Two anaerobic fungi, one a monocentric strain (Piromyces sp. KSX1) and the other a polycentric strain (Orpinomyces sp. 478P1), were immobilized in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. β-glucosidase production with KSX1 was maintained for 45 days over six-repeated batch cultures yielding a maximum level of 107 mIU/mL. For 478P1, β-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/mL. Although repeat-batch cultures of KSX1 produced more β-glucosidase than strain 478P1, the maximum specific β-glucosidase produced from these immobilised cultures was similar. The immobilized polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any viable growth in the culture liquor.
IntroductionAnaerobic fungi isolated from the digestive tract or faeces of herbivorous mammals, both ruminant and non-ruminant, produce all of the cellulolytic enzyme activities needed for complete cellulose degradation, that is, endoglucanases, exoglucanases and β-glucosidases [11].The finding that the cellulose solubilization capabilities of Neocallimastix frontalis and Piromyces communis are better than that of Trichoderma reesei [12,13] provides confidence that the potential for converting cellulose to fermentable sugar may lie in the superior cellulolytic activity of anaerobic fungi.The most studied method of cellulase production by both aerobic and anaerobic fungi has been batch culture. Batch culture can be coupled with the use of immobilized cells. This approach has been little exploited in cellulase production but appears to hold considerable potential for improving it since immobilization enables the microbial cells to be used repeatedly and continuously [3]. The first report on fungal enzyme production by an immobilized biocatalyst was that of Frein et al. [2], who showed that the fungus Trichoderma reesei entrapped in κ-carrageenan gel matrix can produce extracellular cellulolytic enzymes. In another study, glucoamylase production by immobilized, pre-grown mycelia of Aspergillus phoenicus was shown to be sustained over 5 repeated batches in a total time of 19 days [6].In a previous investigation we achieved colonization of alginate beads using either monocentric or polycentric types of anaerobic fungi [7]. We found that the two types had different growth habits in calcium alginate gel, providing further insight into the life cycle and growth patterns particular to monocentric and polycentric fungi. This current study is the first to report on the use of these immobilized anaerobic fungi in repeat-batch fermentations and relates these differences in colonization mode to subsequent enzyme productivity.
Materials and methods
Organisms and culture mediaThe isolation and description of Piromyces sp. strain KSX1 and Orpinomyces sp. strain 478P1 have been described previously [7]. The cultures were maintained at 39°C in a semi define...