Improvements in Drill-Core Headspace Gas Analysis for Samples from Microbially Active Depths

The IsoJarTM container is widely used in headspace gas analysis for gases adsorbed on cuttings or bore cores from oil and gas fields. However, large variations in the carbon isotopic ratios of CH4 and CO2 are often reported, especially for data obtained from depths of <1000m. The IsoJarTMmethod leaves air in the headspace that allows microbial oxidation of CH4 to CO2, meaning that isotopic fractionation occurs during storage. This study employed the IsoJarTM method to investigate the causes of differences in δC data reported by previous studies in the Horonobe area of Japan. It was found that after 80 d storage, δCCO2 values decreased by ~2‰, while δCCH4 values increased by >30‰, whereas samples analyzed within a week of collection showed no such fluctuations. The conventional amount of microbial suppressant (~0.5ml of 10% benzalkonium chloride (BKC) solution) is insufficient to suppress microbial activity if groundwater is used as filling water. The significant variations in carbon isotopic compositions previously reported were caused by microbial methane oxidation after sampling and contamination by groundwater from different depths. To avoid these problems, we recommend the following: (1) if long-term sample storage is necessary, >10ml of 10% BKC solution should be added or >0.3% BKC concentration is required to suppress microbial activity; (2) analyses should be performed within one week of sampling; and (3) for CO2 analyses, it is important that samples are not contaminated by groundwater from different depths.


Introduction
Investigation of the origin of deep hydrocarbons is an important aspect of resource exploration and may lead to an improved understanding of geological environments.In this investigation, gases adsorbed on rock fragments or bore cores were studied by headspace gas analysis (e.g., [1][2][3][4][5]), which provides information on the generation and migration of light hydrocarbons and gases.The IsoJar™ (Isotech Laboratories and Humble Instruments, USA) container, which is widely used in such analyses (e.g., [6,7]) comprises a plastic container of ~600 ml volume with an aluminum screw cap on which there is a rubber septum through which headspace gas can be taken by syringe.The analysis procedure (e.g., [8]) normally involves storage of wet cuttings or cores in the jar with water and an air headspace for several days or weeks, the addition of a microbicide such as benzalkonium chloride (BKC) to minimize bacterial activity, the partitioning of gas into the headspace during storage, and analysis of these gases (e.g., [9][10][11]).The use of distilled or tap water avoids contamination from dissolved gases.The δ 13 C CH4 values of gases from depths of <1000 m, in the biogenic region, are usually in the range of -70‰ to −60‰, with isotopic compositions becoming heavier as depth increases towards the thermogenic region (e.g., [12,13]).Large variations in carbon isotopic ratios in CH 4 and CO 2 are often reported for depths of <1000 m, with δ 13 C CH4 values sometimes reaching −20‰ (e.g., [14][15][16][17]).These variations are associated with the effects of microbial activity on methane production or oxidation in underground environments [18].There are a number of factors that control the rate of methanogenesis [19], including temperature [20], groundwater salinity [21], pH [22], and pore space [23].Peak microbial activity occurs at 35-45 °C, which corresponds to depths of <1000 m [19,24].
At greater depths, microbial action decreases as thermogenic production increases with the onset of catagenesis (subsequent to diagenesis at shallower depths [8]).More importantly, pore diameters of at least 1 μm are necessary for in situ methanogenesis, as microbes are in the 1-10 μm size range [25], which suggests that active methane production occurs at depths of <1500 m [24].At shallow depths (less than several meters) below the ocean floor, where the concentration of dissolved gas is relatively low, considerable care was taken to avoid contamination and microbial activity (e.g., [26]).Hachikubo et al. [27] adjusted the concentration of BKC in samples to ~2.5% using 25 ml vials to obtain precise depth profiles of gases relative to hydrates.While the concentration and/or amount of microbicide normally added to IsoJar™ vessels are often omitted in reports, it is considered that the final concentration in IsoJar™ containers should be of the order of 0.01%, which is two orders of magnitude less than that reported by Hachikubo et al. [27].It is speculated that another possible cause of variations in carbon isotopic composition may be microbial activity in the headspace after sampling, as the amount of microbicide commonly used with samples from microbially active depths might be insufficient to suppress microbial activity.
In a previous study, gas samples from two boreholes (PB-V01 and SAB-1, both ~500 m deep) in the Horonobe area, Hokkaido, were processed using IsoJar™ containers [14].In that study, cores were stored in IsoJar™ containers with water and a few drops of BKC solution [14] for up to three months before headspace analysis.Because sampling date of cores and analysis date of gases, which are necessary for the calculation of the storage period, have not been presented in Funaki et al. [14], these unpublished information are summarized in Tables S1a and S1b.Concentration of the BKC solution and amounts of cores and water also have not been reported in Funaki et al. [14].In the conventional way of using IsoJar™ headspace gas analysis, it is considered that the concentration of BKC solution is lower than 10%, in which case concentrations of BKC in jars are in order of 0.01%.In the construction of the Horonobe Underground Research Laboratory (URL), including two boreholes (PB-V01 and SAB-1), water was collected from groundwater at ~50 m depth, and this was used by Funaki et al. [14] as filling water for the IsoJar™ containers.Gases dissolved in deep groundwater from the URL were also analyzed, using an evacuated-vial (EV) method [28].Measured δ 13 C values for CH 4 and CO 2 from both sets of analyses are plotted against each other in Figure 1.Large variations in δ 13 C CH4 values from the IsoJar™ measurements (Figure 1) were attributed to methane-oxidizing bacterial activity using sulfate ions in the deep underground environment or to isotopic fractionation during gas migration through fractures [14].However, these possible causes are considered unlikely because (a) geochemical studies in the Horonobe area indicate that reducing conditions are maintained deep underground and sulfate ions are either absent or present at very low concentrations [29][30][31][32]; (b) studies of iodine enrichment [33] indicates that any traces of methane oxidation by sulfate in pore waters of sediments would have been erased during upward fluid flow due to compaction during burial; and (c) there is no evidence in the study area of isotopic fractionation in gases during migration [34,35].The δ 13 C CH4 and δ 13 C CO2 values obtained by the EV method (Figure 1) show little scatter plotting in the carbonate reduction field.
Differences between these data sets could be attributed to factors such as aerobic microbial oxidation of methane in the containers after sampling and/or using groundwater from the different depths of core samples.Possible causes were investigated in the present study to improve the methodology of headspace gas analysis using IsoJar™.Gases from the Wakkanai Formation in the Horonobe area were sampled using the methods of Funaki et al. [14] and Miyakawa et al. [28].The effects of the sampling method (storage period, water type, and additives) on carbon isotopic ratios in CH 4 and CO 2 were investigated, and improvements in headspace gas analysis techniques are suggested.

