Anaerobic biodegradation of crotonic acid by a bacterial population specialized in the degradation of butyric acid
Volatile Fatty Acids (VFAs) are intermediate metabolites formed in the anaerobic biodegradation of organic matter. They are commonly found in sewage, municipal sanitary landfill leachate and effluents from agricultural and food-processing industries. A good knowledge of the microorganisms involved in VFA biodegradation is necessary to operate satisfactory biotreatment of those effluents. The objective of the present study is to better understand the metabolism of the anaerobic bacteria responsible for the degradation of butyric acid and one of its metabolites (crotonic acid), which is still poorly known. Syntrophomonas wolfei is one of the few butyrate-degrading acetogenic bacteria that bas been documented. First studios have shown that this microorganism is not capable of degrading crotonic acid (MCINERNEY et al., 1979, 1981). This is surprising since crotonyl-Coenzyme A, in its activated form, is an intermediate metabolite of n-butyrate ß-oxidation, which is the most common mechanism of butyrate biodegradation. In addition, ß-oxidatlon of crotonate is thermodynamically possible, even under standard conditions. These observations are al the origin of the present study, which investigates the anaerobic biodegradation of crotonate. Other Investigators have followed a similar approach and isolated S. wolfei in pure culture on crotonate. The degradation of crotonate was studied in a bench-scale up-flow anaerobic filter of twenty liters, operated in the dark, at 35 °C. A first set of experiments was carried out with a biomass exclusively adapted to the biodegradation of butyrate. Heat-expansed vermiculite was used as a packing medium. Various experimental protocols were successive followed. First, pulses of crotonate were injected into the reactor under conditions of continuous feeding with butyrate, and then, the reactor was continuously fed with crotonate. The objective was to determine whether a bacterial population exclusively adapted to butyrate biodegradation would be capable of degrading crotonate. It was found that crotonate was actually biodegraded in the reactor. Woth the first protocol, when pulses of crotonate were injected into the reactor, crotonate was totally removed in 55 hours (fig. 3). Butyrate and acetate concentrations increased as crotonate was degraded, but no significant increase in biogas production was observed. On the other hand, under the same conditions, it was found that iso-butyrate was not degraded, which is consistent with other published data (MCINERNEY et al., 1979, 1981 ; STIEB and SCHINK, 1985,1989). With the second protocol (continuous feeding with crotonate at 5.2 gg/l), crotonate was totally biodegraded in 48 hours after a 24 hours lag period. This biodegradation resulted in the accumulation of acetate and, in a lower extend, butyrate (fig.4). Following this stage, the reactor was fed with a higher crotonate concentration (12 g/l), and it was observed that crotonate was totally degraded in 20 hours, without any lag period (fig. 5). These results showed that butyrate-degrading bacteria were capable of degrading crotonate effectively after a short period of adaptation. Further experiments were conducted with a biomass previously adapted to the degradation of a mixture of VFAs (acetate, propionate, iso-butyrate, butyrate and caproate). Berl saddles were used as a support for bacterial growth. The reactor was operated in a recirculated batch mode and spiked with crotonate. Finally, the reactor was successively fed for four weeks with propionate and for two weeks with butyrate, before being spiked with crotonate. In all these experiments, crotonate biodegradation was observed, but, in contrast to the results obtained with the “vermiculite reactor”, no butyrate accumulation occured (fig.6). These results show that a bacterial population adapted to the degradation of a mixture of VFAs or to the degradation of individual VFAs such as propionate and n-butyrate, is capable of degrading crotonate. Based on the present study and on literature data, the following mechanism can be proposed for the biodegradation of crotonate (fig.7). The first stage is the activation of crotonate into crotonyl-Coenzyme A by an acetyl-CoA/crotonyl-CoA transferase, as recently isolated from S. wolfei (BEATY and MCINERNEY, 1987). When present at low concentrations, crotonate is probably directly degraded into acetate, as shown by the results obtained with the “selles de Berl reactor”, in which no intermediate metabolite has been detected. At higher concentrations, enzymatic sites may be saturated and an equilibrium be established with butyrate, which is then released into the medium. This has been shown by the accumulation of butyrate under conditions of continuous feeding with crotonate. In addition, another intermediate metabolite has been formed, which has not been identified in the present study. This product is most probably poly-ß-hydroxy-butyrate, which has been found in S.wolfei (MCINERNEY et al, 1979) although if is not very common in chemiotrophic bacteria.