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Influence of methane enrichment by aeration of recirculated supernatant on microbial activities during anaerobic digestion
Bioresource Technology 71 (2000) 217±224
In¯uence of methane enrichment by aeration of recirculated supernatant on microbial activities during anaerobic digestion D.M. O'Keefea, R.L. Brigmonb, D.P. Chynowethc,* a Full Circle Solutions Inc., Gainesville FL 32641, USA Savannah River Technology Center, Aiken SC 29808-0001, USA Agricultural and Biological Engineering Department, University of Florida, Gainesville FL 32611-0151, USA b
c
Received 13 August 1998; received in revised form 12 May 1999; accepted 17 May 1999
Abstract A methane enrichment process (MEP) was evaluated that involved air purging of recycled digester contents to strip CO2 and increase biogas methane content. The objective of this work was to determine if the aeration resulted in oxygen inhibition of microbial activities involved in anaerobic digestion of municipal solid waste. To assess the degree of biological perturbation associated with the MEP, the reactor euent was sampled twice while the MEP was operating and twice while it was not operating. Analyses were run on composite samples (representing several dierent sample ports of the non-mixed reactor) and samples of digester euent entering and leaving the MEP process. The analyses included volatile organic acids (VOA), euent solids content, dehydrogenase activity, speci®c methanogenic activities (SMA), and enzyme-linked immunosorbent assay (ELISA) for methanogenic, sulfate-reducing, and cellulolytic bacterial species. Methane enrichment in these experiments occurred with the methane content of the biogas exceeding 90%. There were no eects of the MEP on euent VOA concentration. The MEP had no eect on volatile solids (VS) levels of composite samples representing the non-mixed digester contents, however, VS was reduced in euent passing through the MEP. Although MEP had an inhibitory eect on anaerobic populations leaving the MEP process, no inhibitory eects were observed in measurements of microbial activities in digester samples, including speci®c methane production rate, dehydrogenase, and numbers of speci®c organisms estimated using ELISA techniques. Ó 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Anaerobic digestion; Methane enrichment; ELISA; Dehydrogenase; Methane production; Microbial inhibition
1. Introduction One of the current goals of biogasi®cation research is to increase the methane content of biogas. The normal methane concentration in biogas is 50±60%, which is too low for pipeline transport and use as vehicular fuel. Methods for removing the major component (CO2 ) from the biogas are therefore required for these end uses. To this end a CO2 -removal methane enrichment process (MEP) was designed and evaluated with benchscale reactors (Hayes et al., 1990). The concept was then tested in the present work using a pilot-scale digester fed municipal solid waste (MSW). This reactor has been described in detail (Chen et al., 1990). The basic concept of the MEP design was the circulation of euent through an aerated, baed column (Fig. 1). In brief, the methane enrichment process as employed here involved *
Corresponding author.
recycle of digester contents from the lower depth where solution of carbon dioxide is encouraged because of higher pressure. Euent containing the dissolved carbon dioxide was removed from the digester and subjected to air stripping to remove carbon dioxide after which it was recycled back to the digester to absorb more carbon dioxide. Continuous operation of this recycle loop removed most of the carbon dioxide resulting in the desired methane enrichment. Inherent in the design of the MEP was the exposure of to oxygen of populations of anaerobic bacteria suspended in the supernatant. We anticipated that this could have deleterious eects on these bacteria and reactor performance. The following research was therefore performed in order to evaluate the impact of operating the MEP on the microbial ecology of the reactor contents and the relative utility of several methods of assessing these biological perturbations. Numerous approaches have been proposed and practised for the monitoring of microbial populations
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D.M. O'Keefe et al. / Bioresource Technology 71 (2000) 217±224
and their activities in anaerobic digesters (Chynoweth and Pullammanappallil, 1996; Chung and Neethling, 1989; Fannin, 1987; Peck and Chynoweth, 1990; Zinder, 1993). We selected methanogenesis and dehydrogenase as measurements of microbial activities and used immunological probes developed for digester isolates to measure population dynamics. 2. Materials and methods 2.1. Reactor design The reactor was a cylindrical, pilot-scale (4500 l working volume) solids-concentrating digester described elsewhere in detail (Chen et al., 1990). Fig. 1 is a schematic of the reactor and MEP device. The MEP was done in a cylindrical tank 4 m tall and 0.3 m diameter with a series of internal baes to increase gas exchange between the euent and the air being blown through the MEP. A solids reduction tank (570 l) was installed in line, preceding the MEP to keep solids from fouling the MEP. A holding tank (570 l) was used to allow facultative bacteria to remove O2 from euent leaving the MEP before returning the euent to the digester. The feedstock was a mixture of the organic fraction of municipal solid waste and primary sewage sludge at a ratio of 15:1 on a volatile solids basis. The feed concentration was 8% total solids and the digester organic loading rate was 3.2 kg VS mÿ3 dÿ1 . The digester was operated at 35°C. During MEP operation, reactor euent was circulated through the MEP at a rate of 3.5 reactor vol-
umes per day. The aeration rate was 46 l air per l of recycled digester euent. 2.2. Reactor sampling Evaluation of the microbial impact of operating the MEP was done by sampling the reactor euent twice while the MEP was operating (12 and 22 February 1990) and twice while it was not operating (19 and 28 February 1990). Composite samples consisting of 750 ml sub-samples from six depths (0.9, 1.2, 1.5, 1.8, 2.3, and 2.9 m from the tank bottom, i.e. Sample Ports 2, 3, 4, 5, 6, and 8, respectively) were taken on the above sampling dates. While the MEP was operating, additional samples were taken at the send (to the MEP ± see Fig. 1) and return (to the reactor) ports. Samples were put into 5 l containers, sealed and transported to the laboratory for immediate analysis. The methods used to evaluate the biological impact of the MEP, and the rationale for using each assay, are summarized in Table 1. The following analyses were done on the euent: volatile organic acids (VOA), total and volatile solids content, dehydrogenase activity, speci®c methanogenic activity (SMA), and enzyme-linked, immunosorbent assay (ELISA) for methanogenic, sulfate reducing, and cellulolytic bacteria. 2.3. Volatile organic acids analysis The VOA concentration of the euent was determined by acidifying samples with the addition of 20% (v/v) H3 PO4 to a ®nal concentration of 2% (v/v). Sam-
Fig. 1. Schematic diagram of the reactor and methane enrichment process.
