Influence of headspace flushing on methane production in Biochemical Methane Potential (BMP) tests

Bioresource Technology 186 (2015) 173–178

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Influence of headspace flushing on methane production in Biochemical Methane Potential (BMP) tests Konrad Koch a,⇑, Yadira Bajón Fernández b, Jörg E. Drewes a a b

Chair of Urban Water Systems Engineering, Technische Universität München, Am Coulombwall 8, 85748 Garching, Germany Cranfield Water Science Institute, School of Applied Sciences, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK

h i g h l i g h t s  Influence of headspace flushing on the specific methane production was studied.  Nitrogen gas (N2), a mixture of N2 and CO2 (80/20 v/v) and no flushing was applied.  Results revealed that removing the oxygen is crucial to avoid aerobic respiration.  CO2 in the flush gas increased significantly the methane production by over 20%.  Flushing with gas similar to the expected biogas is suggested.

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Article history: Received 22 February 2015 Received in revised form 13 March 2015 Accepted 14 March 2015 Available online 19 March 2015 Keywords: Headspace flushing BMP test Nitrogen gas N2/CO2 mixture Headspace correction

a b s t r a c t The influence of headspace flushing on the specific methane (CH4) production of blank samples with just inoculum in Biochemical Methane Potential (BMP) tests was studied. The three most common ways were applied: flushing with nitrogen (N2) gas, flushing with a mixture of N2 and CO2 (80/20 v/v), and no flushing. The results revealed that removing the oxygen is crucial to avoid aerobic respiration, which caused both hindered activity of methanogens and loss of methane potential. Furthermore it was demonstrated that 20% of CO2 in the flush gas increased significantly the methane production by over 20% compared to the flushing with pure N2. In order to mimic the same headspace conditions as in full-scale treatment plants, using a flush gas with a similar CO2 concentration as the expected biogas is suggested. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Anaerobic digestion is seen as one of the key technologies in sustainable waste and wastewater management. Furthermore, biogas production from agricultural wastes and energy crops plays an important role in providing renewable energy worldwide (Lebuhn et al., 2014). As Biochemical Methane Potential (BMP) tests are easier to perform than continuous experiments, they are considered state-of-the-art in determining the methane yield of a specific substrate, which is of great relevance for the design and operation of digesters (Koch and Drewes, 2014). Despite the wide use of BMP tests, no commonly accepted experimental procedure yet exists that is based on a standardized protocol for the execution of the test. In 2006, the Association of German Engineers (Verein Deutscher Ingenieure, VDI) published ⇑ Corresponding author. Tel.: +49 89 289 13700; fax: +49 89 289 13718. E-mail address: [email protected] (K. Koch). http://dx.doi.org/10.1016/j.biortech.2015.03.071 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

the technical guideline VDI 4630 ‘‘Fermentation of organic materials. Characterization of the substrate, sampling, collection of material data, fermentation tests’’ (VDI 4630, 2006). In addition to characterization of substrates, sampling and sample preparation, the guideline also provides information regarding the methodology for performing fermentation tests in batch and continuous mode. The guideline is commonly applied by German researchers, although it is of growing interest also for international research groups (cited by 43 peer-reviewed papers according to Web of Knowledge, February 2015). Probably one of the most applied standard protocols for performing BMP tests is the one published by the ‘‘Task Group for the Anaerobic Biodegradation, Activity and Inhibition’’ of the Anaerobic Digestion Specialist Group of the International Water Association (IWA) in 2009 (Angelidaki et al., 2009), which was cited by 231 papers according to Web of Knowledge (February 2015). A number of other protocols are occasionally applied such as the ISO 11734 (ISO 11734, 1995). However, the scope of this protocol is to determine the ultimate biodegradability

