Chloroform Aerobic Cometabolic Biodegardation in a Continuous-Flow Reactor

Chlorinated Aliphatic Hydrocarbons (CAHs) are common contaminants of groundwaters and industrial wastewaters. Lab-scale assays and field pilot tests showed that aerobic cometabolism with aliphatic and aromatic hydrocarbons as growth substrates can lead to the rapid and complete dechlorination of a wide range of CAHs, including high-chlorinated compounds such as 1,1,2,2-tetrachloroethane. Aerobic cometabolism can therefore be regarded as a promising technology for the treatment of CAH-contaminated sites and wastewaters. However, with specific regard to the implementation of this technology for the remediation of CAH-contaminated sites, several issues still need to be addressed. Of these, two deserve particular mention: i) it is important to avoid the risk of a complete consumption of the supplied growth substrate within a short distance from the injection wells: in other words, the bioreactive zone must be sufficiently long to allow the complete biodegradation of the target contaminants; ii) aquifer clogging near the injection well can occur as a result of an excessive biomass growth on the growth substrate supplied. The supply of alternated pulses of growth substrate and oxygen represents an interesting solution, potentially effective both in the creation of a long bioreactive zone and in the control of aquifer clogging: as a result of hydrodynamic dispersion and substrate sorption, the over-lapping of substrate and oxygen occurs at low concentration, over a wide aquifer portion and, in each point, in a discontinuous way. This study, focused on chloroform (CF) cometabolism by butane-grown bacteria, was conducted in a 2-m continuous-flow column reactor simulating a portion of saturated aquifer. The main goals were: a) to investigate the pulsed injection of growth substrate and oxygen as a tool to control clogging of the porous medium and to attain a wide bioreactive zone; b) to determine the minimum substrate/CAH ratio required to sustain the cometabolic process; and c) to determine the most suitable kinetic and fluid-dynamic model to fit the experimental data of butane utilization and CF cometabolism. A preliminary group of fluid-dynamic tests was aimed at determining the main fluid-dynamic parameters required for the modeling of the process: the reactor effective porosity n_eff and longitudinal dispersivity a_L, and the retardation factors R relative to butane and CF (assuming R = 1 for oxygen). These tests consisted of pulses of only oxygen (with no butane or CF in the column), only butane or only CF. For each compound, different pulses at different interstitial velocities were operated. The results, elaborated by means of a PDE solver (Comsol Multiphysics) yielded the following best-estimates: n_eff = 50%, a_L= 0.35 mm, R_butane = 1.08, R_CF = 1. A kinetic model of aerobic cometabolism, together with a set of kinetic parameters, was derived from a previous batch study conducted with the Rhodococcus bacterial strain that resulted the prevailing species in the soil utilized in this work, after a long period of butane utilization and CF degradation (Frascari et al., Appl. Microbiol. Biotechnol. 73 (2006) 421-428). The complete fluid-dynamic/kinetic model was utilized to run a series of simulations solved with Comsol Multiphysics, with the goal to design three types of injection of alternated pulses of growth substrate (butane) and oxygen, characterized by different values of the ratio of butane utilized to CF degraded (B/CF ratio). Indeed, in a previous batch study conducted with the same soil utilized in this study (Frascari et al., Process. Biochem. 42 (2007) 1218-1228), the minimum B/CF ratio required to sustain the cometabolic process resulted equal to 1.6 mg_B/mg_CF. In a process of CAH cometabolism, the supply of growth substrate is necessary not only to re-produce or re-activate the biomass killed or inactivated by the toxic CAH degradation products, but also to produce the reducing energy (NADH) consumed but not re-generated by the CAH transformation process. Therefore, the above-mentioned three types of butane and oxygen pulsed injection were designed to attain, in comparison with the minimum B/CF ratio estimated in the batch tests, a first ratio largely higher (17 mg_B/mg_CF), a second ratio slightly lower (1.0 mg_B/mg_CF) and a third ratio slightly higher (2.3 mg_B/mg_CF), so as to validate in a continuous-flow saturated reactor the estimate of the B/CF ratio obtained by means of batch slurry assays. The column reactor was then run for three consecutive periods, characterized by the three above-mentioned pulsed injections. In these tests, the interstitial velocity was set to 0.5 m/d (corresponding to a 2-day hydraulic retention time), and the CF inlet concentration to about 0.4 mg/L. The first phase (butane consumed / CF degraded = 17, corresponding to a ratio of butane supplied / CF supplied = 27) was run for 35 days, and yielded and average CF removal equal to 50%. The cometabolic process proved sustainable under these conditions. The designed schedule of pulsed injection (butane pulse: 21 mg/L, 7.5 h; 3 h without butane or oxygen; oxygen pulse: 20 mg/L, 11 h; 2.5 h without butane or oxygen) allowed the development of a bioreactive zone over the entire length of the column (2 m) and prevented any measurable clogging of the porous medium. The second phase (butane consumed / CF degraded = 1.0, corresponding to a ratio of butane supplied / CF supplied = 2.4) was run for 46 days. The corresponding sequence of butane and oxygen pulsed injection was based on a 3.5-day cycle. During the first 20 days the average CF removal was equal to 80%, and the cometabolic process appeared sustainable. A typical representation of the concentration profiles of butane, oxygen and CF over the column during this period is provided in Figure 1, together with the corresponding model simulation performed with the kinetic parameters derived from the single-strain kinetic study. It can be observed that, at a given instant, CF cometabolic degradation occurred in the reactor zones characterized  - as a result of the pulsed injection - by the presence of oxygen. Due to convection and dispersion, these zones shifted through the reactor. We therefore obtained a CF outlet concentration characterized by a fluctuating trend. However, after 20 days, the CF degradation rate decreased and the cometabolic process rapidly halted. The most likely interpretation for this event is that, as a result of the excessively low ratio of butane consumed to CF degraded, the cellular storage of NADH was progressively consumed. A model interpretation of this hypothesis, based on the work of Chang and Alvarez-Cohen (Environ. Sci. Technol. 29 (1995) 2357-2367) is in progress. The third phase (butane consumed / CF degraded = 2.3, corresponding to a ratio of butane supplied / CF supplied = 2.0) was run for 49 days. The corresponding sequence of butane and oxygen pulsed injection was based on a 3.5-day cycle. Under these conditions, the cometabolic process resumed rapidly and remained stable over time, with an average 80% CF removal. No sign of aquifer clogging was observed. In conclusion, the experimental and modeling results show that: a) the pulsed injection of growth substrate and oxygen is an effective tool to prevent aquifer clogging as a result of an excessive biomass growth, and to attain a long bioreactive zone; b) in the specific case of our process of CF cometabolism with butane, the minimum ratio of substrate utilized / CAH degraded ranges between 1.6 and 2, with a good agreement between the results obtained in batch slurry assays and those deriving from the column tests; the corresponding ratio of substrate supplied / CAH supplied depends on several factors, among which the number of substrate and oxygen pulses in each cycle plays an important role; c) the kinetic parameters previously estimated in batch, single-strain assays, combined with the fluid-dynamic parameters evaluated in the first part of this work, allowed the development of an effective modeling tool for the design of the pulsed injection and for the interpretation of the experimental data. Overall, this work provides encouraging indications on the successful application of aerobic cometabolism for the in-situ remediation of sites contaminated by a wide range of CAHs.