D.M. O'Keefe et al. / Bioresource Technology 71 (2000) 217±224
219
Table 1 Summary of methods used to detect biological eects of operating the methane enrichment process with an anaerobic digester fed the organic fraction of municipal solid waste Method
Time required
Target organisms
Comments
pH Volatile organic acids Methane content Biogas production
Minutes Minutes Minutes Minutes
Entire, balanced, methanogenic consortium Entire, balanced, methanogenic consortium Entire, balanced, methanogenic consortium Overall microbial population
1 Day 1 Day
Simple, fast,cause may be non-biological Simple, fast Simple, fast, cause may be non-biological Simple, fast, in conjunction with methane content may be mechanistic Mechanistic Mechanistic
1
SMA 1 acetate Dehydrogenase assay
1 Day Hours
Hydrogen and formate-utilizing methanogens Cellulolytic microorganisms, acid-utilizing methanogens Acetate-utilizing methanogens Overall microbial population
Antibody probes
Days
Species speci®c (see Table 2)
SMA SMA
1
1
formate cellulose
Mechanistic Does not discriminate between aerobic and anaerobic metabolism Mechanistic, does not discriminate between dead, live, active, and inactive bacteria
Speci®c methanogenic activity.
ples were then centrifuged and 1 ll of the supernatant analyzed by gas chromatography (Shimadzu GC Model 9AM, Kyoto, Japan) using a 1.8 m ´ 2 mm glass column of 10% SP-1000 on 100/120 Chromosorb W/AW (Supelco, Bellefonte, PA). The column was maintained at 140°C with nitrogen as the carrier gas at 40 cm3 minÿ1 . The injector was set at 160°C and the ¯ame ionization detector at 200°C. The total VOA concentration reported here represents the sum of acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids. 2.4. Solids Total and volatile solids content of euent samples was analyzed according to Standard Methods (Clesceri et al., 1989). 2.5. Speci®c methanogenic activity A procedure modi®ed from Owen et al. (1979) was used to assess the ability of the reactor microbial populations to produce methane. Unamended euent and euent amended with 0.2 g 100 mlÿ1 acetate, formate, or cellulose (Avicel microcrystalline) were incubated at 35°C in 125 ml-serum bottles ®tted with rubber septums. Gas volume and composition were assessed at 4, 12, 24, and 48 h. Gas volume was measured using a gas-tight syringe. Gas composition was determined using a gas chromatograph (Fisher Gas Partitioner, Mode1200, Pittsburgh, PA) equipped with two matched pairs of thermal conductivity detectors and dual stainless steel columns operated at 55°C. The ®rst column was 2 m ´ 3.2 mm packed with 80±100 mesh Porapak Q (Supelco, Bellefonte, PA), and the second column was 3.35 m ´ 4.8 mm packed with 60±80 mesh molecular sieve 13X (Supelco, Bellefonte, PA). Helium was the carrier gas at a ¯ow of 30 cm3 minÿ1 .
2.6. Dehydrogenase assay The dehydrogenase activity of the euent was assayed according to the method described by Lopez et al. (1986) which uses the redox dye 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyl tetrazolium chloride (INT). Fiveml samples of digester euent were incubated for two hours at 35°C with 0.5 ml of the INT reagent. After incubation the reaction was stopped by adding 1.0 ml of a 37% formaldehyde solution. The formazan was extracted in methanol according to the method of Lee et al. (1988). Formazan concentrations of the extracts were determined by optical density at 480 nm. 2.7. Enzyme-linked immunosorbent assay probes Speci®c polyclonal antibody probes were selected (Table 2) that had been developed for six strains of anaerobic bacteria using methods of Archer (1984). An ELISA was utilized as previously described to test the samples (Brigmon et al., 1992). In brief, fresh samples of euent were centrifuged at 20 000 RPM for 30 min. The clear supernatant was discarded. The pellets were resuspended to original volume in carbonate±bicarbonate buer and the pH adjusted to 9.8 with 4 M NaOH. Duplicate, 200 ll samples were added to Immulon-2, 96well immunoassay plates (Dynatech, Chantilly, VA). Plates were incubated 16 h at 4°C. Fifty ll of PBS containing 0.5% glutaraldehyde was subsequently added to all wells for 5 min. The plates were washed 6´ with a PBS solution containing 0.1% Tween 20 (Sigma, MO) (PBST) after which they were incubated for 1 h with 1% Bovine Serum Albumin (Sigma, MO) (BSA) in PBS (PBSA) at room temperature. Plates were again washed 6´ with PBST. Duplicate samples of primary antibody, mouse or rabbit speci®c anti-bacterial polyclonal antibodies (PAB), diluted 1:1000 in PBSA (100 ll) were
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Table 2 Bacteria used to prepare antibodies for immunological probing of reactor populations Figure I.D.
