Small-Column Experiments

Unsterile straw stems were used in all experiments because steriliza­tion of large quantities of straw for construction of large windrows would be uneconomical and impractical. For this strategy to be effective, it was necessary that the inoculated fungus be able to compete with the indig­enous organisms in the straw. Since white-rot fungi dominate in nature under conditions of nitrogen deprivation (9), experiments were previ­ously performed (4) to determine inoculum production conditions neces­sary to limit nitrogen addition during inoculation of the straw with the fungus. C/N ratios in the media tested ranged from about 29 to 89 and were adjusted by adding yeast extract to 20 g/L glucose solutions. The nitrogen-limited medium finally utilized for straw stem inoculum pro­duction contained 3.0 g/L of yeast extract, for a C/N of 32.6. Mycelial inocula for inoculation of straw stems were produced in this nitrogen — limited medium, using as inoculum the fungal pellets produced at Utah State University in YM broth (4).

Approximately 500 mL of wet fungal pellets of P. ostreatus grown at Utah State University in YM broth were transferred to a sterile blender and blended for 2 min, producing a slurry of finely chopped mycelial fragments. The optical density (OD) at 550 nm was determined for dilu­tions of this slurry using a standard UV/VIS spectrophotometer. The undiluted slurry was then inoculated to 1.0 OD into the fresh nitrogen — limited medium in sterile shake flasks and incubated for 5-7 d at 30°C, 135 rpm. The fungal pellets in the inoculum cultures were then transferred with the spent medium to a sterile blender and blended for 2 min. The OD at 550 nm was determined for dilutions of this slurry, and the concentra­tion of biomass was estimated from a previously measured calibration. The undiluted slurry was transferred to a sterile hand-pump garden sprayer for addition to the straw stems. No extraordinary measures were taken beyond this point to maintain sterility, except the use of initially sterile equipment.

Air-dried straw stems (150 g dry wt at about 9-13 wt% moisture) were weighed onto a clean, dry, tared tray and spread in a thin (5-cm) layer. The homogenized mycelial inoculum slurry was then sprayed onto the stems, with frequent mixing of both the inoculum and the stems. Sufficient inocu­lum was added to reach the desired initial level of fungal inoculum in the stems. Periodically during addition of inoculum, a fan was used to blow nonsterile air across the tray of inoculated straw to evaporate excess water,


Fig. 1. Combinations of inoculum amount and moisture content tested.

with frequent mixing of the straw. After the desired amount of inoculum was added, additional sterile distilled water was sprayed onto the straw as needed to reach the desired initial moisture content for the particular experiment. A separate sample of the well-mixed inoculum slurry was then added to a tared bottle and dried to constant weight at 105°C to determine the actual biomass concentration of the slurry. In addition, sev­eral small samples of the inoculated stems were transferred to tared bottles and dried to constant weight at 105°C to determine the actual moisture content of the inoculated stems.

Combinations of inoculum amounts and moisture contents tested in this exploratory study are shown in Fig. 1; controls lacking inoculum were also conducted at 0.4-0.77 g of H2O/g of stems and are not shown in Fig. 1. The first tests performed (4) are represented by the 12 points in the lower left-hand corner of Fig. 1. When these tests indicated that higher inoculum was needed for better selectivity and that higher moisture was needed for faster degradation (4), parameter testing moved to the combi­nations plotted in the upper middle and right-hand corner of Fig. 1.

The inoculated straw was added to clean, initially sterile columns fabricated from glass process pipe as previously described (4). The col­umns were prepared in triplicate with approx 50 g dry wt of inoculated stems in each column. The loaded columns were supplied with humidified oil-free instrument air at 193 kPa and a flow rate sufficient to turn over the air in the system once per day (about 10 mL/min). Approximately 2.5 g (dry wt) of straw was sampled from the top and bottom of each column initially and approx every 3 to 4 wk thereafter for 12 wk. The samples were com­bined, dried to constant weight overnight at 105°C, and ground to 60 mesh in a Wiley mill for compositional analyses.