Geological Setting
The Horonobe area is located in northwestern Hokkaido, in a Neogene-Quaternary sedimentary basin (Figure 2).Since August 2006, the Japan Atomic Energy Agency (JAEA) has been excavating the URL for a research associated with the development of technologies related to the geological disposal of high-level radioactive waste.Geologically, the URL area comprises marine sediments of the Wakkanai Formation (Neogene siliceous mudstone containing opal-CT) and Koetoi Formation (Neogene-Quaternary diatomaceous mudstone containing opal-A).Burial and subsidence of these formations occurred throughout the Neogene and Quaternary, when they underwent early diagenetic thermal alteration at temperatures of <60 °C [36].The URL and surrounding geology are depicted in Figure 3.The highpressure boreholes have steel casings, with valves allowing the sampling of groundwater from multiple depths or zones Figure 1: Plot of δ 13 C CH4 vs. δ 13 C CO2 in coexisting gases, showing fields relating to different gas sources and isotopic shifts resulting from production and oxidation (adapted from [50]).The data are from Funaki et al. [14] and Miyakawa et al. [28].

Sampling and Analytical Methods
3.1.IsoJar™ Samples 3.1.1.Effect of Storage Period.Core samples including in situ pore water (300-400 g), crushed to pieces roughly 30-50 mm in diameter, were placed in IsoJar™ containers with 250-300 g of "filling" water.HgCl 2 -saturated or 10% BKC aqueous solution (10 drops (~0.5 ml) of either) was added to suppress microbial activity, and the jars were sealed with an air headspace.Final concentrations of both microbicides in the jars were about 0.01%-0.02%.The jars were kept in the dark at room temperature for 5-92 d before analysis.Details of each experiment are summarized in Table 1 and Table S2.
The cores were obtained immediately after drilling of borehole 350-Fz-01 from the bottom of the east shaft (Figure 3).Although distilled or tap water is usually used in the IsoJar™ method, groundwater from depths of 53.5-64.5 m and 350 m in borehole 13-350-C01 (drilled in the 350 m gallery; Figure 3) was used to match the 50 m groundwater used by Funaki et al. [14].The priority in this study was to evaluate the effects of sampling method on the carbon isotopic ratios of CH 4 and CO 2 , rather than to obtain accurate in situ values.A large portion of CH 4 dissolved in groundwater around the URL had already escaped due to the pressure decrease associated with excavation, so it was expected that only small amounts of gas would remain in cores.Groundwater, rather than tap water, was used to compensate for this (with water from borehole 13-350-C01 being used because water was not available from borehole 350-Fz-01).Core samples IJ1-IJ25 were from depths of 384-416 m (Table S2).Core samples IJ26-IJ28 were from a depth of 470 m (Table S2) and kept in vacuum storage for one month after drilling.At these depths, the C isotopic ratios for CH 4 and CO 2 had similar values to those from 350 m depth [28].
3.1.2.Effect of Additives.The effective amount of additives was investigated as follows.IsoJar™ samples were prepared as described in Section 3.1.1,with up to 20 ml of BKC and HgCl 2 solutions per jar (Table 2 and Table S3).All core samples were from a depth of 480 m in 350-Fz-01.Fresh cores taken immediately after drilling were not available for analysis, and the samples used in this study had been kept in vacuum storage for about six months.Groundwater from borehole 13-350-C01 was used as filling water.Headspace gas compositions were determined after storage in IsoJar™ for one month.

