A Buchi Rotavapor R110 evaporator (Flawil, Switzerland) equipped with a Cole Palmer aspirator pump Model 7049-00 (Chicago, Illinois) was used (55 °C) to remove volatiles (vacuum treatment) prior to H 89 adjustment of hydrolysate. Unless otherwise indicated, hydrolysate was evaporated to 50% by weight, restored to original weight by adding deionized water, and adjusted to pH 6.3 before testing for toxicity.
Laccase (Novozymes NS-22127) was generously provided by Novozymes North America, Inc. (Franklinton, North Carolina). For laccase treatment, hydrolysate was adjusted to pH 5.0 with ammonium hydroxide (5 N). Laccase (250 U total) was added to 50 ml hydrolysate in a 250 ml flask. This was incubated in a water bath (50 °C, 150 rpm) for 3 h. A laccase unit, U, is defined as the amount of laccase that catalyzes the conversion of 1 μmol (Leonowicz and Grzywnowicz, 1981) of syringaldazine per minute under standard conditions (pH 7.5, 30 °C). When needed, aeration was provided in tube cultures using an incubator with a rotator (30 rpm, 60° angle, 37 °C).