N ratio of the substrates was 27.8 (see Table 1), which is optimal for methane fermentation [19, 20, 21].

The other vital parameters of anaerobic digestion include VFA and COD. During the process, the production of volatile fatty acids increases and intensifies the bio-gas generation. This enhances fermentation of the substrates [22, 23].

In properly run AD, VFA are removed from each kg of COD loads, and the biogas results from microbial activity. High concentration of VFA may inhibit anaerobic digestion [24].

A comparison of VFA and COD values at the beginning and at the end of the experiment (see Table 1) showed a significant decrease in their concentration. At 20^°C VFA and COD dropped by 86.8 and 87.1%, respectively, at 30^°C the decrease was 87.0 and 96.7%, and at 40^°C it peaked at 91.5 and 91.8%. Data analysis indicates that the use of a higher temperature of AD is associated with a decrease in the concentration of VFA and COD as well as with increase of the yield of biogas production (FA=84.5, FB=101.8 and FC=133.3 dm³/kg VS).

The TOC value decreased from 53.6% (SD 0.42) to FA = 34.3% (SD 0.27), FB = 33.9% (SD 0.87), FC = 33.5 (SD 0.14) at the end of incubation. This indicated partial consumption of organic carbon derived from the substrates during the AD.

The influence of the higher process temperature on biodegradation of organic carbon from the substrates was found. The degree of organic carbon consumption, determined on the basis of TOC, varied from 19.3 to 20.1% for the lowest and the highest temperature (see Table 1). To prove the above statement, ΔVS was calculated and it also showed partial consumption of the substrates during fermentation. Results varied from 23.0 to 25.8% between experiments (see Table 1).

Plant materials most often used for anaerobic digestion include agricultural substrates such as maize silage and other plant silages, slurry, manure, or decoction distillers. Analysis of literature provides information about carbon isotope composition of the above components (see Table 3). In comparison with other raw materials, maize silage (C4 photosynthesis plant product) is significantly more abundant in heavy isotopes of carbon and in our study δ¹³C value was −12.8‰, which is comparable with literature indications [25, 26]. Other raw materials used in ADin this study (cattle manure) show that the composition of the carbon isotope was −23.3‰(see Table 1).

In Table 1 shows the isotopic composition of the fermentation mixture measured at the beginning (F) and residue after the incubation at the end (FA, FB, FC) of AD. Noticeable changes in δ¹³C as a result of microbial activity at different temperatures were observed. In each experiment the residue after the incubation was depleted in heavy carbon isotopes, which was similar to a report published by Bucha et al. [29].

At the highest temperature the difference between the initial and final isotopic composition (Δ13Cb-e) of the fermentation solution was over 7‰, while at the lowest temperature (FA=20°C) it was only 1‰(see Table 1). We expected a reverse tendency, i.e. enrichment of the residue in heavy carbon isotopes as light isotopic compounds reacts faster than heavier. However, this process is often observed in case of degradation of single substrate in closed system. In case of our incubation experiments, the system was closed, but the organic substrates consist of variety of compounds e.g. lignin, cellulose, other constituents of plant organic matter. It is possible that only the heavier fraction of the substrate organic matter was decomposed, resulting in depletion in heavy carbon isotopes. This should be studied on a molecular level by means of e.g. GC-IRMS, GC-MS and HPLC.

Our results of isotopic analysis of carbon in methane were between −43.7‰ (FC) and −19.9‰ (FB), which indicated that methane originated from the decomposition of acetic acid and/or oxidation processes [12]. Also, stable carbon isotopes in the products were significantly enriched in heavy isotopes, which may be caused by bacterial methane oxidation during incubation [45]. David L. et al. [30] indicated importance of the isotopic distribution when characterizing CH₄ source. Therefore, we postulate that this finding is probably due to a strong enrichment of the substrate mixture with ¹³C (F) (see Table 1), and thus carbon sources from which methane was formed [31, 32]. The isotopic composition of the primary organic matter from which methane is produced, affects its final composition in terms of isotope distribution [1, 45].

The value of δ²H in the methane generated as a result of CO₂ reduction varies from −250‰ to −150‰, and via the fermentation of acetic acid from −400‰to −250‰[12, 33]. Our results of isotopic analysis of hydrogen in methane ranged from −410.4‰ (FC) to −253.6‰ (FA). As mentioned above, these outcomes are indicative of methane formation via acetoclastic reaction.

To confirm acetoclastic methanogenesis we calculated the isotopic fractionation coefficient between CH₄ and CO₂ using a formula proposed by Whiticar [12]:

αC = α¹³C(CO₂-CH₄)= (δ¹³C(CO₂) + 1000)/(δ¹³C(CH₄) + 1000)

value <1.06: pathway - acetic fermentation

values> 1.06: pathway - CO₂ reduction

The α C mean values from the experiments FA-FC ranged between 1.036 and 1.034, indicating predominance of the acetic pathway (see Table 4).