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of a single organic compound by applying diluted inoculum to a relatively high substrate concentration. Hence, the protocols of Angelidaki et al. (2009) and VDI 4630 (2006) are more commonly applied. When performing a BMP test, removing the oxygen in the headspace seems to be crucial. Besides the question of whether flushing the headspace is really necessary (Strömberg et al., 2014), mainly two types of flush gas are applied: nitrogen gas (N2) and different mixtures of N2 and carbon dioxide (CO2). The guidelines VDI 4630 and ISO 11734 suggest to either work in a N2 atmosphere (ISO 11734, 1995) or to flush the headspace with N2 prior to incubation (ISO 11734, 1995; VDI 4630, 2006). In contrast, the standard protocol of the IWA suggests to flush continuously with a N2/CO2 mixture of 80% to 20% as volume (Angelidaki et al., 2009). When a N2/CO2 mixture is applied, 80/20 v/v is the dominant ratio reported in the peer-reviewed literature, although some publications also apply a different mixture like 70/30 v/v (Hwu et al., 1997; Tan et al., 1999), very rarely 90/10 v/v (Wagener and Pfennig, 1987), or 75/25 v/v (Boccazzi and Patterson, 2011). When flushing with a portion of CO2 in the flush gas, another phenomenon needs to be considered, which has not been given much interest up to date. Already in 1992, Ochi and Sato (1992) reported an increased methane gas production in anaerobic sewage sludge digesters when maintaining a high CO2 gas concentration in the headspace. At first glance, increasing the methane production by the addition of a byproduct gas (CO2) seems to be conflicting. However, the idea was taken up by several researchers, proving the approach with different substrates and under various conditions (Alimahmoodi and Mulligan, 2008; Bajón Fernández et al., 2014; Salomoni et al., 2011). A compilation of hypotheses on the underlying mechanism can be found for instance in Bajón Fernández (2014). In general, displacement of equilibriums seems to cause an intensified digestion of the sludge by stimulating acetoclastic methanogenesis. While the CO2 was explicitly bubbled in the liquid phase for intensified gas to liquid mass transfer in the mentioned studies, flushing only the headspace of the reactor with a portion of CO2 in the flush gas will likely reduce any potential effect. Finally, a number of very recent studies did not apply any headspace flushing (Mayer et al., 2014; Strömberg et al., 2014). Waiving headspace flushing is mainly justified due to the fact that a headspace correction is needed anyway, as long as the gas composition in the headspace prior and after incubation differs (Strömberg et al., 2014). However, it remains to be demonstrated that the oxygen in the headspace has no negative impact on the anaerobic digestion process; especially for blank samples (samples with just inoculum in order to assess its residual methane potential), where the relation of biogas production to headspace is typically low. In all cases, neither the duration of the gas flushing and flow rates (e.g., 10 L/min for 30 s), nor specifications on the exchange volume are provided (e.g., flush gas flow and duration should guarantee that the total headspace of the system was statistically replaced for at least 10-times). The aim of this study was to compare the effect of different gases applied for headspace flushing (i.e., no gas, N2, N2/CO2) on methane formation in blank samples. Additionally, this contribution should initiate a discussion on how the comparability of BMP tests can be further improved. 2. Methods 2.1. Source and characteristics of inoculum Inoculum for the BMP tests was collected from the wastewater treatment plant (WWTP) Garching (20 km north of Munich, Germany), treating mainly municipal wastewater of approximately

30,000 population equivalents. The digester was fed with raw sludge, which was a mixture of primary and secondary sludge dominated by waste activated sludge. The digester was operated at mesophilic conditions (approximately 40 °C) with a hydraulic retention time of about 25 days.

2.2. BMP tests The BMP tests were conducted with the Automatic Methane Potential Test System II (AMPTS II; Bioprocess Control Sweden AB), which was specially designed for the determination of Biochemical Methane Potential (BMP). The system consists of 15 glass bottles of 650 ml, with a working volume of 400 ml. The units were stirred in cycles of 5 min mixing and 25 min resting. The biogas produced passed through a CO2 capturing unit (consisting of a 3 M sodium hydroxide solution with 0.4% Thymolphthalein as pH indicator) and its volume was automatically converted to standard temperature and pressure (0 °C and 1 bar). A more detailed description of the system can be found in Strömberg et al. (2014). The data were recorded by AMPTS II software and automatically transferred to a MS Excel™ file for subsequent analysis and visualization. All bottles were filled with the same kind of inoculum. No substrate was added in order to mimic blank samples, where any potential effect of headspace flushing will be more intensified due to the low methane production. For each experiment, a sample was taken to analyze the inoculum for the concentration of total solids (TS) and volatile solids (VS) with reference to fresh matter (FM). Their determination followed German Standard Methods for the examination of water, wastewater and sludge (DEV, 2015). On average, the VS concentration of the inoculum was 16 gVS/kgFM. Prior to incubation, the bottles were either not flushed (group Air), flushed with N2 gas at a flow rate of 10 L/min for 30 s (group N2), or flushed with N2/CO2 in a mixture of 80% to 20% as volume at a flow rate of 10 L/min for 30 s (group N2/CO2). With an average headspace volume of 250 ml, the headspace was statically replaced 20 times. All experiments were operated in quintuplicate and have been repeated 4 times.

