Environ. Sci. Technol. 1990, 24, 1642-1648
Behavior of Carbon Monoxide as a Trace Component of Anaerobic Digester Gases and Methanogenesis from Acetate Robert F. Hlckey' and Mlchael S. Switzenbaumt Michigan Biotechnology Institute, Lansing, Michigan 48909, and Department of Civil Engineering, University of Massachusetts, Amherst, Massachusetts 01003
U Carbon monoxide was a normal trace component of the gases produced during anaerobic sludge digestion. The CO concentration increased in response to perturbing the digestion process by increasing organic loading or adding acetate. Reducing the headspace methane level resulted in higher measured CO concentrations. Accordingly, a thermodynamic relationship was developed by dividing the acetoclastic methane reaction into two half-cell reactions, representing production of and subsequent oxidation of CO. A constant fraction of the total free energy available for acetate conversion to methane was assigned to each half-cell based on the basis of experimental observations. It was determined that approximately 54% of the energy available for acetate conversion to methane was consistently associated with the anaerobic oxidation of CO to carbon dioxide. Estimated values compared well for measured concentrations for both mesophilic and thermophilic digesters operating under steady-state conditions. The thermodynamic relationship, based upon gaseous components only, was manipulated to accurately predict acetate concentration in a digester system subjected to an organic overload that was monitored with an on-line data acquisition system. Introduction Recent evidence indicates that carbon monoxide dehydrogenase is relatively common among some of the major trophic groups in anaerobic ecosystems. Many methanogens possess CO dehydrogenase ( I ). High levels of CO dehydrogenase also occur in acetogens (2-7). In the case of hydrogen-utilizing methanogens (8-10) and acetogenic bacteria (11, 12) CO dehydrogenase may be essential for the anabolic fixation of C 0 2 into cell carbon. Recently, sulfate-reducing bacteria (SRB) not possessing an operative citric acid cycle were reported to have high levels of CO dehydrogenase. In SRB, the function of CO dehydrogenase is thought to be essentially a reverse of the anabolic reaction with 2C02 formed from acetate (13). Conrad and Thauer (14) first reported production of gaseous CO under strictly anaerobic conditions. It was subsequently determined that CO was produced by Methanobacterium thermoautrotophicum as a consequence of C 0 2 fixation (15). It had been previously reported that an acetogenic bacterium produced trace levels of CO during carbon dioxide fixation (16). Acetoclastic methanogens also possess high levels of CO dehydrogenase activity (17-19). Up to 5% of the soluble protein of Methanobacterium barkeri is CO dehydrogenase (20). These findings suggest that CO dehydrogenase plays a physiological role in the production of methane from acetate. It is now believed that acetate is first activated and then split into bound methyl and carbonyl groups in a reaction analogous to the reverse of the acetate synthesis *To whom correspondence should be addressed: Michigan Biotechnology Institute, P.O. Box 27609, Lansing, MI 48909. University of Massachusetts. 1642
Environ. Sci. Technol., Vol. 24, No. 11, 1990
by acetogens (18,19,21) and autotrophic methanogens (9, 10,22). It was suggested that the carbonyl or bound CO may equilibrate with CO in solution (23), although free CO does not appear to be an actual intermediate (24). Over the course of experiments designed to investigate the major and minor components of digester gases produced during the anaerobic digestion of waste activated sludge (WAS),carbon monoxide was identified as a normal trace component (25). It was further observed that when batch anaerobic reactors were perturbed, by changes in organic loading or introduction of an inhibitory compound (26), CO concentrations in the gaseous headspace varied in a manner that suggested a possible relationship to metabolic activity levels of one or more trophic groups within the anaerobic consortium. Reported herein are some findings that demonstrate production of trace levels of CO by methanogenic systems catabolizing acetate. Since acetate metabolism accounts for approximately 70% of the methane formed in digester systems (27-29), the relationship between observed gaseous CO and acetate metabolism in digester systems was investigated. Recently, it hass been shown that thermodynamics are extremely important in the formation and consumption of substrates in anaerobic ecosystems (3,31). Although CO is not believed to be a product or substrate but rather equilibrates with a bound form involved in acetate catabolism (23), the possibility that its concentration