Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30
Introduction
Cellulase enzymes have been used in textile and paper processing for finishing applications [1], [2]. This enzyme group has potential applications in cellulosic biofuel production to hydrolyze biomass into fermentable sugars. However, expansion has been limited by relatively high production costs [1]. Members of the branched fungi genus Trichoderma are commonly employed to produce cellulase.
A variety of species, mutants, feeding, pH, temperature, mixing, nutrient supplements, and process control schemes have been identified to improve both volumetric and specific cellulase production [3], [4], [5], [6], [7], [8]. A key advancement was removing the glucose repression of cellulase expression, which allowed for cellulase production on low cost sugars. The Rut C-30 mutant is lacking the cellulase expression dependency on glucose lean conditions [9]. Likewise, T. reesei Rut C-30, MCG-77, and NG-14 strains have all proven to be among the best cellulase producers [7]. The production of cellulase is a complex function of the growth environment. Cellulose, sophorose, and lactose have been shown to be effective cellulase inducing agents for T. reesei [10], [11]. In some cases, improved cellulase production requires a complex feeding scheme to maintain low fungal growth and adequate cellulase production [12], [13]. A simple control scheme is desirable to arrest the growth of T. reesei for continuous cellulase production. Stationary culture conditions may allow for mycelial retention, long-term process stability, and potentially higher product yields with little waste of nutrients on fungal biomass production.
To achieve better process stability, recent process control schemes and chemical additives have been used to modify the morphology from filamentous to pellet for simplified industrial processing [14], [15]. In submerged fermentation, specific shear ranges and surfactants have both been effective for morphology control. These morphological changes may help reduce the cost of cellulase production though improved separation. The use of pellets aids in extended periods of cellulase production with retained fungal biomass. Furthermore, the constant presence of fungal biomass, coupled with minimal nutrient availability may reduce the potential for contamination by competing organisms. Attempts have been made to create pellet T. reesei. However, these are limited by very specific operating conditions, pellet degradation, or overgrowth out of pellet form that leads to mixing failure [15], [16], [17], [18].
It is hypothesized that phosphorus limitation will halt T. reesei Rut C-30 growth without limiting cellulase production. Reduced potassium phosphate levels have been previously shown to improve cellulase production [19]. Further, the application of nitrogen limitation may be effective at halting growth and providing insight into the nutrient allocation for synthesis of cellulose enzymes versus cell mass. Ideally, this non-carbon nutrient limitation can be combined with pellet morphology for continuous cellulase production under stationary phase conditions. Here, the fungal biomass concentration is stable and the cellulase activity is accumulating or continually harvested.
Section snippets
Strain and culture maintenance
Trichoderma reesei Rut C-30 (NRRL 3469) was obtained from the United States Department of Agriculture’s Agricultural Research Service’s culture collection. It was maintained on potato dextrose agar plates (Sigma–Aldrich, St., Louis, MO). These plates were incubated at 30 °C for 4 days before storage at 4 °C. The culture was transferred every 4 weeks by streaking a fresh plate with a loop of cells taken from a mature plate. Fermentation inoculum was prepared by transferring 6 loops of cells, from
Results
A reference fermentation for T. reesei is shown in Fig. 1. This fermentation was stopped when the lactose was depleted and the system reached carbon source limitation. The total duration was 62 h, during which time the cellulase activity increased. By averaging the incremental increase in both fungal biomass and cellulase, the specific rate of cellulase production during this period was determined to be 10 ± 2 FPU per gram of cell per hour. Given 8.3 g/L lactose consumed and 1 g/L proteose peptone,
Discussion
During T. reesei Rut C-30 fermentation under phosphorus limitation, it was observed that the specific cellulase productivity was 5 ± 2 FPU/g-h in stir-tank systems. The use of specific productivity has been periodically reported in previous works. However, in many typical batch fermentations the fungal biomass concentration changes with time. Since the cell concentration is not stable, the apparent cellulase production rate changes during the fermentation, as more biomass is produced, lost, or
Supplementary data
Economic analysis details are provided in the supplementary materials.
Acknowledgments
This work was supported by the U.S. Department of Agriculture under the Biomass Research and Development Initiative (award # 2009-10001-05112). Data collection and sample analysis was greatly assisted by Rebecca Howdyshell and Alexander Dannemiller. Jacob C. Kohl assisted with figure formatting and proofreading.
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