3. Results and discussion 3.1. Headspace correction Due to the mode of operation of the AMPTS II, the system can only distinguish between CO2, which is captured in the CO2 trap, and ‘‘not CO2’’, which is assumed to be CH4. The system is not capable of differentiating between CH4, N2, O2 or other gases. When the concentration of CO2 in the headspace prior and after incubation differs, a mathematical correction has to be applied in order to avoid an over- or underestimation (Strömberg et al., 2014). An overestimation can occur in case of lower concentration of CO2 prior to incubation compared to the end, e.g. when the headspace is flushed with pure N2 gas. An underestimation can only occur in case of higher concentration of CO2 prior to incubation compared to the end, which is only possible when the flush gas contains a high concentration of CO2. Assuming a typical CH4 concentration in sludge digesters of 65% (Tchobanoglous et al., 2003), the methane yield will only be underestimated, if the flush gas has a CO2 concentration of more than 35%. Such a high CO2 concentration in the flush gas in a BMP test, however, seems not to be applied in any of the studies. Hence, the headspace correction is usually applied to eliminate an overestimation. This correction however, is a strictly mathematical procedure and does not take

K. Koch et al. / Bioresource Technology 186 (2015) 173–178

into account any impact of the flush gas on the digestion process itself. In order to consider the different CO2 concentrations in the flush gases, the following MS Excel™ function has been applied to the raw data of the cumulative methane production to calculate the overestimated volume:

V corr;iþ1 ¼ If ðV corr;i < ðX BG;CO2  X FG;CO2 Þ  V HS ; V i  ðX BG;CO2  X FG;CO2 Þ; V corr;i Þ where Vcorr,i+1 is the overestimated volume at the specific measurement point i + 1 [mlCH4], Vcorr,i is the overestimated volume at point i [mlCH4], XBG,CO2 and XFG,CO2 are the CO2 concentrations in the biogas (BG) and in the flush gas (FG), respectively [%], VHS is the total headspace volume including tubes, etc. [ml], and Vi is the cumulative methane production at point i [mlCH4]. The approach is based on the assumption that headspace behaves like a plug flow reactor, where biogas formed replaces the headspace gas. For instance, if between i and i + 1 10 ml of CH4 (or more correctly ‘‘not CO2’’) are recorded by the system and the CO2 concentration in the flush gas and in the produced biogas are 0% and 35%, respectively, the 10 ml are reduced by 3.5 ml to 6.5 ml of real methane production. Application of the If-function guarantees that the correction stops, when the whole initial headspace volume left the system. As already stated above, the higher the biogas production compared to the headspace volume (or the more often the headspace is replaced by biogas formed), the lower the impact of this headspace problem. Blank samples are specially characterized by a low biogas yield, but play an important role in every experiment in order to calculate the net gas production of the substrate by subtracting the gas production of the inoculum itself. To minimize the effect of methane overestimation during a BMP test when CO2 is

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removed prior to volume measurements, two approaches can be considered. Firstly, headspace is reduced to a minimum. One possible system is the Hohenheim Biogas Yield Test (HBT), where a glass syringe acts as fermenter resulting in no headspace volume (Mittweg et al., 2012). If a certain headspace is not avoidable, use of a flush gas with similar CO2 concentration as the produced biogas, for instance, 35% CO2 and 65% ‘‘not CO2’’ (e.g. N2) for sewage sludge is suggested. However, this headspace correction involves rather simple mathematics and can easily be applied. The following experiments have been carried out to study the impact of different headspace flushing procedures on methane formation. In order to consider the special system configuration of the AMPTS II, the above mentioned headspace correction has been applied. 3.2. Influence of different headspace flushings on methane formation The influence of the different headspace flushings (i.e., Air, N2, N2/CO2) on methane formation of blank samples in BMP tests was studied by repeating the experiment 4 times under identical conditions. Fig. 1 depicts the development of the average specific methane production (SMP) of the three groups Air, N2, and N2/ CO2 for the four experiments without (dashed lines) and with (solid lines) application of the mentioned headspace correction. Apart from some minor differences between experiments, all ‘‘raw’’ SMP (without headspace correction; dashed lines) showed similar performances; however, with a narrow lead of the group N2. By applying the headspace correction (solid lines), the relation of the groups Air and N2 remain the same, because both were flushed with 100% ‘‘not CO2’’ prior to incubation. In general, the bottles flushed with N2 gas performed better than those without

Fig. 1. ‘‘Raw’’ specific methane productions (dashed lines) and headspace corrected specific methane productions (solid lines) for experiments A–D.