Bacteria
Source
Biochemical Role
Habitat
A B C D E F G H
Methanothrix concilii strain GP6 Methanobacterium thermoautrophicum Methanobacterium thermoautrophicum Methanosarcina barkerii strain Mb Methanogenic bacterium strain T3 Clostridium acetobutylicum Desulfovibrio desulfuricans Clostridium aldrichii P1
G. Patel, NRC Ontario, Canada Y. Yang, University of Florida Y. Yang, University of Florida Y. Yang, University of Florida Y. Yang, University of Florida ATCC #824 ATCC #29577 Y. Yang, University of Florida
Methanogen Methanogen Methanogen Methanogen Methanogen Acid former, cellulolytic Sulfate reducer, acid former Acid former, cellulolytic
± Sewage-sludge digester Sewage-sludge digester Sewage-sludge digester Termite gut Soil Soil Anaerobic, wood digester
tested. Negative controls employed in the ELISA were PBSA, plasma from normal mice or normal rabbits diluted 1:1000 in PBSA and (PBST). ELISA plates were then incubated for 1 h. Samples were removed and the wells washed 6´ with PBST. 100 ll of secondary antibody, anity-puri®ed alkaline phosphatase goat antimouse or anti-rabbit immunoglobulins (Organon Teknika, Malvern, PA) 1:1000 dilution in PBSA were added to each well. Plates were incubated for 1 h with secondary antibody and then washed. 200 ll of alkaline phosphatase substrate (1 mg mlÿ1 p-nitrophenyl phosphate, in diethanolamine buer (Sigma, MO) prepared just before use) was added to each well. The plates were protected from light and read on a Titertek Multiskan (Flow Laboratories, McLean, VA) at 405 nm after 30 min. Sensitivity assays were done by serially diluting bacteria from 2 ´ 108 to 2 ´ 102 cells mlÿ1 in carbonate± bicarbonate buer and performing the ELISA. Absorbencies from reactor samples were compared to standard curves to estimate bacterial concentrations. Fig. 2. Sampling and MEP operation dates and methane content of biogas during MEP trials.
3. Results 3.1. Methane enrichment Operation of the MEP resulted in methane contents of 90% (Fig. 2) in each of two runs. Similar results have been obtained with glucose (Hayes et al., 1990) and particulate biomass feedstocks (Jewell et al., 1993). Air purging of digester supernatant appears to be an eective technique for incorporation of methane enrichment into the anaerobic digestion process. With the solids concentrating reactor employed for these studies, methane enrichment was not sustainable (for more than a few days) because of solids carryover and clogging of the methane enrichment process. 3.2. Solids and volatile organic acids Volatile solids and VOA concentrations of the reactor and MEP euent are summarized in Table 3. There were no signi®cant (P 6 0.05) eects of the MEP on ef-
Table 3 Means and standard deviations (SD) for volatile organic acid (VOA) concentrations and volatile solids (VS) of euent samples by mode (MEP on or o) and port (composite, MEP-send, and MEP-return). Composite samples consisted of 750-ml samples from 6 depths. Volatile organic acid values are adjusted for solids. All values are means of six replicates VS (mg mlÿ1 )
VOA (mg (gVS)ÿ1 )
Mean
SD
Mean
Mode On O
8.6 6.9
6.0 2.9
8.5 8.1
6.8 6.9
Port Composite Send Return
8.6 2.3 1.1
6.0 1.3 0.2
8.5 17.0 13.7
6.8 10.5 7.8
SD
¯uent VOA concentration. Operation of the MEP had no eect on VS levels of composite samples taken from the reactor. However, there was a reduction in volatile
D.M. O'Keefe et al. / Bioresource Technology 71 (2000) 217±224
solids in euent passing through the MEP which may be attributed to oxidative metabolism, ®ltering, and/or settling of solids. 3.3. Speci®c methanogenic activity Operation of the MEP increased the methane potential of composite samples of the reactor euent for the unamended and three amended treatments (Fig. 3).
Fig. 3. Cumulative methane yields of composite euent samples taken while the MEP was on or o and incubated in sealed jars for 48 h. Euent was left unamended or amended with formate, cellulose, or acetate. Points represent means of three replicates and error bars are standard deviations of the mean.
221
However, comparison of the SMA of MEP in¯uent and euent revealed that the MEP had a localized inhibitory eect on euent methanogen populations as they passed through the device (Fig. 4). This was true for unamended and amended (with acetate formate or cellulose) MEP measurements.
Fig. 4. The eect of the MEP on methanogen populations passing through the device. Methane yields of euent samples taken while the MEP was operating. A composite sample and samples entering (MEP send) and returning (to the reactor) were incubated in sealed jars for 48 h. Euent was left unamended or amended with formate, cellulose, or acetate. Points represent means of three replicates and error bars are standard deviations of the mean.
