Cellulolytic enzymes production guided by morphology engineering

Highlights

• Accumulation of endoglucanase or β-glucosidase is a function of mixing for A. niger.

• The ratio of enzyme activities is indicative of mycelial morphology.

• A form factor may be used to correlate the ratio of enzyme activities.

• Dispersed forms of mycelia are responsible for β-glucosidase accumulation.

Abstract

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. β-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or β-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Introduction

Submerged fermentations of filamentous microorganisms form the foundation of industrial production of fungal enzymes. These microorganisms display a complex morphological life, with forms being classified into pellets and dispersed mycelium. This includes clumps, branched and isolated hyphae [1]. Morphology has a big impact on accumulation of bio-based products [2,3].

Microbial morphology and growth forms are defined by broth rheology, oxygen transfer and shear conditions within a bioreactor, which in turn affect efficiency of enzyme production [4,5,6]. Impellers influence fungal morphology and growth due to shear conditions and oxygen transfer [7,8,9,10]. The environment and interactions during the cultivation of microorganisms are complex, and together with the specific physiology and secretion pathways for a given microorganism, make it difficult to quantify the influence of individual variables on the production efficiency [4,11].

Morphology engineering may be achieved by the use of microparticles [12,13], changes in the spore concentration [14], pH of the inoculum [15] and medium osmolality [16]. However, this poses challenges to defining protocols for selecting appropriate bioprocess conditions, or specific conditions for generation of bio-based products as a function of microorganism and/or the strain. Ahamed and Vermette [17], when cultivating Trichoderma reesei with disperse morphology, concluded that the highest number of hyphae tips by clumps resulted in enhanced production of cellulases. Callow and Ju [18], when cultivating T. reesei in pellet form by addition of surfactants showed an increase in 73% of cellulase production compared to cultivation in the absence of surfactants that caused more dispersed morphology. Dong et al. [19] were able to alter the morphology of Trichoderma viride, reducing the pellet size diameter by three times resulting in an increasing enzyme activity by 17%. Morphology was also found to be associated with higher expression levels of hemicellulase [20].

Reports in literature [21,22] have shown endoglucanase and xylanase are critical enzymes for liquefaction of high solids lignocellulosic biomass, which can facilitate movement of biomass within a biorefinery [23], hence favoring a more cost-effective process. Together with β-glucosidase, these enzymes are also important for the production of desired lignocellulosic biomass derived products [23]. Aspergillus niger is a filamentous fungus recognized as a good producer of both cellulolytic and hemicellulolytic enzymes and has been granted GRAS status from the FDA [24,25,26,27,28,29]. In previous work, Buffo et al. [8], working only with pellet morphology, analyzed the effect of different bioreactor conditions, such as agitation and aeration, in pellet fragmentation of Aspergillus niger and the effect on endoglucanase and β-glucosidase production. The authors verified a higher production of β-glucosidase coincided with an increased pellet fragmentation, while a lower extent of fragmentation favored endoglucanase production.

This paper aims to further understand the relation between fungal morphology for increased production of A. niger cellulolytic (endoglucanase and β-glucosidase) and hemicellulolytic (xylanase). More specifically, it is reported a statistical design of experiments in which spore concentrations and pH were varied, to obtain different morphologies for enzyme production. The impact of the combined effects has not been previously reported, to the best of our knowledge. The data presented here show the different combinations of pH and spore concentrations that were tested influence inoculum morphologies (pellets, clumps and branched/isolated hyphae), and this could be directed to selectively produce one type of enzyme over the other. A morphologic scale (Y) is proposed, based on a form factor that considers the size and frequency of each morphology class, and points to conditions that result in high selectivity for either endoglucanase or β-glucosidase production, with co-production of xylanase. An equation that relates enzyme yield as a function of A. niger morphology provides a useful tool for tuning enzyme production.