Environ. Sci. Technol. 1992, 26, 369-376
Anaerobic Butyrate Degradation in a Fluidized-Bed Reactor: Effects of Increased Concentrations of H, and Acetate Foroozan Lablb,**+John F. Ferguson,* * +Mark M. Benjamin,+ Mohamed Merlgh,z and N. L. Rlckerz
Department of Civil Engineering and Department of Chemical Engineering, University of Washington, Seattle, Washington 98195
rn The microbial degradation of butyrate was studied in a continuous-flow anaerobic fluidized-bed reactor. The reactor characteristics at steady-state butyrate loading, and in response to transient loadings of butyrate, hydrogen, acetate, and formate, were determined using an on-line computerized data acquisition system. The fluidized-bed reactor operated at a steady-state butyrate loading of 10 g of chemical oxygen demand (COD)/L.day and at an unusually high specific loading rate of 8-10 g of COD/g of volatile solids per day, achieving >97 ?& COD removal. It was found that increases in hydrogen and acetate concentrations above their values at steady state can partially inhibit butyrate degradation, even though the free energy change for the butyrate oxidation was still negative. Increases in hydrogen and acetate concentrations that resulted in a positive free energy change stopped butyrate oxidation completely. Thus, there is evidence for a concentration-dependent product inhibition, as well as the expected inhibition when the reaction becomes energetically unfavorable. Due to mass-transfer limitations, adding hydrogen to the reactor had only a small effect on butyrate oxidation. Addition of formate to the reactor inhibited butyrate oxidation probably due to hydrogen generation via microbial degradation of the formate. Possible inhibition by formate could not be distinguished from hydrogen effects.
Introduction In many cases, anaerobic wastewater treatment can have important benefits with few drawbacks compared to conventional aerobic processes. Anaerobic processes convert organic wastes into a usable end product, methane, and do so with much less energy consumption and biological sludge production than aerobic processes. However anaerobic processes may require long start-up times due to the slow growth rate of the bacteria, and they are very sensitive to variations in pH, toxic shocks, and organic overloads. The microbial ecosystem in an anaerobic reactor comprises several groups of microorganisms which must interact effectively to convert the organic matter into methane and carbon dioxide. The product from a reaction carried out by one group of bacteria serves as substrate for the subsequent specialized bacterial groups. When the process is operating efficiently, a balance is established between the rates of formation and removal of intermediates, preventing accumulation of the intermediates in the reactor. The instability associated with the anaerobic processes during an organic overload is often the result of an imbalance in the microbial interactions and reflects unequal abilities of different microbial groups in the community to respond to the change (1,2). When the substrate is particulate, solids hydrolysis is apparently the rate-limiting step in anaerobic processes (3). On the other hand, when soluble substrates are being consumed, the terminal methanogenic reactions usually limit the overall degradation rate (4-7). During periods f
Department of Civil Engineering. Department of Chemical Engineering.
0013-936X192/0926-0369$03.00/0
Table I. Anaerobic Degradation of Butyrate' AG3.ib
+
-
Acetogenesis 2H20 2CH3COO- + 2H2 +
CH3(CH2)2COOHt (1) = +86.0 kJ/mol of butyrate)
Methanogenesis 2CH3COO- + 2Ht 2CH4 + 2C02 (2) (AG370= -159.1 kJ/mol of butyrate) 2H2 + 0.5c02 0.5CH4 H 2 0 (3) = -63.0 kJ/mol of butyrate)
-
4
+
Overall Butyrate Degradation CH3(CH2)2COO- HzO + HC 2.5CH4 1.5C02 = -136.1 kJ/mol of butyrate)
+
4
+
-15
-45 -10
(4) -70
"The standard free energy changes are calculated at 37 OC using values from Thauer et al. (20). *Typical AG37 in the reactor at steady state (kJ/mol of butyrate), using concentrations measured in the bulk gas and liquid phases.