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3.4. Dehydrogenase activity Operation of the MEP had no eect (P 6 0.01) on dehydrogenase activity of composite digester samples while euent exiting the MEP had increased activity compared to composite digester samples and MEP in¯uent (Fig. 5). This result was expected as dehydrogenase activity is a measure of anaerobic and aerobic microbial activity and aeration of the euent should stimulate over-all microbial activity. As with the SMA assay, however, the eect was localized and the overall reactor population was not similarly aected. 3.5. ELISA probes The ELISA analysis of the reactor revealed no signi®cant (P 6 0.01) eects of the MEP on the reactor microbial populations (Fig. 6). The reason that the antibody analysis did not reveal treatment eects may be that antibodies bind to live and active, live and inactive, and dead cells. The results of this measurement demonstrated that, for short-term experiments, antibody probe analysis may not be appropriate for detecting microbial changes. This would be particularly true if the changes were shifts in metabolic activity more than
Fig. 5. The eect of the MEP on dehydrogenase activity of microbial populations passing through the device. Bars represent means of 10 replicates and error bars are standard deviations of the mean.
shifts in population composition. It is noteworthy that every antibody used in this probe gave a positive result. This demonstrates the rich microbial diversity of the methanogenic consortium in MSW-fed anaerobic digesters. 4. Discussion We had expected the MEP to inhibit anaerobic microorganisms since 3.5 volumes of reactor euent were being vigorously aerated each day. However, this treatment had no apparent negative eect on overall anaerobic microbial populations. Microbial populations in the euent leaving the MEP, on the other hand, were in¯uenced by aeration in ways anticipated. Solids content of the reactor was four times higher than in the euent stream being circulated through the MEP. This suggests that, for the most part, microorganisms were not being circulated through the MEP but were retained in the reactor, probably sorbed to solids. Any dissolved oxygen in euent returning to the MEP was probably consumed in the O2 -removing tank before reaching the reactor. Therefore microbial populations within the reactor were not exposed to oxygen introduced during the MEP operation. The choice of amendments used in this assay allowed us to draw some conclusions as to the preferential inhibition of the basic biochemical components (acetogens and methanogens) of the methanogenic consortium. Had the methanogens been inhibited in the absence of inhibition of the acetogens, the amended treatments would have shown an initial ¯ush of biogas followed by a drop in production rate from VOA inhibition and little or no methane production. As this was not the case, we concluded that oxygenation of the euent inhibited all components of the methanogenic consortium. We do not know why operation of the MEP stimulated the methanogenic activity of the composite samples. Solids and VOA concentration of the euent do not explain this dierence as they were not in¯uenced by the MEP operation. In addition, dissolved methane in the euent due to increased methane content of the reactor headspace during the MEP operation was not sucient to account for the increase (data not presented). Evidence of inhibition of anaerobic microbial populations was only found in euent exiting the MEP system. On a practical level, therefore, monitoring of reactor performance alone would have been sucient to test the biological impact of this device. A summary of the relative utility of the methods used in this experiment is contained in Table 1. The localized inhibition allowed us to make several generalizations concerning the relative utility of some of the methods used. First, antibody probes are of little use in detecting short-term microbial perturbations. This is due to the inability of antibody
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Fig. 6. The eect of the MEP on antibody probing of overall reactor microbial populations (a) and populations in the euent stream passing through the MEP (b). Bars represent means of duplicate samples. Letters refer to isolates listed in Table 2.
probes to discriminate between live, dead, active, and inactive microbes. For this technique to be useful, suf®cient time is required for degeneration of the antigenic, cell wall constituents. The primary drawback to the dehydrogenase assay in this application was its inability to discriminate between aerobic and anaerobic metabolic activity. All the SMA tests clearly showed that oxygenation of the euent stream compromised acetogens and methanogens in that stream. We conclude that the SMA assay was the most instructive method employed in this study.
Acknowledgements This work was supported by US Department of Energy Contract DE-ACO2-83CH10093, Biological Assessment of Anaerobic Digestion of Solid Waste, and the University of Florida Institute of Agricultural and Food Science.
References Archer, D.B., 1984. Detection and quanti®cation of methanogens by enzyme-linked immunosorbent assay. Appl. Environ. Microbiol. 48, 797±801.
Brigmon, R.L., Zam, S.G., Farrah, S., Bitton, G., 1992. Detection of Salmonella enteritidis in environmental samples by monoclonal antibody-based ELISA. J. Immunol. Methods 152, 135±147. Chen, T., Chynoweth, D.P., Biljetina, R., 1990. Anaerobic digestion of municipal solid waste in a nonmixed solids concentrating digester. Appl. Biochem. Biotech. 24/25, 533±544. Chung, Y.C., Neethling, J.B., 1989. Microbial activity measurements for anaerobic sludge digestion. J. W. P. C. F. 16, 343±349. Chynoweth, D.P., Pullammanappallil, P., 1996. Anaerobic digestion of solid wastes. In: Palmisano, A.C., Barlaz, M.A. (Eds.), Microbiology of Solid Waste. CRC Press, Boca Raton, pp. 71± 113. Clesceri, L.S., Greenberg, A.E., Trussell, R.R., 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, Washington, DC. Fannin, K.F., 1987. Start-up, operation, stability, and control. In: Chynoweth, D.P., Isaacson, H.R. (Eds.), Anaerobic Digestion of Biomass. Elsevier, New York, pp. 171±196. Hayes, T.D., Isaacson, H.R., Pfeer, J.T., Liu, Y.M., 1990. In-situ methane enrichment in anaerobic digestion. Biotech. Bioeng. 35 (1), 73±86. Jewell, W.J., Cummings, R.J., Richards, B.K., 1993. Methane fermentation of energy crops: maximum conversion kinetics and in situ biogas puri®cation. Biomass Bioenergy 5 (3/4), 261±278. Lee, C.W., Koopman, B., Bitton, G., 1988. Evaluation of the formazan extraction step of INT-dehydrogenase assay. Toxicity Assessment: An International Journal 3, 41±54. Lopez, J.M., Koopman, B., Bitton, G., 1986. INT-dehydrogenase test for activated sludge process control. Biotech. Bioeng. 28, 1080± 1085. Peck, M.W., Chynoweth, D.P., 1990. On-line monitoring of the methanogenic fermentation by measurement of culture ¯uorescence. Biotech. Lett. 12, 17±22.
