ARTICLE Expression of merA, trxA, amoA, and hao in Continuously Cultured Nitrosomonas europaea Cells Exposed to Cadmium Sulfate Additions Tyler S. Radniecki, Lewis Semprini, Mark E. Dolan School of Chemical, Biological and Environmental Engineering, Oregon State University, 102 Gleeson Hall, Corvallis, Oregon 97331-3212; telephone: 541-231-9351; fax: 541-737-4600; e-mail: tyler.radniecki@oregonstate.edu Received 13 March 2009; revision received 19 June 2009; accepted 23 June 2009 Published online 2 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22454 ABSTRACT: The effects of CdSO 4 additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia-monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cul- tured N. europaea cells. The reactor was fed 50 mM NH þ 4 and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO 4 were made with increasing maximum concentrations ranging from 1 to 60 mM Cd 2þ . The expression of merA was highly correlated with the level of Cd 2þ within the reactor (Rs ¼ 0.90) with significant up-regulation measured at non- inhibitory Cd 2þ concentrations. Cd 2þ appears to target AMO specifically at lower concentrations and caused oxi- dative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up-regulation of trxA. Since Cd 2þ inhibition is irreversible and amoA was up-regulated in response to Cd 2þ inhibition, it is hypothe- sized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd 2þ inhibition than previously examined batch cultured cells due to the presence of Mg 2þ and Ca 2þ in the growth media, suggesting that Cd 2þ enters the cell through Mg 2þ and Ca 2þ import channels. The up-regulation of merA during exposure to non-inhibitory Cd 2þ levels indicates that merA is an excellent early warning signal for Cd 2þ inhibition. Biotechnol. Bioeng. 2009;104: 1004–1011. ß 2009 Wiley Periodicals, Inc. KEYWORDS: Nitrosomonas europaea; chemostat; CdSO4 ; merA; amoA; trxA Introduction The oxidation of ammonia to nitrite by ammonia oxidizing bacteria (AOB) in wastewater treatment plants (WWTPs) is often considered the most sensitive step in the nitrification process (U.S.EPA, 1993). Heavy metals, such as Cd 2þ , enter WWTP influents primarily from industrial discharges and urban storm water runoff (Davis et al., 2001; Sorme, 2002; U.S.EPA, 2005; Wang, 2005) and are potent inhibitors of the nitrification process (Hu et al., 2003; U.S.EPA, 1993). Cd 2þ , a representative divalent cation, has been shown to be highly inhibitory towards pure cultures of Nitrosomonas europaea, the model AOB (Chandran and Love, 2008; Park and Ely, 2008a). N. europaea is an obligate chemolithoautotroph that derives all of its energy for growth solely from the oxidation of ammonia (NH 3 ) to nitrite ðNO  2 Þ (Wood, 1986). This two-step process utilizes ammonia-monooxygenase (AMO) to oxidize NH 3 to hydroxylamine (NH 2 OH) and hydro- xylamine oxidoreductase (HAO) to oxidize NH 2 OH to NO  2 . A net gain of 2 mol of electrons is generated for every mole of NH 3 oxidized, which are shuttled further down the electron transport chain for cell growth and maintenance (Arp et al., 2002). The relatively low number of electrons generated from ammonia oxidations leads to a slow doubling time of 8–12 h even under ideal conditions (Watson et al., 1981). The sensitivity of AOB to metal toxicity and inhibition combined with the slow growth rate can result in failures in the nitrification process. The early detection of Cd 2þ -mediated nitrification inhibition in AOB may be useful in preventing and minimizing nitrification failures at WWTPs. In previous batch studies, the expression of merA,a mercury resistance protein, and trxA, an oxidative stress protein, were found to be up-regulated in batch grown N. europaea cells and short time exposures to Cd 2þ (Park and Ely, 2008a). This study builds on that work by determining Correspondence to: T.S. Radniecki Contract grant sponsor: National Science Foundation’s Division of Bioengineering and Environmental Systems Genome-Enabled Environmental Sciences and Engineering Program 1004 Biotechnology and Bioengineering, Vol. 104, No. 5, December 1, 2009 ß 2009 Wiley Periodicals, Inc.