Earlier work has found that Methylocystis strain SB2 can indeed degrade chlorinated ethenes via pMMO activity when grown on acetate (Yoon et al., 2011). Here, we extend these findings to show that Methylocystis strain SB2 can also degrade a variety of chlorinated alkanes and alkenes when grown on either methane or ethanol via pMMO activity. Methylocystis strain SB2 was initially grown at 30 °C in 200 mL of nitrate mineral salt medium (Whittenbury et al., 1970) in 2 L Erlenmeyer flasks shaken at 225 r.p.m. in a methane-to-air ratio of 1 : 2
at 1 atm of pressure. Copper (10 μM) was added as CuCl2. Highest-purity methane (99.99%) and acetylene (99.6%) were obtained from Airgas Great Lakes (Lansing, MI). Ethanol (>99.5%), trichloroethylene (TCE, >99.5%), trans-dichloroethylene (t-DCE, >98%), and dichloromethane (DCM, >99.5%) were purchased from Fisher Scientific (Fair Lawn, NJ). t-DCE Lenvatinib nmr (>98%), vinyl chloride (VC, >99.5%), 1,1,1-trichloroethane (1,1,1-TCA, >99.5%), and chloroform
(CF, >99%) were purchased from Aldrich (Milwaukee, WI). Sodium formate (>99%, ACS grade) was purchased from Alfa Aesar (Ward Hill, MA). For chlorinated hydrocarbons that MI-503 purchase are liquid at room temperature (i.e. TCE, DCM, 1,1,1-TCA, and CF), saturated stock solutions were prepared according to a method developed by Chang & Alvarez-Cohen (1996). Aliquots were taken using Hamilton 1700 series gas-tight syringes (Hamilton, Reno, NV) with care taken to exclude any non-aqueous-phase liquids. For methane, acetylene, and VC, which are gaseous at room temperature, aliquots were added to vials using Precision Lok gas-tight syringes
(Precision Sampling Corp., Baton Rouge, LA). Each chlorinated compound was injected at an initial concentration of 40 μM. The amount added was calculated using the following dimensionless Henry’s constants: VC, 1.262 (Morel & Hering, 1991); TCE, 0.458; t-DCE, 0.474; 1,1,1-TCA, 0.804; DCM, 0.125 (Tse et al., 1992); and CF, 0.189 (Gossett, 1987). Distilled deionized water (>18 MΩ cm) from a Corning Millipore D2 system was used for all experimental setups. All glassware was washed with detergent and then soaked in 2 N HNO3 overnight Selleck ZD1839 to remove trace metals, including copper. Nitric acid was subsequently removed by repeated rinses with distilled deionized water. The growth rates of Methylocystis strain SB2 in the presence of different chlorinated hydrocarbons were measured using the procedure described previously by Lee et al. (2006). Briefly, the cells were grown to the mid-exponential phase on methane at 30 °C [OD600 nm of 0.3–0.4 as measured using a Spectronic 20 spectrophotometer (Milton Roy Company)]. Before transferring for growth on either ethanol or methane, strain SB2 was harvested by centrifuging 100 mL of the culture at 2160 g for 5 min, and washed twice with a fresh nitrate mineral salt (NMS) medium to remove residual methane.