EV Samples.
The EV sampling procedure involved the preliminary evacuation of septum-topped 50 ml glass vials containing ~1 ml phosphoric acid (85 wt%).Acidification removed any inorganic carbon as CO 2 , with the CO 2 concentration being measured as total inorganic carbon (TIC).Groundwater (15-30 ml, from borehole 09-V250-M02#1 drilled from the 250 m gallery; Figure 3) was introduced by a syringe through a 0.22 μm membrane filter to remove microsized carbonate grains and microbes.Samples were stored in the dark at room temperature for 5-98 d, after which ultrapure He was added by a syringe to equalize headspace gas and atmospheric pressures.The sample was left to stand overnight for gas exchange equilibrium to be established between the headspace and dissolved gases.The composition of the headspace gas was determined by GC, with the concentration of dissolved gas being calculated using Henry's law and the ideal gas equation.and δ 13 C CO2 ) were determined by GC combustion isotoperatio mass spectrometry (GC-C-IRMS), using an IsoPrime GC-MS system (GV Instruments, UK), and are expressed in the usual VPDB δ notation.The lower limit of determination of carbon isotope ratios (δ 13 C CH4 and δ 13 C CO2 ) requires concentrations of 0.01%.Details of GC and GC-C-IRMS procedures can be found in Waseda and Iwano [43].

Results
Headspace concentrations of the gases analyzed and δ 13 C CH4 and δ 13 C CO2 values for the IsoJar™ samples prepared as described in Section 3.1.1are listed in Table 1 and Table S2, and the results for the EV samples are listed in Table 3 and Table S4.Sample contamination by air does not affect the present discussion of isotopic ratios because concentrations of CH 4 and CO 2 in air are much lower than those in the free gas from groundwater (where CH 4 = 74%-100%; CO 2 = 1%-20%; [28,34]).
CO 2 in the free gas undergoes isotopic fractionation during exchange with dissolved inorganic carbon (e.g., CO 2(aq) , HCO The δ 13 C CO2 and δ 13 C CH4 ratios for the EV samples are relatively constant, although δ 13 C CH4 values show a small variation (<4‰) after 98 d (Figure 4(b)), which is consistent with a previous study [28].It is possible that some microbes are not removed by 0.22 μm groundwater filtration (e.g., [45]), and this might have caused the slight variation in δ 13 C CH4 values.Differences between carbon isotopic values  S2).
of CH 4 and CO 2 (δ 13 C CO2 − δ 13 C CH4 = isotopic separation factor; [46]) in the Horonobe area decrease slightly (<1‰) with increasing depth and temperature according to isotopic equilibrium values, with values at depths of 250 m and 350 m being similar to each other within the natural variation of ~2‰ (1σ) [28].Therefore, δ 13 C CO2 and δ 13 C CH4 values of 250 m groundwater obtained by the EV method (Table 3) were used as reference values for the IsoJar™ samples with 350 m groundwater.The δ 13 C CO2 values for IsoJar™ samples show two separate trends for 350 m and 50 m groundwater, with both showing a slight decrease over time, from +15‰ at 5 d to +13‰ at 92 d and from +5‰ at 17 d to −1‰ to +3‰ at 79 d, respectively (Figure 4(a)).The patterns are the same for both BKC and HgCl additives.The two trends suggest that the δ 13 C CO2 ratios represent dissolved gases from different depths.The concentration of CO 2 in the groundwater, as HCO 3

