General

[quote=Fajsak]...What? xD Should this really be in the fighter section?[/quote]

Yep. That is truly what the monkey said.

...What? xD Should this really be in the fighter section?

That said: "To protect the plant from a variety of chemical shocks but there is no known process to specifically protect reactors from chloroaniline (CA) shocks. CAs are released during the production of polyurethanes, rubber, azo-dyes, drugs, photographic chemicals, varnishes, and pesticides. The effect of CA on activated-sludge microbial communities is not known, but the nonchlorinated compound aniline inhibits nitrification. We hypothesized that 3-CA shock loads in wastewater treatment plants will have a negative impact on wastewater treatment processes. The spontaneous removal of aromatic pollutants proceeds slowly and, in reactors, is not rapid enough to prevent reactor upset. In our previous work, a 3-CA-degrading strain, Comamonas testosteroni I2, was isolated from activated sludge, marked with the gfp gene, and used in a semicontinuous activated-sludge (SCAS) system to biodegrade a continuous stream of 3-CA-contaminated wastewater. For more than 14 days, strain I2 gfp removed all 3-CA. Although strain I2 gfp was never tested in a wastewater treatment system where a shock load had occurred, we used it in this work I2 gfp to protect SCAS reactors from 3-CA shocks. Few reports have investigated the recovery of disrupted reactor functions after bioaugmentation. Therefore, the aim of this work was to evaluate the short-term effects of a 3-CA shock load and bioaugmentation with a 3-CA-degrading inoculant on reactor functions such as nitrification, carbon removal, and sludge compaction. Denaturing gradient gel electrophoresis (DGGE), FISH, and real-time PCR were used to examine the changes in the structure, abundance, and activity of the nitrifying community. The experiments were conducted with sludge freshly collected from a domestic wastewater treatment plant (Bourgoyen-Ossemeersen, Ghent, Belgium), by using a modified SCAS procedure (12). In brief, every 2 days, 200 ml of the mixed liquor was removed from the reactors (in 2-liter plastic Erlenmeyerflasks with an active volume of 1.2 liters) and 1.0 liter was allowed to settle for 30 min in an Imhoff cone, of which the upper 400 ml of the effluent was removed for analysis. The remaining settled sludge was poured back into the respective reactor, together with 600 ml of synthetic influent (skim milk powder [Gloria, Nestle, Brussels, Belgium]; volumetric loading rate of 1 g of chemical oxygen demand [COD] liter1 day1; COD/N/P ratio of 100:6:1) to bring the active reactor volume back to 1.2 liters. The SCAS reactors operated with a hydraulic retention time of 4 days and a sludge retention time of 12 days and contained 4 g of suspended solids liter1. To analyze sludge volume (SV), 1 liter of the mixed liquor was allowed to settle for 30 min in an Imhoff cone. The sludge volume index (SVI) was calculated by dividing the SV by the suspended solids. All reactors were operated for 6 days before the experiment in order to allow the reactors to stabilize. On day 0, reactors 1 (n 2) continued to receive only milk powder and were control reactors. In addition to the milk powder, reactors 2 (n 2) and 3 (n 2) both received on day 0 a shock load of 300 mg of 3-CA (Fluka AG Chemische Fabrik, Buchs, Switzerland; 99% pure), resulting in a final concentration of 250 mg liter1 in the reactor mixed liquor. The bioaugmentation experiment was performed in reactors 3, in which C. testosteroni I2 gfp was inoculated. This strain, which has been chromosomally marked with gfp (the gene encoding green fluorescent protein), mineralizes 3-CA, fluoresces green under UV light and is rifampin (100 g/ml) as well as kanamycin (50 g/ml) resistant (12, 13). Luria broth (LB) agar supplemented with rifampin (100 mg liter1) and kanamycin (50 mg liter1) (both from Ducheva Haarlem, The Netherlands) was used to count I2 gfp (12). The C. testosteroni I2 gfp inoculum was grown overnight at 28°C in 5 ml of LB medium (1 liter contains 5 g of NaCl, 10 g of tryptone, and 5 g of yeast extract) containing 100 mg of 3-CA liter1. Subsequently, 5-ml