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Owen, W.F., Stuckey, D.C., Healy, Jr. JB., Young, L.Y., McCarty, P.L., 1979. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 13, 485±492.
Zinder, S.H., 1993. Physiological ecology of methanogens. In: Ferry, J.G. (Ed.), Methanogensesis: Ecology, Physiology, Biochemistry, and Genetics. Chapman and Hall, New York, pp. 128±206.
In¯uence of methane enrichment by aeration of recirculated supernatant on microbial activities during anaerobic digestion D.M. O'Keefea, R.L. Brigmonb, D.P. Chynowethc,* a Full Circle Solutions Inc., Gainesville FL 32641, USA Savannah River Technology Center, Aiken SC 29808-0001, USA Agricultural and Biological Engineering Department, University of Florida, Gainesville FL 32611-0151, USA b
c
Received 13 August 1998; received in revised form 12 May 1999; accepted 17 May 1999
Abstract A methane enrichment process (MEP) was evaluated that involved air purging of recycled digester contents to strip CO2 and increase biogas methane content. The objective of this work was to determine if the aeration resulted in oxygen inhibition of microbial activities involved in anaerobic digestion of municipal solid waste. To assess the degree of biological perturbation associated with the MEP, the reactor euent was sampled twice while the MEP was operating and twice while it was not operating. Analyses were run on composite samples (representing several dierent sample ports of the non-mixed reactor) and samples of digester euent entering and leaving the MEP process. The analyses included volatile organic acids (VOA), euent solids content, dehydrogenase activity, speci®c methanogenic activities (SMA), and enzyme-linked immunosorbent assay (ELISA) for methanogenic, sulfate-reducing, and cellulolytic bacterial species. Methane enrichment in these experiments occurred with the methane content of the biogas exceeding 90%. There were no eects of the MEP on euent VOA concentration. The MEP had no eect on volatile solids (VS) levels of composite samples representing the non-mixed digester contents, however, VS was reduced in euent passing through the MEP. Although MEP had an inhibitory eect on anaerobic populations leaving the MEP process, no inhibitory eects were observed in measurements of microbial activities in digester samples, including speci®c methane production rate, dehydrogenase, and numbers of speci®c organisms estimated using ELISA techniques. Ó 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Anaerobic digestion; Methane enrichment; ELISA; Dehydrogenase; Methane production; Microbial inhibition
1. Introduction One of the current goals of biogasi®cation research is to increase the methane content of biogas. The normal methane concentration in biogas is 50±60%, which is too low for pipeline transport and use as vehicular fuel. Methods for removing the major component (CO2 ) from the biogas are therefore required for these end uses. To this end a CO2 -removal methane enrichment process (MEP) was designed and evaluated with benchscale reactors (Hayes et al., 1990). The concept was then tested in the present work using a pilot-scale digester fed municipal solid waste (MSW). This reactor has been described in detail (Chen et al., 1990). The basic concept of the MEP design was the circulation of euent through an aerated, baed column (Fig. 1). In brief, the methane enrichment process as employed here involved *
Corresponding author.