of organic overload, the terminal reactions may be incapable of removing organic intermediates as rapidly as they are produced, so the intermediates accumulate. Their concentration and composition are thought to reflect the sensitivity of the process to H2 concentration, another intermediate (8-11). High H2 concentrations may both increase methane production and inhibit the use of reduced organic intermediates such as ethanol, propionate, and butyrate. The effects of H2are probably related to the thermodynamics of the reactions (12-15). Acetate also has an important regulatory role (14). Thus the kinetics of methanogenesis from H2 and acetate may affect the rates of the H2-producing acetogenic reactions. Understanding the factors that cause reactor instability and give rise to elevated concentrations of organic acids can help in formulation of process control strategies. Such strategies would be used to prevent reactor souring due to acid accumulation, which, if unchecked, leads to process failure in reactors treating butyrate, propionate, fatty acids, or fats. Butyrate and propionate metabolism has been known to be initiated by different microbial species, since the studies of McInerney (16, 17) and Boone (18) elucidated the consortia carrying out their conversions to methane. The propionate consortium has been studied extensively in recent years because of interest in the severe requirements for partitioning energy among the three metabolic groups participating in methanogenesis (19). In the present study, butyrate has been used as the model substrate in order to apply our reactor-based methodology for studying anaerobic microbial communities and to study energy partitioning and inhibition, analogously with propionate, in a system that is somewhat more representative of metabolism of long-chain fatty acids and fats. Methanogenesis from butyrate involves the sequential formation of acetate and H2 by H,-producing acetogens and their utilization by metanogens, as shown by the reactions in Table I. Various loading perturbations were employed on the reactor to elucidate the response of the major microbial groups involved in the anaerobic degradation of butyrate and to understand the significance of
0 1992 American Chemical Society
Environ. Sci. Technol., Vol. 26, No. 2, 1992 369
Table 11. Composition of Nutrient and Buffer SolutionD compds
concn, mg/L
concn, pg/L
trace compds
concn, r g / L
653 24 24 24 24
Na,MoO,dH,O H~BOI CaCI,.2H10 vitamin B-12
24 24 24 2.4
compds
concn, mg/L
trace compds
CoCI26H20 MnC12.4H,0 NH,VO, NiCI,6H20 ZnC1,
#Adjusted to pH 1.0, and comprising approximately 90% of the feed flow to the reactor.
&buffer SOlUDO"
Flgure 1. Schematic
diagram of tlm reactor setup.
H, and acetate on the reactor stability. Experimental Setup A 3-L fluidized-bed reactor was used in this study (Figure 1). About 3.2 kg of Ottawa sand (2C-30 mesh; 0.7-0.8-mm diameter) was the support medium for microbial growth. The sand grains were rounded and quite smooth. The bed was expanded 15% by recycling the reactor liquid at a flow rate of 3 L/min. Thii high recycle rate completely mixed the reactor contents. The reactor was seeded with anaerobic digester sludge and was initially operated in a batch mode, using butyrate as the sole organic carbon source. After growth on the sand was observed, the reactor was fed continuously, and the loading rate was gradually increased to 10 g of chemical oxygen demand (COD)/L.day. The feed was made up from a nutrient and buffer solution and a solution containing the organic substrates. The compounds and the concentrations are shown in Table 11. The feed chemical oxygen demand concentration was 5 g/L, and the hydraulic residence time was 12 h. The ionic strength of the combined feed was 0.047. A t steady state, the amount of total volatile solids (VS) in the reactor was about 3-4 g, corresponding to 1.2 g of VS/L of reactor volume and a specific organic loading rate of 6-10 g of COD/g of VS per day. Calculations assuming spherical grains of 0.75 mm estimated a uniform biofilm thickness to be 6.4 pm. However, m i c r m p i c examination (at 2OOX and 400X) indicated the biofilm was very thin and not continuous, occurring mainly in slight indentations on the relatively smooth sand grains. It could be seen on no more than 10% of the sand surface. The volatile suspended solids (VSS) concentration in the reactor was -70 mg/L. Based on this VSS and the effluent flow rate of 6 L/day, the estimated solids retention time of volatile solids in the reactor was less than 10 days. 370 Environ. Sci. Technol., VoI. 26. No. 2, 1992
Data collection was automated, as described in detail by Slater et al. (21). Gas produced in the process passed through a mass flow meter after passing through a 100-mL bed of desiccant to remove water vapor. The mass flow meter is insensitive to temperature fluctuations. Gases were then analywd in a dual gas chromatographic system, which was assembled by John Booker Co. (Austin, TX). CH, and COP were separated on a HaySep Q packed column and detected by a thermal conductivity detector (TCD). H, and CO were separated on a silica gel packed column and detected by a reduced gas detector (Trace Analytical, Palo Alto, CA). Reactor gas was analyzed every 3 min. Temperature, pH, and oxidation-reduction potential (ORP) were measured with probes in the recycle line every 1min. Volatile fatty acids (VFAs) were analyzed off-lineusing a gas chromatograph with a flame ioiniition detector (FID). This analysis was subsequently carried out on-line. Theae analytical functions are designed so that experiments investigating imposed input transients can be carried out on a reactor unit by placing it in the testing system. Reactors were maintained a t steady state when they were not in the testing system. The reactors and the test systems were housed in a temperature-controlled environmental chamber a t 37 OC. In experiments in which it was suspected that H,in the reactor gas was not at equilibrium with H2 dissolved in liquid, H, concentration in the liquid was also measured off-line by head space gas analysis. In such cases, reactor samples were collected in test tubes containing mercuric chloride and were sealed. They were then shaken vigorously to allow the dissolved H, to equilibrate with the gas phase. The gas in the test tube was analyzed for H,, and estimates of H2 concentration in the liquid were made.