As reported in the literature, in the freshwater sediments the temperature is not a direct factor affected the methanogenesis pathways [34]. The isotopic composition of CH₄ is mostly dependent on carbon sources, their availability and microbial processes. However, in our experiments at higher AD temperature the isotope signal from the tested gas components is richer with light isotopes (Table 4). Szynkiewicz et al. [33] indicate that temperature changes affect the activity of various groups of methanogenic microorganisms, and thus changes in the dominance of methanogenesis pathways can be observed. In case of our experiments, the role of acetoclastic methanogenesis pathway was dominant in all temperature conditions, but the lower α¹³C_CO2−CH4 fractionation factor was observed at 30°C and 40°C (α¹³C_CO2−CH4= 1.033 and 1.034, respectively), than at 20°C (α¹³C_CO2−CH4= 1.036). It should be pointed that maize silage is a substrate rich in lactic acid, which can be used for acetate production. Detman A., et al. [35] suggested that the energy output on lactate degradation to the substrates for methanogenesis is the lowest in comparison to e.g. oxidation of acetate, butyrate, and propionate. So, attractiveness of lactate as an intermediate during AD is the highest. In acetogenesis lactate is oxidized mainly to acetate, which is used for methane production in acetotrophic pathway of methanogenesis.

To better understand the processes occurring during methanogenesis in the systems composed of many substrates, a correlation between δ¹³C(CH₄) and δ¹³C(CO₂) and between δ¹³C(CH₄) and δ²H(CH₄) (see Figure 7 and 8) was also analysed.

Figure 7

Variability of δ¹³C(CH₄) and δ¹³C(CO₂) values in FA, FB and FC experiments (all days).

Figure 8

Variability of δ¹³C(CH₄) and δ²H(CH₄) values in FA, FB and FC experiments (all days).

Analyzing the correlation graph (see Figure 7), it is clearly visible that with the application of a higher incubation temperature, the isotopic composition of both gases presents increasingly lighter isotope values.

Isotopic composition of methane and carbon dioxide shows the general tendency of enriching in time, both gases in the light carbon isotopes, in each temperature variant of the experiment. Surprisingly in the case of FB and FC experiments, after the 30 days of incubation, we can observe a sudden enrichment of both gases in heavy carbon isotopes. This may indicate the substrate consumption and switching the carbon source which has been decomposed during AD process [8, 13]. The highest correlation coefficient r = 0.93 occurred for the FC variant and there are statistically significant differences p = 0.02 (see Figure 7). This proves stability of the process at 40^°C as well as may also indicate that the process was carried out with one path of methanogenesis.

Interpretation of correlation between δ¹³C(CH₄) and δ²H(CH₄), it’s more complicated (see Figure 8). Nevertheless, a similar tendency to the one described above can be noticed. It can be seen that the values of carbon and hydrogen in methane, with increasing temperature of incubation are more depleted in heavy isotopes. In addition, particularly for the experiment FB and FC, it can be observed depletion of methane in heavy isotopes of carbon and hydrogen in the early days of incubation until the about 30 days, where the values abruptly change their course. The authors explain this by switching the source of carbon in digestion residues from which methane was generated.

5 Conclusions

In the course of a well-designed AD, the yield of biogas production rose along with temperature increase (FA 20ºC=3.44, FB 30°C=4.14, and FC 40°C=5.42 [mM]/C_org[g]). At the highest temperature of anaerobic digestion the consumption of organic carbon from the fermentation mixture was also the highest and amounted to 20.1% for TOC and 25.8% for VS.

Isotopic composition of maize silage was enriched in ¹³C. This could affect the isotopic composition ratio of the gases generated during AD. We observed enrichment of the fermentation mixture and the gas products in ¹³C. The isotopic composition of carbon in methane depends on the degree of selective decomposition of substrates and the share of individual components in total methane production.

At the highest experimental temperature we noted the highest isotopic fractionation of the fermentation mixture of more than 7‰, as compared with its initial and final isotopic composition.

Isotopic studies allow us to identify methanogenic pathways with high probability. Higher AD temperature resulted in the enrichment of gas products in light carbon and hydrogen isotopes. Our findings indicate that methane was generated in the acetoclastic pathway. The calculated carbon isotopic fractionation coefficient between CH₄ and CO₂ also indicated this type of methanogenesis.

It was observed that during the fermentation of acetic acid, as the temperature of the experiment increased, the isotopic fractionation between CH₄ and CO₂ was lower.

We found no strong correlations between δ¹³C(CH₄) and δ¹³C(CO₂), for bioreactors FA and FB, which may reflect complexity of the AD process. The correlation coefficient value turned out to be significant only for the highest incubation temperature (r= 0.93).

Performing an isotopic mass balance could provide additional insight and will be the subject of our future research. Moreover, as the biomass we used for AD consists of many potential and complex carbon sources, the experiments should be supplemented with molecular level studies involving simple carbon sources characteristic of agricultural biomass as well as research in the field of genomics.