−
, is relatively high even when degassed at atmospheric pressure (30-50 mmol kg −1 ; [47]), compared with that of adsorbed CO 2 , so carbon isotopic values should be largely dependent on dissolved CO 2 .The δ 13 C CO2 values of samples with 50 m groundwater (dashed line in Figure 4(a)) are distinct from those of samples with 350 m groundwater, indicating that the former samples are strongly contaminated.
The δ 13 C CH4 values for IsoJar™ samples (Figure 4(b)) increase markedly with time regardless of additives and fluctuate by more than 30‰ after 80 d; separate trends for different depths are not evident.The concentration of CH 4 remaining in the groundwater at atmospheric pressure after sampling is relatively low (~3 mmol kg −1 ; [47]), and δ 13 C CH4 values for the IsoJar™ samples mainly represent gases adsorbed on cores.Therefore, while the relatively large amounts of dissolved CO 2 reduced the effects of isotopic fractionation on δ 13 C CO2 values, δ 13 C CH4 values were strongly affected.
The results of the effect of additives as described in Section 3.1.2are shown and discussed in Section 5.2.  5 Geofluids with carbon isotopic fractionation, resulting in enrichment of 13 C in unreacted CH 4 and depletion in CO 2 produced.However, there is no clear relationship between headspace gas concentrations and storage period, possibly because of the variability of adsorbed gas levels in natural samples.An apparent carbon isotopic fractionation factor, α, defined as α = δ 13 C CO2 + 1000 / δ 13 C CH4 + 1000 , was calculated using the δ 13 C CO2 and δ 13 C CH4 values in Table 1 (Figure 5).Initial values of α in the Horonobe samples, calculated using the results of the EV method, were around 1.06-1.08, in good agreement with those determined by Miyakawa et al. [28].The values of α for the IsoJar™ samples decreased from ~1.07 to 1.04 with increasing storage time (Figure 5).An earlier study reported a similar trend, where microbial methane oxidation in marine sediments gave an α value of ~1.08 in the methanogenesis zone, decreasing to ~1.02 in the methane oxidation zone [48].

Discussion
A bivariate plot of δ 13 C CH4 vs. δ 13 C CO2 (Figure 6) indicates two trends for the IsoJar™ samples (Figure 6(b)), as in Figure 4(a).Thus, data plotted in Figures 4-6 indicate that δ 13 C CH4 values in samples stored for more than one week were affected by CH 4 oxidation to CO 2 .δ 13 C CH4 and δ 13 C CO2 values reported by Funaki et al. [14] are plotted between the two trends (Figure 6(b)).It seems, therefore, that the variations can be explained by the mixing of the two trends, indicating effects of both methane oxidation and contamination of 50 m groundwater in the IsoJar™ container after sampling.

Effect of Additives.
Significant isotopic fractionation occurred in the IsoJar™ samples, strongly affecting δ 13 C CH4 values despite the addition of BKC or HgCl 2 (Figure 4), suggesting that the amounts of additives used were insufficient to suppress microbial activity.Results of effect of additives as  7.With <10 ml BKC solution or <0.3% BKC concentration, δ 13 C CH4 values fluctuated significantly, and isotopic compositions became lighter.This is opposite to the effect of microbial carbonate reduction and may be due to low CH 4 concentrations (Table 2) resulting from storage of the cores for about six months in a vacuum container (to avoid oxidation by the air and drying), possibly with significant removal of adsorbed gases.An instrumental mass bias of carbon isotope ratio was reported in a very low concentration of hydrocarbons with respect to mass spectrometry [49].In this study, all the data of CH 4 concentrations were above the lower limit of determination of 0.01% (Table 2) indicating that large fluctuations of δ 13 C CH4 values (Figure 7) are not due to instrumental mass bias.The mechanism of any reaction opposing fractionation is not clear.In a low CH 4 concentration, the carbon isotope ratio may be easily disturbed by complex microbial metabolism (e.g., methane oxidation and carbonate reduction).With >10 ml BKC solution or >0.3% BKC concentration, the δ 13 C CH4 values were relatively constant at about −56‰ (Figure 7), which is in good agreement with the values for EV samples.Although the δ 13 C CH4 values for IsoJar™ samples with HgCl 2 solution are relatively constant (Figure 7), they are consistently 5‰-6‰ lower than those with BKC solution.Concentrations of CO 2 in the headspace of samples IJ47 and IJ48, to which 20 ml HgCl 2 solution was added, are considerably higher than those in the other samples (Table 2).A possible cause of the decrease in δ 13 C CH4 values may be isotopic fractionation associated with mercuric reactions with methane, which generate HCl and lead to CO 2 outgassing.Although this mechanism is not clear, considerable care should be required using HgCl 2 as a microbicide with respect to carbon isotope fractionation.