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headspace flushing in all the experiments. However, the difference is moderate and is likely caused by the remaining oxygen in the headspace resulting in a loss of methane potential due to aerobic metabolism. For a better visualization, the absolute differences between the two specific methane productions (SMP) of the groups Air and N2 (SMPAir–SMPN2) are plotted for each experiment in Fig. 2. Although scaling of the ordinate differs, all four figures have a common shape characterized by a negative trend, especially in the first 50 h. After a more or less pronounced plateau phase, another downwards trend is noted. Only in Experiment B, a slight recovery phase was observed after about 100 h. The described shape indicates that the inhibition of methanogenesis due to the presence of remaining oxygen took place throughout the experimental trial, rather than only in the initial phase. It is likely that the oxygen also accounted for the degradation of degradable material, which was lost from the system for additional methane production. In general, this finding put the approach of waiving the headspace flushing of Mayer et al. (2014) and Strömberg et al. (2014) into question and is an argument to continue the commonly applied N2 gas headspace flushing as for instance also specified by the technical guideline VDI 4630 (2006). As already noted in Fig. 1, the corrected SMPs of the groups N2 and N2/CO2 (solid lines) revealed a significant difference. From a mathematical point of view this is not surprising, because both groups exhibited a similar performance in the ‘‘raw’’ SMP and the correction attributed to the different content of CO2 in the headspace was higher for N2 than for N2/CO2. From a scientific point of view, the over 20% higher specific methane yield is a really remarkable difference. This also provides another piece of evidence for the phenomena of increased methane productivity at higher CO2 partial pressure as described by Ochi and Sato (1992). Furthermore, the results indicate that an alternation of headspace’s

Fig. 3. Quotient of the specific methane productions (SMP) of the groups N2/CO2 and N2 (SMPN2/CO2/SMPN2) for experiments A–D.

concentration leads to a modified methane production, with an extensive injection of the CO2 in the liquid phase (e.g., by bubbling) not being necessary for obtaining an effect. This observation might be of great interest for exploiting the effect of CO2 enrichment on the methane production in full-scale due to omission of gas compression. In addition, these findings suggest that CO2 in the mixture for headspace flushing has an influence on the digestion process. For a better visualization, the quotient of SMP for the groups N2/CO2 and N2 (SMPN2/CO2/SMPN2) is plotted for each experiment in Fig. 3. Although experiments A and D differ slightly from B and C, all graphs are characterized by a similar shape. Only during the first five (experiment D) to 24 h (experiment A), the quotient is

Fig. 2. Absolute differences between the specific methane productions (SMP) of the groups Air and N2 (SMPAir–SMPN2) for experiments A–D.

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K. Koch et al. / Bioresource Technology 186 (2015) 173–178 Table 1 Specific methane yields of the quintuplicates (with headspace correction applied) in each of the four experiments as base for significance test (see Table 2). Test

A B C D

Air

N2

N2/CO2

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5

31.25 36.45 29.54 40.99

32.64 36.43 34.11 41.08

35.29 36.42 34.10 38.80

33.74 36.43 34.07 36.49

28.56 28.43 34.06 35.32

38.04 40.49 43.20 43.29

41.30 43.10 35.66 44.41

42.12 40.44 35.62 42.22

37.95 36.42 34.06 36.44

31.22 35.10 35.65 39.80

40.25 49.13 42.41 47.99

45.42 49.26 47.16 52.67

40.34 46.66 41.11 50.38

37.42 46.62 40.96 46.90

34.87 42.46 41.01 46.76

Table 2 Likelihood of significant difference between the specific methane yields of each group and in each of the four experiments (see Table 1 for dataset). Test

N2/CO2 vs. N2 (%)

N2/CO2 vs. Air (%)

N2 vs. Air (%)

