Long-Term Performance of Bioreactors Cleaning Mercury-Contaminated Wastewater and Their Response to Temperature and Mercury Stress and Mechanical Perturbation H. von Canstein, 1,* Y. Li, 2 I. Wagner-Do ¨ bler 1 1 Division of Microbiology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany; telephone: 49-531-618-1406; fax: 49-531-618-1411; e-mail: hfc@gbf.de 2 Division of Biochemical Engineering, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany Received 12 August 2000; accepted 18 February 2001 Abstract: The long-term performance of bioreactors re- taining mercury from contaminated industrial wastewa- ter was analyzed at the laboratory scale, and its response to mechanical perturbations (gas bubbles and shaking) as well as to physical (increased temperature and hy- draulic load) and chemical stresses (increased mercury concentration) likely to occur during on site operation was studied. Two packed-bed bioreactors with 80-cm 3 lava chips as biofilm carrier were inoculated with nine Hg(II)-resistant natural isolates of - and -proteobacte- ria. Chloralkali wastewater containing ionic mercury (3.0 to 9.7 mg/L Hg 2+ ), amended with sucrose and yeast ex- tract, flowed through the bioreactors at 160 mL/h. During the 16-month investigation the bioreactors showed no sign of depleted performance in terms of mercury- retaining capacity. After 16 months, both bioreactors still retained 96% of the mercury load. The performance of the bioreactors was sensitive to mechanical perturba- tions (e.g., sheer forces of gas bubbles). Shifts to higher Hg 2+ inflow concentrations initially decreased the mer- cury retention efficacy slightly. However, the bioreactors could adapt to Hg 2+ concentrations of up to 7.6 mg/L within several days. Old biofilms were less affected than the younger ones. The performance of the bioreactors was not affected by an increase in temperature up to 41°C and an increased volumetric load (up to 240 mL/h). The bioreactors regained activity spontaneously after the stress had stopped. Recovery could be accelerated by increased nutrient concentration, although this may lead to blocking of the packed bed. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 212–219, 2001. Keywords: bioreactors; mercury-contaminated wastewa- ter; mercury stress; mechanical perturbation INTRODUCTION Mercury and mercury compounds are extremely toxic (Langford and Ferner, 1999), yet they continue to be used widely in industry. Industrial dumping of mercury into riv- ers, the combustion of coal, and solid waste incineration have led to significant mercury pollution of the environment (Bryan and Langston, 1992; Zilloux et al., 1993). The effi- cient elimination of the dilute mercury from large volumes of contaminated waste streams (e.g., chloralkali electrolysis wastewater) by conventional physical or chemical methods is technically difficult and expensive. Therefore, a biotech- nological approach was pursued, which is based on the ac- tive enzymatic reduction of ionic mercury (Hg 2+ ) to metallic mercury (Hg 0 ), a transformation carried out by mercury- resistant microorganisms as a detoxification mechanism (Izaki et al., 1974; Robinson and Tuovinen, 1984; Summers, 1986). It is encoded by the microbial mer operon, and in- volves regulatory proteins, transport proteins, and the en- zyme mercuric reductase, encoded by the merA gene (Hob- man and Brown, 1997; Silver and Misra, 1988; Silver and Phung, 1996). The removal of mercury from wastewater by Hg(II)- reducing biofilms was first demonstrated by Brunke et al. (1993) using synthetic Hg(II) solutions. We showed that this process is also applicable to industrial wastewater of the chloralkali industry (von Canstein et al., 1999). The process took place in laboratory-scale packed-bed bioreactors, where the bacteria were immobilized as active biofilms on carrier chips and fed continuously with wastewater amended with carbon sources, because mercury reduction is a process that consumes metabolic energy. The bacteria import the ionic mercury into the cytoplasm in an active manner. Subsequently, the mercury is reduced by the cyto- solic enzyme mercuric reductase to Hg(0), whereby Nico- tinamide Adenine Dinucleotide Phosphate (NADPH) is consumed (Silver and Misra, 1988). The metallic mercury atoms diffuse out of the cells where they stick together, resulting in steadily increasing mercury droplets of several microns in diameter, which remain in the matrix of the bioreactor (Brunke et al., 1993). Previous fixed-bed column experiments have focused on the performance of communities and pure cultures of natu- Correspondence to: H. von Canstein * Present address: German Research Centre for Biotechnology. Dept. Microbiology, Mascheroder Weg 1, D-38124 Braunschweig. Phone +49- 531-6181406; fax +49-531-6181411; email hfc@gbf.de Contract grant sponsor: European Union’s LIFE Program © 2001 John Wiley & Sons, Inc.