could be described via a thermodynamic relationship was investigated. The concentrations of trace levels of CO in the headspace during the anaerobic digestion of waste activated sludge do appear to be redox regulated, with CO levels increasing with either increasing acetate concentrations or decreasing methane concentrations. A thermodynamically oriented relationship was developed based on the free energy available during conversion of acetate to methane. The concentration of acetate in mesophilic and thermophilic digesters operating at steady state and a digester subjected to an organic overload of sufficient magnitude to induce upset of the process could be estimated by using the developed thermodynamic relationship. Materials and Methods Reserve Digesters, Operation and Monitoring. Two digesters with working volumes of 55 and 110 L were used to supply inocula with 10- and 20-day hydraulic retention times, respectively, for the serum bottle testa and 5-L test digesters. The digesters were housed in constant-temperature incubators maintained a t 35 "C. The reactors were operated in a fill and draw mode. The units were fed daily a mix (5.5 L) of waste activated sludge (WAS) and dissolved air flotation waste activated sludge from the Amherst, MA, wastewater treatment plant. The feed to each unit was maintained at a constant level of 1.25-1.50% total solids (1.0-1.2% volatile solids). The digesters were initially filled with screened sludge from a municipal digester (Northampton, MA) operated a t a hydraulic residence time of approximately 15 days on a combination of
0013-936X/90/0924-1642$02.50/0
0 1990 American Chemical Society
primary and secondary treatment sludges. Gas monitoring of the 10- and 20-day hydraulic residence time (HRT) digesters reported in this study was done after the units equilibrated to the WAS feed and were performing at steady state (4 months after employing constant feed). Subsequent monitoring of these units over 18 months demonstrated no significant change in gas composition compared to results presented herein. A 2-L (working volume) digester was constructed from a glass aspirator bottle. This unit was seeded with sludge from a thermophilic unit operating on heat-treated WAS. The digester was operated at a 10-day HRT under steady-state conditions for approximately 3 months prior to obtaining the gas composition information reported in this paper. The unit was operated in a daily draw and fill mode with the 1.25-1.5% (12.5-15.0 g/L) total solids WAS feed used for the mesophilic digesters. Sampling of the gaseous headspace was performed either just prior to or immediately after feeding. The digester was housed in a 55 "C constant-temperature incubator and maintained completely mixed with a magnetically drive Teflon stir bar. A 1-in.-thick sheet of expanded Styrofoam was placed between the magnetic stirrer and the digester to ensure no heat was transferred from the stirring apparatus to the digester. Daily gas production of the reserve digesters was measured with wet test meters. Total volatile fatty acids concentration was performed by a titration technique (32). Methane and carbon dioxide levels were analyzed via thermal conductivity (Gow-Mac 550, Bridgewater, NJ). Separation was accomplished with Porapak Q (2 m X 4 mm i.d. stainless steel column) using helium as a carrier. Area integration was performed with a HP-3390A integrator. Methane and COPanalyses had relative standard deviations of 0.4 and 1.3%, respectively. Carbon monoxide and hydrogen concentrations were analyzed with a mercury reduction based chromatographic system (Trace Analytical, Stanford, CA) equipped with a 0.1-mL sample loop. Separation was accomplished with molecular sieve 5A (2 m X 4 mm i.d.) using catalytically purified nitrogen as a carrier as previously reported (6,32). Relative standard deviations for hydrogen and carbon monoxide analyses were 0.48 and 0.38%, respectively. The detection limits for hydrogen and CO were 1 ppm and 100 ppb, respectively. Shock Organic Load Serum Bottle Assay. Batch serum bottle assays were conducted for this study using inocula from the reserve digesters. The protocol used for the serum bottle assays was an adaptation of the serum bottle technique developed by Miller and Wolin (33)and later modified by Owen et al. (34). A total of 90 mL of inocula from the 10-day HRT reserve digester was anaerobically transferred to each serum bottle with a syringe pump and mixed with 10 mL of WAS supplied at 1,2, and 3 times the normal concentration (1.25% total solids) that the reserve digester inocula normally received. The assay, which was conducted for 24 h, was designed to mimic a typical daily draw and fill operation employed at many wastewater treatment plants. All samples were gassed with an 02-free (
