Materials and Methods

Maintenance and Preculture of Ganoderma lucidum

The strain G. lucidum was maintained on potato-agar-dextrose slants. The slant was inoculated and incubated at 28°C for 7 days, then stored at 4°C for about two weeks. Preculture medium and conditions were described elsewhere (10-13).

02 Supply Experiments

The effects of 02 supply were preliminarily investigated by changing the medium loading volume of shake flask on a rotary shaker (30°C, 120 rpm). By setting the loading volume at 30, 50, 70 and 100 mL in 250-mL conical flasks, an initial K\?l value of 32.6, 18.5, 13.2 and 6.4 h"1 was obtained, respectively. For bioreactor cultures, a 3.5-L (working volume) agitated bioreactor with two six-bladed turbine impellers (6.5 cm ID) was used. Cultivation was conducted at 30°C in the dark. The Kl?l value was determined using the dynamic gassing-in and gassing-out method (21). The cultures were all agitated at the same speed (200 rpm), and the aeration rate was set at 220, 1050, 1750 and 3500 mL/min to obtain the desired K\?l values of 16.4, 60.0, 78.2 and 96.0 h"1, respectively.

Lactose Feeding in Shake Flasks

The fermentation medium (with an initial pH of 5.5) was composed of 35 g L"1 of lactose, 5 g L"1 of peptone, 5 g L"1 of yeast extract, 1 g L"1 of KH2P04, 0.5 g L"1 of MgS047H20 and 0.05 g L*1 of vitamin Bi. The medium was inoculated with 5-mL second preculture broth (with ca. 350-400 mg DW of cells L1) in 45-mL medium in a 250-mL flask. The fermentation was conducted in the dark at 30°C on a rotary shaker at 120 rpm. Fed-batch fermentation was operated by feeding 15 g L"1 of lactose when its residual concentration was below 10 g L"1. Samples were taken every 2-3 days. In all fermentations, there were 2-3

identical cultivation vessels operated under each condition, and the data shown represent average values with standard deviations.

Determination of Dry Weight, Residual Medium Sugar, and Lactate Concentration

For sampling, three flasks were sacrificed each time; for the bioreactors, about 20-30 mL of cell culture was taken once from each reactor. Dry cell weight (DW) and residual sugar concentration was measured by gravimetric method and phenol-sulfuric acid method, respectively (11). Lactate concentration in the fermentation broth was determined by using a conventional enzymatic method, and lactate dehydrogenase and NAD* were purchased from Sigma Chemical Co. (USA).

Measurements of Ganoderma Polysaccharide and Ganoderic Acid

The concentration of extracellular polysaccharide (EPS) in the fermentation broth, contents of intracellular polysaccharide (IPS) and ganoderic acid (GA) in mycelia were analyzed. Details of these analyses were reported earlier (10-13).

Preparation of Cell Extracts

Samples taken from fermentation broth were centrifuged (12,000xg, 10 min, 4°C) and the supernatant was decanted. The cell pellet was washed twice with 0.85% NaCl, and suspended in 20 mM phosphate buffer (pH 6.5) containing 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol. The cells were disrupted ultrasonically at 0°C for 72 cycles of 5s. Cell debris was removed by centrifugation (12,000xg, 10 min, 4°C). The protein concentration of the cell extract was determined by the method of Bradford (22) and compared with a bovine serum albumin standard.

Assay of (3-Galactosidase, a-PGM and PGI Activities p-Galactosidase (EC activity was estimated by detecting the rate of hydrolysis of o-nitrophenyl-p-D-galactopyranoside (ONPG) at 30°C (20). The amount of ONPG hydrolyzed was determined by measuring the solution's absorbance at 420 nm. Assay of a-PGM (EC and PGI (EC was performed at 30°C in a total volume of 1.0 mL with freshly prepared cell extracts, and the formation or consumption of NAD(P)H was determined by measuring the absorbance change at 340 nm (19). The means of the results from at least two independent measurements were calculated.

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