Activity Associated With Microbial Flocs

Goel et al.36 used p-nitrophenolphosphate as the surrogate substrate for quantifying alkaline and acid PO4ase activity, p-nitrophenol a-D-glucopyranoside as the surrogate substrate for a-glucosidase activity, and azocasein as the model substrate for protease activity in activated sludge. After an appropriate incubation period to allow enzymatic release of Pi, glucose, or peptide from the substrate, the activated sludge samples were centrifuged and the absorbance of the supernatant fraction was measured spectropho-tometrically at 410 nm for p-nitrophenol (alkaline and acid PO4ases, a-glucosidase), and at 440 nm, for trichloroacetic acid (TCA) soluble peptides (protease activity). Whiteley et al.37 used the same substrates to measure exoprotease and extracellular alkaline PO4ase activity in samples from two large-scale stirred tank laboratory reactors operated in series: the first seeded with a mixed culture of methanogenic bacteria from a standing digester at a sewage works, and the second seeded with a mixed culture of sulfate-reducing bacteria.

Cadoret et al.12 assayed L-Leu-aminopeptidase, a-glucosidase, protease, and «-amylase activities in whole and dispersed activated sludges, as well as activities associated specifically with the floc EPS fraction. Enzyme activities were based on the rate of release of a chromogenic enzyme substrate following hydrolysis of synthetic substrates over an appropriate incubation period. L-Leu-p-nitroanilide, p-nitrophenyl-a-D-glucopyranoside, azocasein, and amylose azure were used as substrates for L-Leu-aminopeptidase, a-glucosidase, protease, and a-amylase, respectively. Nitroanaline and p-nitrophenol hydrolysis were quantified spectrophotometrically at 410 nm. Azocasein hydrolysis was determined by measuring the amount of cold TCA soluble material produced at 340 nm using a spectrophotometer. a-Amylase hydrolysis was determined by the amount of brilliant blue recovered in the cold TCA soluble material following release from amylose azure and treatment with cold TCA.

Pletschke et al.38 monitored adenosine triphosphate sulfurylase (ATPS) in meth-anogenic and sulfidogenic stirred tank reactors operated in series using primary sludge from an anaerobic digester at a sewage works. ATPS catalyzes the first step in dis-similatory sulfate reduction: the formation of adenosine 5'-phosphosulfate (APS) and inorganic pyrophosphate (PPi). ATPS activity was assayed by measuring ATP produced by reaction of APS with PPi. The rate of ATP production was measured in the coupled spectrophotometric assay involving phosphorylation of glucose and the subsequent oxidation of glucose-6-phosphate to D-6-p-glucono-S-lactone coupled to the reduction of NADP.

Boczar et al.15 assayed esterase activity in the supernatant (cell free) fraction of filtered activated sludge. Esterase activity was determined by measuring the release of fluorescein from fluorescein diesters of acetate, butyrate, caproate, and caprylate, or the release of p-nitrophenol or a-napthol from p-nitrophenol or a-napthol esters of acetate, butyrate, capriate, caprylate caprate, laurate, myristate, palmitate, and stearate. The amount of p-nitrophenol and a-napthol produced was determined spectrophotometrically at 400 nm, and the amount of fluorescein produced was determined spectrophotometrically at 495 nm. Frolund et al.39 monitored sludge bulk extracellular esterase activity using fluorescein diacetate as an enzyme substrate. A spectro-fluorimeter was used to quantify the fluorescence resulting from the formation of fluorescein.

Whiteley et al.40 monitored lipase activity in methanogenic and sulfido-genic stirred tank reactors operated in series using primary sludge from an anaerobic digester at a sewage works. The assay measured enzymatic cleavage of glycerol from the lipid triacetin. Sludge samples from the reactors were sonicated to release enzymes from the particulate fraction and then centrifuged to separate the enzymes from the particulate material. Triacetin was added to the supernatant fraction, and following an incubation period, the enzyme reaction was terminated by addition of sulfuric acid and sodium periodate. Following addition of NaHSO3 and chromotropic acid reagent and heating, the reaction mixture was cooled and the glycerol released was quantified spectrophotometrically at 570 nm, Since particulate matter interfered with the lipase assay, the sample had to be sonicated and the particulate fraction removed by centrifugation prior to assaying for enzyme activity. As a result, it was not possible to determine whether the lipases were free in solution, bound to the floc matrix, or associated with the cells.

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