【文章內(nèi)容簡介】 。 F, UASB reactor。 G, granular sludge blanket。 H, waste bottle。 1 and 2, sample ports 1 and 2. Source of inoculum. The granular sludge was taken from a lab scale UASB reactor fed PCE and ethanol for 1 year (4). Originally, the granules came from a fullscale UASB reactor treating paper mill effluent. The granules were stored at 4176。C for approximately 2 months prior to use. The test and reference 2 (R2) reactors were filled with 20 ml of wet granules, gassed with N2CO2 (4:1), and filled with anaerobic medium. The reference 1 (R1) reactor was also filled with 20 ml of wet granules but was autoclaved three times at 140176。C before being gassed with sterile N2CO2 (4:1) and filled with anaerobic medium. The test and R1 reactors, containing living and autoclaved granules, respectively, were further inoculated with 50 ml of a pure culture of D. multivorans, kindly provided by H. ScholzMuramatsu (Institute for Sanitary Engineering, Department of Biology, University of Stuttgart, Stuttgart, Germany) and allowed to acclimate for 3 days before feeding of the reactors was initiated. As an abiotic control, a 118ml serum vial was filled with 5 ml of wet granules and 50 ml of anaerobic medium. The bottle was gassed (with N2CO2 [4:1]), autoclaved three times, and supplemented with formate and acetate to final concentrations equivalent to those in the reactor system. PCE was added as an aqueous sterile solution. The batch was tested for dechlorination activity every 4 to 5 days during the experimental period. Sterility check. The sterility of the autoclaved reactor inoculated with D. multivorans was checked daily, and that of the sterile control bottle was checked every 4 to 5 days, by lightmicroscopic inspection of a liquid sample. Medium. A basal medium was prepared as previously described (12), except that mg of resazurin/liter, g of Na2S 7H2O to 9H2O/liter, mM acetate, and 2 mM formate were added from anaerobic stock solutions. The final pH of the medium ranged from to . Immunological methods. Granules were sampled from each reactor after reactors were operated at an HRT of h. The granules and inoculum were fixed in 4% formalin, embedded in paraffin, cut into 5181。m slices, and placed on glass slides. Paraffin was removed with xylene, and the granules were hydrated in decreasing concentrations of ethanol. Polyclonal rabbit antiserum against D. multivorans labeled with FLUOS (56carboxyfluoresceinNhydroxysuccinimide ester) was obtained from the Institute for Sanitary Engineering, Germany. The hydrated granules were incubated with normal rabbit serum, washed with bovine serum albumin in phosphatebuffered saline (pH ), and incubated with the antiserum for 25 min. As a control, granules were prepared without antiserum incubation. The slides were investigated by immunofluorescence microscopy. Analytical methods. PCE, trichloroethene (TCE), and dichloroethene (DCE) were measured with a mass spectrometer (MS QMG 4211。 Balzers, Liechtenstein) for membrane inlet mass spectrometry (MIMS) (11). PCE, TCE, and DCE were analyzed at masstocharge ratios (m/z) of , , and atomic mass units, respectively. The detection limit for the MIMS is 1 181。g/liter (11a). The range of concentrations tested was 1 to 500 181。M for PCE, TCE, and DCE, corresponding to the range of the standards. Standards were prepared by weighing exact amounts of chloroethenes into doubledistilled water, stirring overnight for plete mixing, and diluting into serum bottles. The liquid phase was then measured. In order to detect unknowns, reactor liquid was pumped from the reactor through the MIMS and discarded, by using viton tubing. The same flow rate was used as that for measuring the standards (60 ml/h). Formate was analyzed by highpressure liquid chromatography (Lambda Max 410。 Waters). An acidified sample (20 181。l) was injected into an Aminex HPX87H column heated to 60176。C. The mobile phase was N sulfuric acid with a flow rate of ml/min. Formate was detected by UV detection at 190 nm. Standards were tested in a range from to 10 mM formate. The detection limit was mM. Samples and standards were acidified to below pH 2 with 20 181。l of sulfuric acid (10%) and were centrifuged. The supernatant was filtered (45181。m poresize filter。 Millipore) and injected. Acetate was measured by gas chromatography as previously described (14). The stereoisomeric position of DCE was determined by gas chromatography. Chloroethenes were extracted by the addition of 150 181。l of pentane containing mg of bromotrichloromethane/liter as the internal standard for a sample. Three microliters of the pentane phase was then injected into a gas chromatograph (HRGC 5300 Mega series。 Carlo Erba Instruments, Milan, Italy) connected to an electron capture detector. The detection limit was 500 181。g of cis1,2DCE/liter. Standards were prepared in the range from 500 to 2,540 181。g/liter. Unknowns were determined by using a liquid sample from the reactor known not to contain any DCE. RESULTS Reactor studies. The test reactor inoculated with D. multivorans transformed an average of 96% of the ining PCE, up to a PCE loading rate of mmol liter of sludge 1 day 1 (Fig. 2). The main product of the reactor was DCE, averaging 93% (mole/mole) of effluent chloroethenes (Fig. 3a). DCE effluent concentrations sometimes exceeded the PCE influent concentration. Only small amounts of PCE and TCE (5 181。M) could occasionally be detected in the effluent. Samples taken at day 61 showed that the stereoisomeric position was 100% cis1,2DCE. The conversion of PCE to DCE was almost equimolar at different HRTs. However, when the HRT was reduced from to h, the PCE effluent concentration increased to a maximum of 25% (mole/mole) of the sum of chloroethenes in the effluent. Nine days after the HRT was reduced to h, PCE dro