Experimental Results and Discussion Five experiments are presented, which investigated degradation of butyrate under various loadings. In experiment 1,the performance of the reactor at steady-state butyrate loading and its response to a series of changes in the butyrate loading were characterized. Experiments 2 and 3 explored, respectively, the effects of increased acetate concentration and increaesd PH on butyrate utilization in the reactor. Due to the high utilization rate by the methanogens, the transfer rate of H, from the sparged gas into solution limited H, use, and this method of increasing PH,had a very small effect on butyrate utilization. In was increased by adding formate to the experiment 4, PH1 reactor influent. Formate metabolism produces H,, thus eliminating the need for H2 transfer from the gas phase. Finally, in experiment 5, the effects of increased concentrations of H, and acetate were studied. The combined
k,
100 50-
d
B 0.4 4 0.2 9s ._7
'4
6e 8 5 -
(PH2) at 50-100 ppm (Figure 2c) in the reactor gas is a characteristic of the H2-utilizingmethanogens. They have a high affinity for H2 (6,22,23),which is necessary for the oxidation of butyrate to proceed. Carbon monoxide was detected in the gas at a partial pressure of 0.1-0.2 ppm (Figure 2d). Carbon monoxide has been detected by Krzycki (24)and Hickey (25)during methanogenesis from acetate. Since acetate is an intermediate produced in the anaerobic degradation of butyrate, methanogenesis from acetate could be a source of CO formation in this reactor. At 11.6 h, the butyrate feed concentration was dropped to zero for 0.5 h by shuting off the butyrate feed pump while maintaining the flow of nutrients and buffer solution to the reactor. At -12.1 h, a step increase in butyrate loading, equivalent to 2.9 times the base-line loading, was imposed on the reactor. The elimination of the butyrate feed reduced the hydraulic flow rate to the reactor by -7%, from 6.3 to 5.8 L/day. The subsequent high loading period increased the flow rate to -7.3 L/day. When the butyrate feed was stopped, the reactor concentrations of butyrate, acetate, and H2started to decrease (Figure 2f), and the gas production rate dropped (Figure 2b). Following the loading increase, the butyrate concentration in the reactor increased, as did the rates of the microbial degradation reactions. As butyrate was oxidized, acetate concentration and H2partial pressure in the reactor increased. Both butyrate and acetate concentrations continued to rise during the period of high loading. By contrast, the H2 partial pressure increased only for 15 min, after which it stabilized and then gradually decreased due to dilution with CHI and C02,as their production rates gradually increased. At 14.1 h the butyrate loading was stopped again, and the butyrate concentration decreased abruptly. However, because butyrate oxidation continued in the reactor, the acetate concentration did not decrease as rapidly. As the concentration of butyrate decreased, its reaction rate was reduced, acetate was not formed as rapidly, and continued acetate utilization lowered the acetate concentration in the reactor. There were no significant changes in CH4 concentration in the gas phase. Gaps in CHI, CO, and H2data are due to lack of sufficient gas flow through the sampler, which was caused by decreased gas production in the reactor. Isobutyrate and propionate were also produced in the reactor (data not shown). In experiments involving step increases in butyrate loading, propionate was always present at 10 mg of COD/L and did not increase during the butyrate loading. However, isobutyrate, usually below detection limit at steady state, increased in response to the butyrate loading. In our experiments, when butyrate oxidation was substantially saturated, isomerization of 10% of the n-butyrate to isobutyrate continued; in this experiment, the isobutyrate concentration reached a maximum of 40 mg of COD/L at 14.5 h before decreasing back to zero. Isobutyrate could be formed and used by the butyrate-degrading microbial consortium in this reactor. Isobutyrate is thought to be converted to butyrate for oxidation (26,27). Experiment 2. Acetate Loading to the Butyrate-Fed Reactor. This experiment was performed to investigate the effect of increased acetate concentration on the butyrate oxidation rate. The reactor was operating at a constant butyrate loading of 8.9 g of COD/L.day. Panels a-e of Figure 3 show the applied loadings and the experimental results. At this loading, the COD removal efficiency was -96%, the acetate concentration was -150 mg
-
10
11
12
13
14
15
16
17
18
Time fir)
Figure 2. Reactor characteristics at steady state, and their response to transient loading of butyrate.
