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Chlorinated Aliphatic Hydrocarbons (CAHs) are common contaminants of groundwaters and industrial wastewaters. Several lab- and field-scale studies 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 tetrachloroethylene. In this study, a process of chloroform (CF) aerobic cometabolic biodegradation by a butane-growing consortium was investigated for 354 days in a 2-m continuous-flow sand-filled reactor. The study was aimed at a) investigating the oxygen/substrate pulsed injection technique as a tool to control the clogging of the porous medium, to attain a long bioreactive zone and to reduce substrate inhibition on CF cometabolism; b) identifying guidelines for optimizing the oxygen/substrate supply schedule; c) developing a reliable model of CF cometabolism in porous media. While the continuous supply of butane rapidly led to the clogging of the porous medium due to excessive biomass growth near the reactor inlet, the testing of six types of oxygen/substrate pulsed feeding led to the identification of a feeding schedule capable to prevent aquifer clogging and to lead to the development of a long bioreactive zone and to satisfactory CF degradation performances (CF removal = 82%; CF degradation rate = 0.035 mg Lreactor d-1; ratio of CF degraded to butane consumed (CF/B) = 0.27 mg mg-1). The tested model of aerobic cometabolism in porous media led to a suitable interpretation of the experimental data (average percent error = 19%) as long as the CF/B ratio was ≤ 0.27 mg mg-1. A long-term simulation of the best-performing schedule of pulsed oxygen/substrate supply indicated the attainment of a steady state condition characterized by lower bioremediation performances than those obtained experimentally, evidencing the need for a further, model-based optimization of the pulsed injection technique.

La biodegradazione aerobica cometabolica di cloroformio (CF) tramite microrganismi autoctoni butanotrofi è stata studiata in una colonna a flusso riempita di sabbia. Gli obiettivi sono stati: i) mettere a punto un‘alimentazione pulsata per evitare l’intasamento delle macroporosità del terreno, ii) creare e mantenere un’ampia zona bioreattiva nella colonna, iii) massimizzare la rimozione di CF tramite differenti modalità di conduzione. E’ stata testata la bio-sostenibiltà del processo con rapporti variabili di ossigeno alimentato su butano alimentato (1.06-20 mg ossigeno/mg butano) e di butano consumato su cloroformio degradato (1.03-17 mg butano/mg CF). Dopo 5 mesi di conduzione non si è verificato intasamento del mezzo poroso, è stata ottenuta una distribuzione della biomassa uniforme lungo la colonna, e l’abbattimento di cloroformio è risultato compreso tra il 50% e l’80%. I risultati sperimentali sono stati interpretati con un modello fluidodinamico accoppiato ad un modello cinetico aerobico cometabolico precedentemente testato.

CF aerobic cometabolic biodegradation by the indigenous biomass of a sandy aquifer was studied in slurry microcosms and in a 2-m continuous-flow reactor simulating a portion of saturated aquifer. Butane was utilized as the growth substrate. The slurry microcosms tests allowed to estimate the lag-times for the onset of butane utilization and CF degradation under different experimental conditions. The CF lag-time proved to be strongly influenced by temperature and by the condition of biomass growth on butane. A 2-month operation period of the flow reactor resulted in the onset of CF cometabolism after about 1 month, and in an average CF removal equal to 50%. As a result of the successful implementation of a pulsed injection of butane and oxygen, biomass growth resulted quite uniform throughout the column, and no sign of pore clogging due to biomass growth was detected.

An aerobic cometabolic CF degradation process by butane-grown biomasses was studied by means of slurry microcosm tests. The lag-phase for the onset of butane utilization by the indigenous biomass of the studied sandy soil was lower than 2 weeks in all the experimental conditions tested. The lag-time for the onset of CF depletion was strongly affected by temperature, with no CF degradation after several weeks in the tests conducted at 15°C. Bioaugmentation treatments performed with 2 types of microbial inocula led, even at the smallest dose tested (0.15 grams of dry cell / m^3 of aq. phase), to a marked decrease of the butane lag-time. Both the initial CF degradation rate and the amount of CF depleted in the absence of butane proved to depend on the type of inoculum utilized, rather than on its dose. The minimum butane/CF molar ratio required to carry out a sustainable CF degradation process proved to be comprised between 1.6 and 14.

In this microcosm study we have monitored the performances of 12 butane-utilizing consortia during a long-term aerobic cometabolic degradation of chloroform (CF). After approximately 100 days of continuous CF depletion, a sudden improvement of the biodegradative performances (7-fold increase of chloroform degradation rate) was observed in two microcosms in which the highest amount of depleted CF (> 70 mg / kg of dry soil) was observed. Two bacterial strains, isolated from the CF-degrading microcosms, were identified as belonging to the genera Rhodococcus and Stenotrophomonas, respectively. A strain, named F, was further characterized to establish the involvement of the butane-monooxygenase in CF degradation.

The effect of several operational conditions on the adaptation of indigenous biomasses was investigated in a microcosms study of chloroform (CF) cometabolic degradation with butane. The objective was to minimize the duration of the adaptation phase and the amount of growth substrate to provide in field applications. The adaptation to butane and CF was monitored at 5 concentrations of butane (1.50, 1.00, 0.50, 0.25 and 0.10 mg/L), at 3 concentrations of CF (1, 5 and 10 mg/L), at 4 temporal sequences of exposition to butane (continuous, 5 h/d, 7 h/d, 24 h every other day) and at 3 temperatures (15, 25 and 30°C). The results indicate that the most favorable condition for the adaptation of indigenous biomasses consists in the continuous exposition to a very low concentration of growth substrate (0.1 mg/L). In all the tests the degradation of CF started within 48 days. The microbiological characterization of the consortia indicated that the prolonged degradation of CF led to the selection of 3 specific butane-utilizing strains.

Chlorinated solvents are toxic and poorly biodegradable pollutants frequently found in contaminated aquifers. Experimental data of chloroform (CF) aerobic cometabolic biodegradation in a sand column with butane as growth substrate were simulated with a system of non‐stationary second‐order partial differential equations with non‐linear kinetic terms. A MATLAB optimization code based on the Gauss‐Newton method and coupled with the Comsol Multiphysics finite elements solver was developed to calibrate the model. For each experimental phase, the best‐fit quality was evaluated by an innovative multi‐variable model adequacy test. The proposed code solved systems of up to 5 partial differential equations and optimized up to 6 unknown parameters, leading to statistically acceptable best‐fits. The optimization of the butane/oxygen pulsed feed led to an 82 % CF biodegradation and to a 0.27 gCF/gbutane transformation yield. When the substrate/pollutant ratio was minimized, the standard model of aerobic cometabolism initially tested required additional terms aimed at taking into account the depletion of reducing energy, in order to attain a statistically acceptable best‐fit. This is the first work in which a model of aerobic cometabolism taking into account reducing energy availability was applied to a continuous‐flow process. The proposed optimization code can be used for model calibration in a wide range of physical problems described by non‐stationary, non‐linear partial differential equations, a task that no commercial software can perform. The developed code is made available in the Supplementary Material.