recycle of digester contents from the lower depth where solution of carbon dioxide is encouraged because of higher pressure. Euent containing the dissolved carbon dioxide was removed from the digester and subjected to air stripping to remove carbon dioxide after which it was recycled back to the digester to absorb more carbon dioxide. Continuous operation of this recycle loop removed most of the carbon dioxide resulting in the desired methane enrichment. Inherent in the design of the MEP was the exposure of to oxygen of populations of anaerobic bacteria suspended in the supernatant. We anticipated that this could have deleterious eects on these bacteria and reactor performance. The following research was therefore performed in order to evaluate the impact of operating the MEP on the microbial ecology of the reactor contents and the relative utility of several methods of assessing these biological perturbations. Numerous approaches have been proposed and practised for the monitoring of microbial populations
0960-8524/00/$ ± see front matter Ó 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 7 3 - 5
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D.M. O'Keefe et al. / Bioresource Technology 71 (2000) 217±224
and their activities in anaerobic digesters (Chynoweth and Pullammanappallil, 1996; Chung and Neethling, 1989; Fannin, 1987; Peck and Chynoweth, 1990; Zinder, 1993). We selected methanogenesis and dehydrogenase as measurements of microbial activities and used immunological probes developed for digester isolates to measure population dynamics. 2. Materials and methods 2.1. Reactor design The reactor was a cylindrical, pilot-scale (4500 l working volume) solids-concentrating digester described elsewhere in detail (Chen et al., 1990). Fig. 1 is a schematic of the reactor and MEP device. The MEP was done in a cylindrical tank 4 m tall and 0.3 m diameter with a series of internal baes to increase gas exchange between the euent and the air being blown through the MEP. A solids reduction tank (570 l) was installed in line, preceding the MEP to keep solids from fouling the MEP. A holding tank (570 l) was used to allow facultative bacteria to remove O2 from euent leaving the MEP before returning the euent to the digester. The feedstock was a mixture of the organic fraction of municipal solid waste and primary sewage sludge at a ratio of 15:1 on a volatile solids basis. The feed concentration was 8% total solids and the digester organic loading rate was 3.2 kg VS mÿ3 dÿ1 . The digester was operated at 35°C. During MEP operation, reactor euent was circulated through the MEP at a rate of 3.5 reactor vol-
umes per day. The aeration rate was 46 l air per l of recycled digester euent. 2.2. Reactor sampling Evaluation of the microbial impact of operating the MEP was done by sampling the reactor euent twice while the MEP was operating (12 and 22 February 1990) and twice while it was not operating (19 and 28 February 1990). Composite samples consisting of 750 ml sub-samples from six depths (0.9, 1.2, 1.5, 1.8, 2.3, and 2.9 m from the tank bottom, i.e. Sample Ports 2, 3, 4, 5, 6, and 8, respectively) were taken on the above sampling dates. While the MEP was operating, additional samples were taken at the send (to the MEP ± see Fig. 1) and return (to the reactor) ports. Samples were put into 5 l containers, sealed and transported to the laboratory for immediate analysis. The methods used to evaluate the biological impact of the MEP, and the rationale for using each assay, are summarized in Table 1. The following analyses were done on the euent: volatile organic acids (VOA), total and volatile solids content, dehydrogenase activity, speci®c methanogenic activity (SMA), and enzyme-linked, immunosorbent assay (ELISA) for methanogenic, sulfate reducing, and cellulolytic bacteria. 2.3. Volatile organic acids analysis The VOA concentration of the euent was determined by acidifying samples with the addition of 20% (v/v) H3 PO4 to a ®nal concentration of 2% (v/v). Sam-
Fig. 1. Schematic diagram of the reactor and methane enrichment process.
D.M. O'Keefe et al. / Bioresource Technology 71 (2000) 217±224
219
Table 1 Summary of methods used to detect biological eects of operating the methane enrichment process with an anaerobic digester fed the organic fraction of municipal solid waste Method
Time required
Target organisms
Comments
pH Volatile organic acids Methane content Biogas production
Minutes Minutes Minutes Minutes
Entire, balanced, methanogenic consortium Entire, balanced, methanogenic consortium Entire, balanced, methanogenic consortium Overall microbial population
1 Day 1 Day
Simple, fast,cause may be non-biological Simple, fast Simple, fast, cause may be non-biological Simple, fast, in conjunction with methane content may be mechanistic Mechanistic Mechanistic
1
SMA 1 acetate Dehydrogenase assay
1 Day Hours
Hydrogen and formate-utilizing methanogens Cellulolytic microorganisms, acid-utilizing methanogens Acetate-utilizing methanogens Overall microbial population
Antibody probes
Days
Species speci®c (see Table 2)
SMA SMA
1
1
formate cellulose
Mechanistic Does not discriminate between aerobic and anaerobic metabolism Mechanistic, does not discriminate between dead, live, active, and inactive bacteria
Speci®c methanogenic activity.
ples were then centrifuged and 1 ll of the supernatant analyzed by gas chromatography (Shimadzu GC Model 9AM, Kyoto, Japan) using a 1.8 m ´ 2 mm glass column of 10% SP-1000 on 100/120 Chromosorb W/AW (Supelco, Bellefonte, PA). The column was maintained at 140°C with nitrogen as the carrier gas at 40 cm3 minÿ1 . The injector was set at 160°C and the ¯ame ionization detector at 200°C. The total VOA concentration reported here represents the sum of acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids. 2.4. Solids Total and volatile solids content of euent samples was analyzed according to Standard Methods (Clesceri et al., 1989). 2.5. Speci®c methanogenic activity A procedure modi®ed from Owen et al. (1979) was used to assess the ability of the reactor microbial populations to produce methane. Unamended euent and euent amended with 0.2 g 100 mlÿ1 acetate, formate, or cellulose (Avicel microcrystalline) were incubated at 35°C in 125 ml-serum bottles ®tted with rubber septums. Gas volume and composition were assessed at 4, 12, 24, and 48 h. Gas volume was measured using a gas-tight syringe. Gas composition was determined using a gas chromatograph (Fisher Gas Partitioner, Mode1200, Pittsburgh, PA) equipped with two matched pairs of thermal conductivity detectors and dual stainless steel columns operated at 55°C. The ®rst column was 2 m ´ 3.2 mm packed with 80±100 mesh Porapak Q (Supelco, Bellefonte, PA), and the second column was 3.35 m ´ 4.8 mm packed with 60±80 mesh molecular sieve 13X (Supelco, Bellefonte, PA). Helium was the carrier gas at a ¯ow of 30 cm3 minÿ1 .