results illustrate the dynamics of a highly enriched butyrate culture a t organic loadings near or beyond the maximum substrate utilization rates of the subpopulations. Experiment 1. Reactor Characteristics at Steady State and under Butyrate Loading Changes. The base-line, steady-state operating conditions in the reactor included an organic loading rate of 8.4 g of COD/L-day, feed COD of 4300 mg/L, and a hydraulic flow rate of 6.3 L/day, yielding a hydraulic retention time in the reactor of -12 h. The reactor had been operated under these conditions for several weeks prior to any experiments, with performance characteristics similar to those shown during the time period 10-11.6 h in Figure 2a. The soluble COD in the reactor effluent was -120 mg/L, corresponding to >97% COD removal. Gas with a methane content of 84% was produced at a rate of 7.6 mL/min (Figure 2b,e), indicating that almost 97% of the COD was converted to CH,. Based on volatile acid analysis of the reactor effluents (Figure 2f), almost all (>99%) of the butyrate in the feed was utilized, with butyrate concentrations less than 20 mg/L and acetate 100 mg/L. The butyrate is primarily converted into acetate and Hz via catabolic reaction 1 (Table I), and some may be used in anabolic reactions leading to the formation of biomass. The acetate removal efficiency can be calculated based on the production of acetate COD from butyrate, assuming the stoichiometry of reaction 2 in Table I. About 97% of the acetate generated in the reactor is removed; however, acetate represents nearly all the effluent soluble COD. A similar calculation indicates that >99.98% of the H2 generated in the reactor is removed. This high H2 removal efficiency and the maintenance of the H2 partial pressure
-
-
-
Environ. Sci. Technol., Vol. 26,
No. 2, 1992 371
Acetate
Pulse
Butyrate Loading 8.92p C O D L - d
Hydraulic Flow = 6.5 Ud
8 8ooo
I
1
1
d
8 95
I
I
(experiment 5) discussed below shows that small increases in the steady-state acetate concentration (with very small changes in Na+ and ionic strength) inhibited butyrate conversion, supporting the interpretation based on product inhibition rather than effects of inorganic ions. The total gas flow rate and its CH4 content are shown in panels d and e of Figure 3, respectively. In response to the increase in acetate concentration, the total gas production rate increased from -8.1 to -9.4 mL/min. However, the CHI production rate increased only from 6.8 to -7.5 mL/min, indicating that the acetate-utilizing capacity in the reactor was near saturation. The CHI content in the gas decreased from 86 to 77% following the acetate loading, while the C02 concentration increased. The changes in CHI and COP concentration were expected, since methanogenesis by acetate splitting produces CHI and CO, at equal molar rates. However, as the butyrate oxidation rate increased, the increased production of H2 and its subsequent utilization by the methanogens removed C02 and produced CH, at an increasing rate. This resulted in a gradual increase in CH, percentage. The Gibbs free energy changes associated with the major reactions (Table I) involved in the degradation of butyrate to CH4 and COz are shown in Figure 3f. The calculations were based on the measurements of volatile acids in the liquid and the partial pressure of the gas constituents (using eqs 1-4 at 37 "C). Prior to 11.5 h, acetate was 150 mg of COD/L, butyrate was 2 mg of COD/L, PH,was 30 ppm, and pH was 7.1, resulting in a rather unequal partitioning of the total energy (AGTot = -72 kJ/mol of butyrate) among butyrate oxidation (AGB = -12 kJ/mol of butyrate), hydrogen utilization (AHG= -5 kJ/mol of butyrate), and acetate splitting (-55 kJ/mol of butyrate). At 11.5 h, butyrate and acetate concentrations were -42 and 6900 mg of COD/L, respectively, pH was 7.05, and PH was 20 ppm. Based on these analyses, AGTotwas --85 kJ/mol, and AGB was -2.1 kJ/mol, which was the highest value for AGE during the experiment. At this time the butyrate reaction was strongly inhibited. Considering the uncertainties in the estimation of AG,, the reaction could have been thermodynamically unfavorable at the reaction sites where butyrate oxidation occurred. Other experiments, where the acetate concentration was increased gradually while AGE remained negative, showed that butyrate oxidation was inhibited and that the degree of inhibition increased with higher acetate concentrations. An example is shown later in experiment 5. Experiment 3. Reactor Response to a H2Loading. This experiment was performed to study the reactor response to H2 loading and to characterize inhibition of the butyrate reaction by H2. This experiment demonstrated that inhibition by H2 lasted through the duration of the H2 loading. The butyrate oxidation rate returned to normal following the end of the Hz loading. Prior to the experiment, the reactor was operating at a constant loading of 10 g of COD/L.day. Panels a-e of Figure 4 show the applied loadings and the experimental results. Acetate was the major constituent of the VFAs and was present at -150 mg of COD/L (Figure 4d). Butyrate concentration was
