A continuous fibrous-bed bioreactor for BTEX biodegradation by a co-culture of Pseudomonas putida and Pseudomonas fluorescens

Abstract

A co-culture of Pseudomonas putida and P. fluorescens immobilized in a fibrous-bed bioreactor was used to degrade benzene, toluene, ethylbenzene and xylenes (collectively known as BTEX), present as sole carbon sources in contaminated water. The kinetics of BTEX biodegradation in the fibrous-bed bioreactor operated under the liquid-continuous condition was studied. Biodegradation rates of BTEX increased with increasing BTEX concentration and reactor loading rate. For benzene, the maximum biodegradation rate was 38 mg/l/h at a loading rate of 265 mg/l/h. For toluene, the rate was 45 mg/l/h at a 100 mg/l/h loading rate. Aeration was not used in the process and the addition of hydrogen peroxide (H₂O₂) as an additional oxygen source improved benzene and toluene biodegradation for the high strength synthetic wastewater feeds. When benzene, toluene, ethylbenzene and para-xylene were present as a mixture in the feed, they were concurrently and completely biodegraded under hypoxic conditions (no addition of air or H₂O₂). The total BTEX biodegradation rate was as high as 600 mg/l/h at the highest BTEX loading rate, 1000 mg/l/h, studied. Individual BTEX compounds were efficiently and concurrently degraded at a retention time of less than 15 h. Immobilized cells adapted in the bioreactor showed no preferential degradation of BTEX present as mixtures. The bioreactor also had a stable long-term performance, maintaining its ability for efficient BTEX degradation without requiring additional nutrients (e.g. glucose) for more than 1 year. The good performance of the fibrous-bed bioreactor was attributed to the high cell density and unique cell immobilization process provided by the fibrous matrix, which allowed use of the reactor for continued regeneration, adaptation and selection of efficient BTEX degraders in the bioreactor environment.

Introduction

Benzene, toluene, ethylbenzene and xylenes, collectively known as BTEX, are the major aromatic components in petroleum. BTEX are widely used in industry as solvents for organic synthesis, equipment cleansing and other downstream processing purposes. They are frequently found in groundwater as a result of leaks in underground storage tanks and pipelines, improper waste disposal practices, inadvertent spills and leaching from landfills. BTEX are classified as priority pollutants regulated by the U.S. Environmental Protection Agency (EPA) and were among the target compounds in EPA's 33-50 program. Benzene is teratogenic and may be associated with the development of leukemia, and toluene is a suspected depressant of the central nervous system. Because of these health concerns, a maximum contaminant level of 5 μg/l for benzene is set as the standard for drinking water by USEPA. The U.S. Public Health Service (1989) has also recommended that drinking water contain no more than 2 mg/l of toluene for lifetime exposure.

Among all remediation technologies for treating BTEX-contaminated water, bioremediation appears to be an economical, energy efficient and environmentally sound approach. Microorganisms are able to degrade BTEX under aerobic, microaerobic or hypoxic, as well as anaerobic conditions (Chaudhuri and Wiesmann, 1996, Dararat and Riffat, 1999, Deeb and Alvarez-Cohen, 1999, Deeb and Alvarez-Cohen, 2000, Dyreborg et al., 1996, Gersberg et al., 1995, Kukor and Olsen, 1996, Langenhoff et al., 1996, Mikesell et al., 1994, Wilson and Bouwer, 1997). Biodegradation of BTEX has been studied extensively, both in the subsurface (soil and groundwater) environments (Eganhouse et al., 1996, Fan and Scow, 1993, Gibson et al., 1998, Meier-Lohr et al., 1998, Morgan et al., 1993, Williams et al., 1997) and in bioreactors (Choi et al., 1992, Goudar et al., 2000, Lu et al., 2000, Mallakin and Ward, 1996, Mason et al., 2000, Neufeld et al., 1994). In general, aerobic biodegradation is considered much faster than anaerobic processes. However, aerobic treatment processes usually result in losses of the volatile organic compounds to air, rather than complete biodegradation, and hypoxic and anaerobic conditions often exist in natural ecosystems, which greatly limit the efficiency of bioremediation.

Most prior BTEX biodegradation studies used either bacterial consortia from sewage sludge or indigenous soil and groundwater microorganisms. Several pure cultures have also been studied, but they usually could not degrade all BTEX compounds simultaneously and efficiently (Baggi et al., 1987, Chen and Taylor, 1995, Cruden et al., 1992, Deeb and Alvarez-Cohen, 1999, Keener and Arp, 1994, Kitayama et al., 1996, Löser and Ray, 1994, Paje and Couperwhite, 1996, Utkin et al., 1992). Recently, several genetically engineered strains have been cloned to enhance their abilities to degrade all BTEX compounds (Bertoni et al., 1996, Lee et al., 1994, Lee et al., 1995a). However, no work has been done with a defined co-culture of Pseudomonas putida and P. fluorescens immobilized, degrading all BTEX compounds efficiently and concurrently under hypoxic conditions from synthetic wastewater containing high concentrations of BTEX.

The main objective of this research was to develop an efficient bioreactor for treating BTEX contaminated water. In general, immobilized cell systems are considered better than free cell systems, in terms of biodegradation rate and operating costs. However, conventional immobilized cell systems usually suffer from problems such as: high pressure drop; flow channeling and plugging; and loss of microbial activity over time. A fibrous-bed bioreactor has been developed to overcome these problems and is successfully used in several fermentations with improved reaction rates, concentration tolerance and long-term stability (Silva and Yang, 1995, Yang et al., 1994). The ability of the fibrous-bed bioreactor to continuously renew the cell population and adapt cells to the reactor environment, as demonstrated in our previous fermentation studies (Huang and Yang, 1998), would be advantageous to the process treating water containing toxic BTEX compounds.

In this work, BTEX biodegradation by the co-culture of P. putida and P. fluorescens immobilized in the fibrous-bed bioreactor was studied under liquid-continuous conditions. Both Pseudomonas species are known aerobes, but P. fluorescens can also use other electron acceptors such as nitrate under oxygen-limited conditions. In this study, no aeration was applied to the bioreactor system to eliminate potential evaporative losses of BTEX. The effects of: hydrogen peroxide (H₂O₂) as a supplemental oxygen source; substrate concentration; single substrate vs. multiple substrates; and co-metabolism with glucose were studied. The long-term stability of the fibrous-bed bioreactor for remediating BTEX-contaminated water was also studied and is discussed.