2.6. Dehydrogenase assay The dehydrogenase activity of the euent was assayed according to the method described by Lopez et al. (1986) which uses the redox dye 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyl tetrazolium chloride (INT). Fiveml samples of digester euent were incubated for two hours at 35°C with 0.5 ml of the INT reagent. After incubation the reaction was stopped by adding 1.0 ml of a 37% formaldehyde solution. The formazan was extracted in methanol according to the method of Lee et al. (1988). Formazan concentrations of the extracts were determined by optical density at 480 nm. 2.7. Enzyme-linked immunosorbent assay probes Speci®c polyclonal antibody probes were selected (Table 2) that had been developed for six strains of anaerobic bacteria using methods of Archer (1984). An ELISA was utilized as previously described to test the samples (Brigmon et al., 1992). In brief, fresh samples of euent were centrifuged at 20 000 RPM for 30 min. The clear supernatant was discarded. The pellets were resuspended to original volume in carbonate±bicarbonate buer and the pH adjusted to 9.8 with 4 M NaOH. Duplicate, 200 ll samples were added to Immulon-2, 96well immunoassay plates (Dynatech, Chantilly, VA). Plates were incubated 16 h at 4°C. Fifty ll of PBS containing 0.5% glutaraldehyde was subsequently added to all wells for 5 min. The plates were washed 6´ with a PBS solution containing 0.1% Tween 20 (Sigma, MO) (PBST) after which they were incubated for 1 h with 1% Bovine Serum Albumin (Sigma, MO) (BSA) in PBS (PBSA) at room temperature. Plates were again washed 6´ with PBST. Duplicate samples of primary antibody, mouse or rabbit speci®c anti-bacterial polyclonal antibodies (PAB), diluted 1:1000 in PBSA (100 ll) were
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Table 2 Bacteria used to prepare antibodies for immunological probing of reactor populations Figure I.D.
Bacteria
Source
Biochemical Role
Habitat
A B C D E F G H
Methanothrix concilii strain GP6 Methanobacterium thermoautrophicum Methanobacterium thermoautrophicum Methanosarcina barkerii strain Mb Methanogenic bacterium strain T3 Clostridium acetobutylicum Desulfovibrio desulfuricans Clostridium aldrichii P1
G. Patel, NRC Ontario, Canada Y. Yang, University of Florida Y. Yang, University of Florida Y. Yang, University of Florida Y. Yang, University of Florida ATCC #824 ATCC #29577 Y. Yang, University of Florida
Methanogen Methanogen Methanogen Methanogen Methanogen Acid former, cellulolytic Sulfate reducer, acid former Acid former, cellulolytic
± Sewage-sludge digester Sewage-sludge digester Sewage-sludge digester Termite gut Soil Soil Anaerobic, wood digester
tested. Negative controls employed in the ELISA were PBSA, plasma from normal mice or normal rabbits diluted 1:1000 in PBSA and (PBST). ELISA plates were then incubated for 1 h. Samples were removed and the wells washed 6´ with PBST. 100 ll of secondary antibody, anity-puri®ed alkaline phosphatase goat antimouse or anti-rabbit immunoglobulins (Organon Teknika, Malvern, PA) 1:1000 dilution in PBSA were added to each well. Plates were incubated for 1 h with secondary antibody and then washed. 200 ll of alkaline phosphatase substrate (1 mg mlÿ1 p-nitrophenyl phosphate, in diethanolamine buer (Sigma, MO) prepared just before use) was added to each well. The plates were protected from light and read on a Titertek Multiskan (Flow Laboratories, McLean, VA) at 405 nm after 30 min. Sensitivity assays were done by serially diluting bacteria from 2 ´ 108 to 2 ´ 102 cells mlÿ1 in carbonate± bicarbonate buer and performing the ELISA. Absorbencies from reactor samples were compared to standard curves to estimate bacterial concentrations. Fig. 2. Sampling and MEP operation dates and methane content of biogas during MEP trials.
3. Results 3.1. Methane enrichment Operation of the MEP resulted in methane contents of 90% (Fig. 2) in each of two runs. Similar results have been obtained with glucose (Hayes et al., 1990) and particulate biomass feedstocks (Jewell et al., 1993). Air purging of digester supernatant appears to be an eective technique for incorporation of methane enrichment into the anaerobic digestion process. With the solids concentrating reactor employed for these studies, methane enrichment was not sustainable (for more than a few days) because of solids carryover and clogging of the methane enrichment process. 3.2. Solids and volatile organic acids Volatile solids and VOA concentrations of the reactor and MEP euent are summarized in Table 3. There were no signi®cant (P 6 0.05) eects of the MEP on ef-
Table 3 Means and standard deviations (SD) for volatile organic acid (VOA) concentrations and volatile solids (VS) of euent samples by mode (MEP on or o) and port (composite, MEP-send, and MEP-return). Composite samples consisted of 750-ml samples from 6 depths. Volatile organic acid values are adjusted for solids. All values are means of six replicates VS (mg mlÿ1 )
VOA (mg (gVS)ÿ1 )
Mean
SD
Mean
Mode On O
8.6 6.9
6.0 2.9
8.5 8.1
6.8 6.9
Port Composite Send Return
8.6 2.3 1.1
6.0 1.3 0.2
8.5 17.0 13.7
6.8 10.5 7.8
SD
¯uent VOA concentration. Operation of the MEP had no eect on VS levels of composite samples taken from the reactor. However, there was a reduction in volatile
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solids in euent passing through the MEP which may be attributed to oxidative metabolism, ®ltering, and/or settling of solids. 3.3. Speci®c methanogenic activity Operation of the MEP increased the methane potential of composite samples of the reactor euent for the unamended and three amended treatments (Fig. 3).