A B C D

86.7617 99.9945 99.9521 99.9954

99.9903 99.9997 99.9994 99.9997

99.9462 99.6777 99.5613 98.1753

below one, meaning that group N2 performed better than group N2/ CO2. Afterwards, an increased methane production is observed for the units flushed with 20% of CO2, which reached a surplus of 20% (except for experiment A, where a lower benefit was achieved). This enhanced methane production might be related to a stimulation of the methanogenic activity due to the presence of CO2 in the flush gas at the beginning of the trials. However, after about 100 h, the quotient is constant meaning that both groups performed in the same way. This was also the time period until the entire headspace volume was exchanged (not shown) and hence, when the headspace in all bottles is dominated by produced biogas instead of the headspace. Nevertheless, any recovery of the group N2 did not take place indicating that the digestion process was not accelerated by the presence of CO2 in the headspace, but rather initiated digestion of additional substances, which were not degraded at all in the N2 atmosphere. Table 1 contains the specific methane yields of the quintuplicates (with headspace correction applied) in each of the four experiments. For each experiment, the dataset was successfully tested for normal distribution with a Kolmogorov–Smirnov test and significance level a of 0.01. Based on the dataset presented in Table 1, Table 2 provides the likelihood of significant difference between the specific methane yields of each group and in each of the four experiments. Except for the comparison of group N2/CO2 and group N2 in experiment A, all differences are highly significant (p 6 0.005). Especially the comparison of group N2/CO2 and Air shows a significant difference, although a headspace correction has been applied. 3.3. Concluding remarks The experiments presented revealed a moderate, but reproducible and significant difference between the specific methane production of blank samples (inoculum) without headspace flushing and the commonly applied headspace flushing with N2 gas. It is therefore recommended to apply the headspace flushing as suggested, for instance, by the technical guideline VDI 4630 (2006). However, more information on the flushing procedure should be reported in specific studies (e.g. flow rate of flush gas and duration) in order to ease comparability. In contrast to the discussion on the necessity of a headspace flushing, an ultimate answer is harder to find to the question of whether CO2 should be present in the flush gas or not. One reason against CO2 in the headspace is the observation that CO2 has a significant effect on methane formation. This seems not to be the case when pure N2 gas is applied. However, one reason for flushing with

CO2 is definitely the fact that it is part of the normal anaerobic atmosphere. When flushing with a gas that mimics the typical anaerobic atmosphere at steady state (e.g., 35% CO2 and 65% ‘‘not CO2’’, i.e. N2 or CH4 for sewage sludge), the composition of the headspace is not subjected to a change anymore. This would also supersede the headspace correction, because it is only necessary when the initial headspace composition differs from the one at the end. However, this approach requires both knowledge of the expected biogas composition and more effort on providing the flush gas. But if a mixture with CO2 is applied anyway, a share of CO2 similar to the expected biogas composition (e.g., 30–40% for sewage sludge) should be chosen. Hence, flushing with gas with a CO2 concentration below the expected concentration of the biogas, might underestimate the methane potential of a continuously operating plant, although a headspace correction has been applied. Further systematic studies on the effect of CO2 in the flushing gas on methane formation in BMP tests should follow. 4. Conclusions The results suggest that headspace flushing of a BMP test does have an influence on the digestion process. Removing the oxygen initially in the headspace is recommended to avoid aerobic respiration. CO2 in the flush gas can significantly stimulate methane production compared to pure N2 gas. In order to mimic real headspace conditions in the bottles, the flush gas should have a similar CO2 concentration as the expected biogas. Hence, flushing with N2 gas might underestimate the methane potential in a continuously operated plant. Acknowledgements Stefan Wagner is kindly acknowledged for his excellent technical assistance. The authors would furthermore thank Nils Horstmeyer for statistical analysis of the data. References Alimahmoodi, M., Mulligan, C.N., 2008. Anaerobic bioconversion of carbon dioxide to biogas in an upflow anaerobic sludge blanket reactor. J. Air Waste Manag. Assoc. 58, 95–103. http://dx.doi.org/10.3155/1047-3289.58.1.95. Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J., Kalyuzhnyi, S.V., Jenicek, P., Van Lier, J.B., 2009. Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci. Technol. 59, 927–934. http://dx.doi.org/10.2166/ wst.2009.040. Bajón Fernández, Y., 2014. Carbon Dioxide Utilisation in Anaerobic Digesters as an On-site Carbon Revalorisation Strategy (Ph.D. thesis). Cranfield Water Science Institute, School of Applied Sciences, Cranfield University, Cranfield, UK. Bajón Fernández, Y., Soares, A., Villa, R., Vale, P., Cartmell, E., 2014. Carbon capture and biogas enhancement by carbon dioxide enrichment of anaerobic digesters treating sewage sludge or food waste. Bioresour. Technol. 159, 1–7. http:// dx.doi.org/10.1016/j.biortech.2014.02.010. Boccazzi, P., Patterson, J.A., 2011. Using hydrogen-limited anaerobic continuous culture to isolate low hydrogen threshold ruminal acetogenic bacteria. Agric. Food Anal. Bacteriol. 1, 33–44. DEV, 2015. Deutsche Einheitsverfahren zu Wasser-, Abwasser- und Schlammuntersuchung (German standard Methods for Examination of Water, Wastewater and Sludge). Wiley-VCH, Weinheim. Hwu, C.-S., van Beek, B., van Lier, J.B., Lettinga, G., 1997. Thermophilic high-rate anaerobic treatment of wastewater containing long-chain fatty acids: effect of

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