Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency.

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, ß-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.

Addition of metal ions to a (hemi)cellulolytic enzymatic cocktail produced in-house improves its activity, thermostability, and efficiency in the saccharification of pretreated sugarcane bagasse.

High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes.

Validation of a novel sequential cultivation method for the production of enzymatic cocktails from Trichoderma strains.

The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.

Liquefaction of sugarcane bagasse for enzyme production.

The objective of this paper is to report liquefaction of pretreated and sterilized sugarcane bagasse for enhancing endoglucanase production through submerged fermentation by Aspergillus niger. After initial solid state fermentation of steam pretreated bagasse solids by A. niger, fed-batch addition of the substrate to cellulase in buffer over a 12h period, followed by 36h reaction, resulted in a liquid slurry with a viscosity of 0.30±0.07Pas at 30% (w/v) solids. Addition of A. niger for submerged fermentation of sterile liquefied bagasse at 23% w/v solids resulted in an enzyme titer of 2.5IUmL(-1) or about 15× higher productivity than solid-state fermentation of non-liquefied bagasse (final activity of 0.17IUmL(-1)). Bagasse not treated by initial solid-state fermentation but liquefied with enzyme gave 2IUmL(-1). These results show the utility of liquefied bagasse as a culture medium for enzyme production in submerged fermentations.

Gas hold-up and oxygen mass transfer in three pneumatic bioreactors operating with sugarcane bagasse suspensions.

Sugarcane bagasse is a low-cost and abundant by-product generated by the bioethanol industry, and is a potential substrate for cellulolytic enzyme production. The aim of this work was to evaluate the effects of air flow rate (QAIR), solids loading (%S), sugarcane bagasse type, and particle size on the gas hold-up (εG) and volumetric oxygen transfer coefficient (kLa) in three different pneumatic bioreactors, using response surface methodology. Concentric tube airlift (CTA), split-cylinder airlift (SCA), and bubble column (BC) bioreactor types were tested. QAIR and %S affected oxygen mass transfer positively and negatively, respectively, while sugarcane bagasse type and particle size (within the range studied) did not influence kLa. Using large particles of untreated sugarcane bagasse, the loop-type bioreactors (CTA and SCA) exhibited higher mass transfer, compared to the BC reactor. At higher %S, SCA presented a higher kLa value (0.0448 s−1) than CTA, and the best operational conditions in terms of oxygen mass transfer were achieved for %S < 10.0 g L−1 and QAIR > 27.0 L min−1. These results demonstrated that pneumatic bioreactors can provide elevated oxygen transfer in the presence of vegetal biomass, making them an excellent option for use in three-phase systems for cellulolytic enzyme production by filamentous fungi.

Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulase.

Sequential solid-state and submerged cultivation with sugarcane bagasse as substrate for cellulase production by Aspergillus niger A12 was assessed by measuring endoglucanase activity. An unconventional pre-culture with an initial fungal growth phase under solid-state cultivation was followed by a transition to submerged fermentation by adding the liquid culture medium to the mycelium grown on solid substrate. For comparison, control experiments were conducted using conventional submerged cultivation. The cultures were carried out in shake flasks and in a 5-L bubble column bioreactor. An endoglucanase productivity of 57 ± 13 IU/L/h was achieved in bubble column cultivations prepared using the new method, representing an approximately 3-fold improvement compared to conventional submerged fermentation. Therefore, the methodology proposed here of a sequential fermentation process offers a promising alternative for cellulase production.