Fig. 3. Cumulative methane yields of composite euent samples taken while the MEP was on or o and incubated in sealed jars for 48 h. Euent was left unamended or amended with formate, cellulose, or acetate. Points represent means of three replicates and error bars are standard deviations of the mean.
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However, comparison of the SMA of MEP in¯uent and euent revealed that the MEP had a localized inhibitory eect on euent methanogen populations as they passed through the device (Fig. 4). This was true for unamended and amended (with acetate formate or cellulose) MEP measurements.
Fig. 4. The eect of the MEP on methanogen populations passing through the device. Methane yields of euent samples taken while the MEP was operating. A composite sample and samples entering (MEP send) and returning (to the reactor) were incubated in sealed jars for 48 h. Euent was left unamended or amended with formate, cellulose, or acetate. Points represent means of three replicates and error bars are standard deviations of the mean.
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3.4. Dehydrogenase activity Operation of the MEP had no eect (P 6 0.01) on dehydrogenase activity of composite digester samples while euent exiting the MEP had increased activity compared to composite digester samples and MEP in¯uent (Fig. 5). This result was expected as dehydrogenase activity is a measure of anaerobic and aerobic microbial activity and aeration of the euent should stimulate over-all microbial activity. As with the SMA assay, however, the eect was localized and the overall reactor population was not similarly aected. 3.5. ELISA probes The ELISA analysis of the reactor revealed no signi®cant (P 6 0.01) eects of the MEP on the reactor microbial populations (Fig. 6). The reason that the antibody analysis did not reveal treatment eects may be that antibodies bind to live and active, live and inactive, and dead cells. The results of this measurement demonstrated that, for short-term experiments, antibody probe analysis may not be appropriate for detecting microbial changes. This would be particularly true if the changes were shifts in metabolic activity more than
Fig. 5. The eect of the MEP on dehydrogenase activity of microbial populations passing through the device. Bars represent means of 10 replicates and error bars are standard deviations of the mean.
shifts in population composition. It is noteworthy that every antibody used in this probe gave a positive result. This demonstrates the rich microbial diversity of the methanogenic consortium in MSW-fed anaerobic digesters. 4. Discussion We had expected the MEP to inhibit anaerobic microorganisms since 3.5 volumes of reactor euent were being vigorously aerated each day. However, this treatment had no apparent negative eect on overall anaerobic microbial populations. Microbial populations in the euent leaving the MEP, on the other hand, were in¯uenced by aeration in ways anticipated. Solids content of the reactor was four times higher than in the euent stream being circulated through the MEP. This suggests that, for the most part, microorganisms were not being circulated through the MEP but were retained in the reactor, probably sorbed to solids. Any dissolved oxygen in euent returning to the MEP was probably consumed in the O2 -removing tank before reaching the reactor. Therefore microbial populations within the reactor were not exposed to oxygen introduced during the MEP operation. The choice of amendments used in this assay allowed us to draw some conclusions as to the preferential inhibition of the basic biochemical components (acetogens and methanogens) of the methanogenic consortium. Had the methanogens been inhibited in the absence of inhibition of the acetogens, the amended treatments would have shown an initial ¯ush of biogas followed by a drop in production rate from VOA inhibition and little or no methane production. As this was not the case, we concluded that oxygenation of the euent inhibited all components of the methanogenic consortium. We do not know why operation of the MEP stimulated the methanogenic activity of the composite samples. Solids and VOA concentration of the euent do not explain this dierence as they were not in¯uenced by the MEP operation. In addition, dissolved methane in the euent due to increased methane content of the reactor headspace during the MEP operation was not sucient to account for the increase (data not presented). Evidence of inhibition of anaerobic microbial populations was only found in euent exiting the MEP system. On a practical level, therefore, monitoring of reactor performance alone would have been sucient to test the biological impact of this device. A summary of the relative utility of the methods used in this experiment is contained in Table 1. The localized inhibition allowed us to make several generalizations concerning the relative utility of some of the methods used. First, antibody probes are of little use in detecting short-term microbial perturbations. This is due to the inability of antibody
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Fig. 6. The eect of the MEP on antibody probing of overall reactor microbial populations (a) and populations in the euent stream passing through the MEP (b). Bars represent means of duplicate samples. Letters refer to isolates listed in Table 2.
probes to discriminate between live, dead, active, and inactive microbes. For this technique to be useful, suf®cient time is required for degeneration of the antigenic, cell wall constituents. The primary drawback to the dehydrogenase assay in this application was its inability to discriminate between aerobic and anaerobic metabolic activity. All the SMA tests clearly showed that oxygenation of the euent stream compromised acetogens and methanogens in that stream. We conclude that the SMA assay was the most instructive method employed in this study.
Acknowledgements This work was supported by US Department of Energy Contract DE-ACO2-83CH10093, Biological Assessment of Anaerobic Digestion of Solid Waste, and the University of Florida Institute of Agricultural and Food Science.
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