Preparation of bacterial inoculum Bacterial stock culture was prepared in nutrient broth, incubated at 32C for 24 h.

The cultures were diluted to turbidity yielding between 0.05 0.1 at absorbance of 600 nm. The turbidity was measured again to make sure all cultures had the same amount of cells (Sittidilokratna et al., 2007).

Submerged fermentation of bacterial isolates Flasks containing 100 ml of nutrient broth were prepared. Each flask was supplemented with substrate depending on the enzyme assayed. Substrates were carboxymethylcellulose (1%), Whatman No. 1 (0.1%), filter xylan paper birchwood (1%), (0.25%),

p-nitrophenyl--D-glucopyranoside

p-nitrophenyl--D-xylopyranoside (0.1%) and polygalacturonic acid (1%). The pH of the medium was maintained between 5 and 6. Bacterial cultures were transferred into flasks containing medium and substrate. Flasks were incubated in rotary shaker at 32C and 120 rpm.

Samples were drawn out hourly for the first 12 h. Sampling for the next 12 h carried out every two hours. At each sampling, 10 ml of sample was withdrawn and transferred into autoclaved nalgene tube. The samples were then centrifuged at

10,000 rpm, 4C for 15 min (Sittidilokratna et al., 2007). Supernatant were assayed immediately for cellulase, xylanase and pectinase activities.

Determination of pectinase activity Substrate, polygalacturonic acid (PGA) was prepared by dissolving 1 g of PGA in 100 ml of distilled water. Reaction mixture was prepared by adding 2 ml of citrate phosphate buffer, pH 5, 0.2 ml PGA and 1 ml sample. The reaction mixtures were incubated at 35C for 25 minutes. After incubation, 1 ml of reaction mixture were withdrawn and added to tubes containing 0.5 ml of 1 M sodium carbonate solution. Each test tube was added with 3 ml of DNS reagent and heated in boiling water bath for 10 min (Bhardwaj and Garg, 2010).

Substrate control tubes were prepared by adding 3 ml of 0.05 M citrate phosphate buffer and 0.2 ml substrate. Enzyme control tubes were prepared by mixing 2.2 ml of buffer with 1 ml of sample. Similar as reaction mixture, 1 ml of content were withdrawn from each control and enzyme tubes and placed into separate tubes containing 1 ml of sodium carbonate solution. 3 ml of DNS were added to the tubes and placed in boiling water bath for 10 min.

The tubes were cooled and absorbance was read at 570 nm against blank. Samples were diluted in 20 ml of distilled water when it reads very high absorbance. Standard galacturonic acid (0.1%) was prepared by dissolving 0.1 g of galacturonic acid in 100 ml of distilled water. Calibration curve was prepared by plotting absorbance against galacturonic acid concentration.

Enzyme activity (g/ml/min) = (g galacturonic acid) / T x S x 194.14

incubation

time

(minutes),

volume

of

sample

taken

(ml)

and

194.14 = molecular weight of galacturonic acid