Incubation, slurries

The second patent describes the use of a microbial mixed culture (Hansenula sydowiorum, Hansenula ciferrii, Hansenula lynferdii, and/or Cryptococcus albidus) in coal desulfurization [160], In this process, the raw mined coal is ground to a particle size smaller than 200 mesh forming a slurry with water, at a solids concentration of less than 40wt%. The bacterial cultures are then inoculated into the feedstock slurry. An incubation step is carried out at a temperature near 25°C and at a pH close to neutral. The highest removal achieved was in the range of 46% S removal. 

There is a need to measure the activities of enzymes and to correlate these measured activities with microbial diversity in soil. It is conceptually wrong to assume a simple relationship between a single enzyme activity and microbiological activity in soil (Nannipieri et al. 2003). Most of the assays used to determine microbiological activities in soil present the same problem measuring potential rather than real activities (Nannipieri et al. 1990). Indeed, assays are generally made at optimal pH and temperature and at saturating concentration of substrate. Furthermore, synthetic rather than natural substrates are often used, and soil is incubated as a slurry (Nannipieri et al. 1990). 

Bedard et al. [245] reported that PCB dechlorination was stimulated by adding 2,5,3, 4 -tetrachlorobiphenyl (25-3 4 -CB) to slurries (incubated under methanogenic conditions) of sediments contaminated with Aroclor 1260 from Woods Pond (MA). The 25-3 4 -CB was converted stoichio-metrically to 25 - 3 -CB and stimulated a selective para-dechlorination which decreased the penta- through heptachlorobiphenyls containing 234-, 245-, or 2345-chlorophenyl groups by up to 83% in 12 weeks. 

Wu et al. (1998) investigated the microbial reductive dechlorination of PCB-1260 in anaerobic slurries of estuarine sediments from Baltimore Harbor, MD. The slurries were amended with 800 ppm PCB-1260 with and without the addition of 2,3,4,5-tetrachlorobiphenyl or 2,3,5,6-tetrachlorobiphenyl and incubated at 30 °C under methanogenic conditions. Without the addition of the tetrachlorobiphenyls, chlorine atoms at the meta and ortho positions on the PCB congeners decreased by 45 and 9%, respectively. When 2,3,4,5-tetrachlorobiphenyl and 2,3,5,6-tetrachlorobiphenyl were added, chlorine atoms at the meta position decreased by 65 and 55% and chlorines at the ortho positions decreased by 18 and 12%, respectively. After 181 d, hexa- and nonachlorobiphenyls decreased by 65, 75, and 88% In PCB-1260 alone, PCB-1260 + 2,3,5,6-tetrachlorobiphenyl, and PCB-1260 + 2,3,4,5-tetrachlorobiphenyl. The investigators concluded that the addition of a single congener stimulated the dechlorination of PCB-1260. 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

Total viable counts (TVCs) were immediately carried out on duplicate controls with no biocide added. The slurries were incubated at 37 °C for 4 hours and TVCs were performed at that time point and at several points over the following 6 weeks. Re-inoculations were carried out after 3 weeks. The resulting bacterial numbers were recorded as colony forming units (cfu)/ml of slurry. 

Incubate 6 min in boiling water bath, stirring tube vigorously after 2 min and 4 min in order to ensure complete homogeneity of the slurry. 

Add the reverse transcription reaction mix (100 pL) directly to the tube containing the washed 25 pL slurry of silica, vortex briefly, and incubate at 25 °C with constant inversion for 10 min. 

Figure 4.2. Carbon released as C02 from unamended soil and soils amended with pig slurry (PS), poultry manure (PM), cattle farmyard manure (FYM), aerobic sewage sludge (SS), municipal solid waste fuse compost (RC), and rye straw (RS) at a rate of lOgkg1 during incubation at 22 °C. Reprinted from Levi-Minzi, R., Riffaldi, R., and Saviozzi, A. (1990). Carbon mineralization in soil amended with different organic materials. Agric. Ecosyst. Environ. 31, 325-335, with permission from Elsevier.

The DM content of the steam-pretreated SFF was adjusted to 5% and the pH set to 5.0 with NaOH. Enzymatic hydrolysis was performed using a Celluclast + Ultraflo mixture (1 1) at a ratio of 2 g of enzyme/100 g of slurry (10). Hydrolysis was carried out for 72 h in 100-mL shake flasks maintained at 50°C and shaken at 200 rpm in a laboratory rotary shaker-incubator (LSR/L-V Adolf Kiihner AG) for 72 h. Samples were withdrawn after 0,2, 4.5, 7.5,11,14, 24,31, 38,48, 60, and 72 h for analysis of monosaccharides. Direct enzymatic hydrolysis of a 5% DM SFF slurry was also performed as a reference to evaluate the effect of steam pretreatment on the yield. 

Suspend 2 g of bacterial cell paste in 25 ml of 0.15 M NaCl, 0.1 M Na2EDTA solution. The bacterial paste is most easily suspended by first adding a small amount of the NaCl-EDTA solution, making a slurry, and then slowly adding the rest of the NaCl-EDTA solution. After suspension, add 1 ml of a 10-mg/ml lysozyme solution and incubate the suspension at 37°C for 30 min, gently agitating occasionally. 

Insulin. Twelve glass test tubes are labeled, and 0.2 ml of phosphate buffer 1 is added to each tube. The two blank tubes receive 0.1 ml of phosphate buffer 2, and the remaining 10 tubes receive in duplicate 0.1 ml of five increasing dilutions of anti-insulin serum in phosphate buffer 2. A constant amount (0.1 ml) of [ I]insulin in phosphate buffer 1 is added to each tube, and the tubes are incubated at 37° for 45 min. The reaction is stopped by the addition to each tube of 5 ml of the Z-gel slurry followed by 10 ml of 0.1 Af NH Ac (pH 6.25). The contents of the tubes are mixed by inversion, and the Z-gel is collected by centrifugation (1000 g for 5 min) at room temperature. The supernatant is decanted, and the pellet is washed by resuspension in 10 ml of 0.1 M NH4AC followed by recentrifugation. The supernatant is decanted, and the amount of associated with the pellet of Z-gel is measured. Approximately 80% of the I in the preparation of [ I]insulin binds to Z-gel in the presence of excess antibody, but only 10% binds in the absence of antibody. A dilution of the... 

The entire assay can conveniently be carried out in a glass scintillation vial [82]. The reaction volume contains Tris buffer (pH 7.5), Mg, [ H]cyclic nucleotide, and snake venom 5 -nucleotidase. After a 10-min incubation at 37°C, the reaction is stopped by the addition of a slurry of AG1-X2 anion exchange resin. The resin binds substrate cyclic nucleotides but does not bind nucleosides. Scintillation fluid is added and the amount... 

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