Conclusions
This study investigated the possible causes of carbon isotopic variations (in δ 13 C CH4 and δ 13 C CO2 values) in borehole drillings to 500 m depth, as reported by Funaki et al. [14].The results have led to improvements in the IsoJar™ method for the determination of carbon isotopic ratios in CH 4 and  7 Geofluids CO 2 adsorbed on bore cores.It was found that with air in the IsoJar™ headspace, microbes oxidize CH 4 to CO 2 during storage, accompanied by isotopic fractionation, especially for samples from depths of <1000 m where microbes are more active.Isotopic fractionation resulted in δ 13 C CH4 and δ 13 C CO2 values reaching >30‰ and >2‰ after 80 d storage, respectively, while samples analyzed within a week of sampling showed no such effect.The significant isotopic fractionation in CH 4 was due to its low concentration in the sampling container, while the weaker fractionation in CO 2 was due to its relatively high concentration.The conventional amount of BKC additive (~0.5 ml of 10% solution) was insufficient to suppress microbial activity at least when using in situ groundwater as filling water.The large variations in isotopic compositions reported by Funaki et al. [14] thus appear to have been caused by microbial methane oxidation in the Iso-Jar™ containers after sampling and contamination with groundwater from different depths.Important technique improvements are summarized as follows: (1) if long-term sample storage is necessary, >10 ml of 10% BKC solution   should be used or >0.3% BKC concentration is required; (2) analysis within a week of sampling is strongly recommended; and (3) for CO 2 analysis, groundwater from different depths should not be used.

Figure 2 :
Figure 2: Maps showing the location of the Horonobe URL site and the boreholes: (a) location map and (b) geological map (after [51]).The plate boundaries and the direction of plate movement in (a) are from Wei and Seno [52].

3 − 3 −
being the dominant aqueous species at around neutral pH.To compare δ 13 C CO2 values of the IsoJar™ samples with those of the EV samples, a fractionation correction (intrinsic isotopic fractionation factor) of +7.9‰ between CO 2 gas and HCO 3 − at 25 °C [44] was added to δ 13 C CO2 values measured for the IsoJar™ samples.The range of room temperatures was 20 °C-25 °C, giving an error of up to −0.6‰.Variations in δ 13 C CO2 and δ 13 C CH4 values with storage time are shown in Figures 4(a) and 4(b), respectively.IsoJar™ samples with groundwater (350 m) stored for less than one week give values similar to the EV samples.Storage periods of ≤7 d and ≥20 d were considered, as no data are available for the interval of 7-20 d.

Figure 4 :
Figure 4: Temporal variations in (a) δ 13 C CO2 and (b) δ 13 C CH4 after sampling.Solid arrow indicates the trend for samples with 350 m groundwater; dashed arrow indicates the trend for samples with 50 m groundwater.

Figure 5 :
Figure 5: Temporal variations in the apparent carbon isotopic fractionation factor, α, between CO 2 and CH 4 .

Figure 6 :
Figure 6: Plot of δ 13 C CH4 vs. δ 13 C CO2 in coexisting gases.(a) Grey areas show fields relating to different gas sources and isotopic shifts resulting from production and oxidation (adapted from [50]).The area indicated by the dashed rectangle is enlarged in (b).(b) Solid arrow indicates a methane oxidation trend for samples with 350 m groundwater; dashed arrow indicates the trend for samples with 50 m groundwater.

Figure 7 :
Figure 7: Plot of δ 13 C CH4 values vs. (a) amount of microbial suppressant used and (b) concentration of additives.
3.3.Analytical Procedure.Gases adsorbed on rock fragments in the IsoJar™ containers were desorbed into the headspace by ultrasonic shaking.Concentrations of O 2 , N 2 , CO 2 , CH 4 , C 2 H 6 , and C 3 H 8 in headspace gas were determined by gas chromatography (GC) using a GC7890A Valve System (Agilent Technologies, USA).Carbon isotopic values (δ 13 C CH4

Table 1 :
Information on IsoJar™ samples and analytical results of headspace gases.

Table 2 :
Information on IsoJar™ samples and analytical results of headspace gases for Section 3.1.2.