cultures were used to inoculate 200 ml of LB medium plus 3-CA (100 mg liter1). After overnight incubation at 28°C in a shaker (140 rpm; New Brunswick Scientific), the cultures were centrifuged (1 min at 5,000 g), washed twice with saline (0.85% NaCl), and finally resuspended in saline. Reactors 3 were inoculated with C. testosteroni I2 gfp to a final concentration of (5.4, 0.37) 108 cells/ml. Sampling. Every 2 days, a sample was taken for high-performance liquid chromatography (HPLC) analysis, for DNA and RNA extraction, for FISH analysis, for plate counts of strain I2 gfp, and for determination of the SS and SVI. For DNA and RNA isolation, aliquots of the samples were immediately frozen at 20 and 80°C, respectively. For FISH, a subsample of activated sludge was fixed overnight with 4% paraformaldehyde. Analytical methods. The effluent was analyzed for 3-CA content by reversedphase HPLC after centrifugation at 5,000 g for 10 min. The Summit HPLC system (Dionex, Wommelgem, Belgium) consisted of a Dionex pump series P580, a Dionex autosampler model ASI-100 (injection volume, 20 l), an STH585 column oven (at 28°C), a Dionex UV/VIS UVD 340S detector, and Chromeleon software system version 6.10. A Hypersil Green Env column (150 by 8 mm [inner diameter]; 5-m particle size [Alltech, Deerfield, Ill.]) was used. The mobile phase consisted of CH3OH-0.1% H3PO4 (ratio, 70:30), with a flow rate of 0.8 ml/min. The UV detector was used at 210 nm. The effluent was analyzed for nitrite, nitrate, and ammonium content by ion chromatography after centrifugation at 5,000 g for 10 min and filtration through a 0.45-m-pore-size filter. The DX-600 system (Dionex) consisted of a Dionex pump series GP50, a Dionex autosampler model AS50 (injection volume, 100 l), a Dionex ED50 electrochemical detector, and PeakNet 6 software system version 6.10. Ionpac AS9-HC (250 by 4 mm [inner diameter]; 9-m particle size [Dionex]) column and Ionpac CS12-HC (250 by 4 mm [inner diameter]; 8-m particle size [Dionex]) were used for anion and cation separation, respectively. The mobile phase consisted of Na2CO3 (9 mM) and methanesulfonic acid (20 mM) for anion and cation analysis respectively, with a flow rate of 1 ml/min. The residual COD and SVI of the effluent were determined by standard methods. Total DNA extraction from the sludge samples and PCR conditions were based on the protocols described previously. The total RNA extraction protocol was adapted from those of Griffiths et al. 25 and Kowalchuk et al. 34 . Briefly, in a 2-ml Eppendorf tube, 0.5 g of RNase-free 0.1-mm-diameter zirconia/silica beads (B. Braun Biotech International, Melsungen, Germany), 0.5 ml of activated sludge, 0.5 ml of CTAB buffer (5% [wt/vol] hexadecyltrimethylammonium bromide, 0.35 M NaCl, 120 mM potassium phosphate buffer [pH 8.0]), and 0.5 ml of phenol-chloroformisoamyl alcohol mixture(25:24:1) were homogenized three times for 30 s each at 5,000 rpm in a Beadbeater (B. Braun Biotech International) with 10 s between shakings. Eppendorf tubes were spun centrifuged (5 min at 3,000 g), and 300 l of the supernatant was transferred to an RNase-free Eppendorf tube. Another 500 l of CTAB buffer was added to the sludge suspension and homogenized again three times for 30 s each at 5,000 rpm in the Beadbeater with 10 s between shakings. Then 300 l of the supernatant was added to the 300 l taken from the first extraction, for a total of 600 l. The phenol was removed by mixing with an equal volume of chloroform-isoamyl alcohol (24:1), inverting the tube, and centrifuging for 10 s. The upper, aqueous phase was transferred to a new Eppendorf tube, and nucleic acids were precipitated with 2 volumes of 30% (wt/vol) polyethylene glycol 6000-1.6 M NaCl for 2 h at room temperature. The Eppendorf tube was then centrifuged at 18,000 g in a refrigerated centrifuge at 4 °C for 10 min. The nucleic acid pellet was subsequently washed in ice-cold 70% (vol/vol) ethanol and dried under vacuum for 10 min before being resuspended in 100 L of RNase-free water. To obtain pure RNA a RQ1 RNase-Free DNase treatment was performed as specified by the manufacturer (Promega, Madison, Wis.). The average RNA yield was 3.4 ng ml of activated sludge1."