Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for Agave Atrovirens lignocellulosic biomass hydrolysis.

In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.

Agave biotechnology: an overview.

Agaves are plants of importance both in Mexican culture and economy and in other Latin-American countries. Mexico is reported to be the place of Agave origin, where today, scientists are looking for different industrial applications without compromising its sustainability and preserving the environment. To make it possible, a deep knowledge of all aspects involved in production process, agro-ecological management and plant biochemistry and physiology is required. Agave biotechnology research has been focusing on bio-fuels, beverages, foods, fibers, saponins among others. In this review, we present the advances and challenges of Agave biotechnology.

Comparison of dilute acid pretreatment of agave assisted by microwave versus ultrasound to enhance enzymatic hydrolysis.

A comparison between microwave and ultrasound irradiations in the agave pretreatment using dilute sulfuric acid as catalyst was assessed for the first time. Pretreatments were performed using a Taguchi Orthogonal Array L9 (34) to improve the hemicellulose removal and the agave digestibility. The results showed that under optimal conditions, the hemicellulose removal was superior in the pretreatment assisted with microwave (77.5%) compared to ultrasound (28.2%). Enzymatic hydrolysis yield of agave pretreated with microwave (MWOC) was 2-fold higher than agave pretreated with ultrasound (USOC). The relatively mild conditions of pretreatment with MWOC allowed to obtain a hydrolyzed free of inhibitors with a high glucose concentration (47.7 g/L) at low solids loading (10% w/v). However, these conditions did not have a significant effect over the agave pretreated with ultrasound. The pretreatment assisted with MWOC allowed to reduce time and temperature of the process compared to pretreatment with conventional heating.

Clostridium strain selection for co-culture with Bacillus subtilis for butanol production from agave hydrolysates.

In this work, three Clostridium strains were tested for butanol production from Agave lechuguilla hydrolysates to select one for co-culturing. The agave hydrolysates medium was supplemented with nutrients and reducing agents to promote anaerobiosis. Clostridium acetobutylicum ATCC 824 had the highest butanol production (6.04 g/L) and was selected for further analyses. In the co-culture process, Bacillus subtilis CDBB 555 was used to deplete oxygen and achieve anaerobic conditions required for butanol production. The co-culture was prepared with C. acetobutylicum and B. subtilis without anaerobic pretreatment. Butanol production in co-culture from agave hydrolysates was compared with experiments using synthetic medium with glucose and a pure culture of C. acetobutylicum. The maximum butanol concentration obtained was 8.28 g/L in the co-cultured hydrolysate medium. Results obtained in the present work demonstrated that agave hydrolysates have the potential for butanol production using a co-culture of B. subtilis and C. acetobutylicum without anaerobic pretreatment.

Solid bioprocess of tarbush (Flourensia cernua) leaves for ß-glucosidase production by Aspergillus niger: initial approach to fiber-glycoside interaction for enzyme induction.

Commercial cellulase production has increased in recent years and consistent research has been carried out to improve levels of ß-glucosidase. Bioprocesses have been successfully adapted to produce this enzyme, with solid-state fermentations as the best-suited technique involving fungi. The aim of this study was to use leaves of tarbush (Flourensia cernua), an abundant shrub of the Chihuahuan Desert, as a carbon source for ß-glucosidase production by Aspergillus niger. During the solid bioprocess, this enzyme reached its peak production at 36 h of culture with 3876.6 U/L. There is a particular interest in the substrate composition because of the possibility of phenolic glycosides having an important role in ß-glucosidase production. HPLC-MS analyses showed that glycosides were present with the highest accumulation at 36 h of fungal culture. Luteolin and apigenin glycosides [1.8 and 2.4 absorbance units, respectively] were also detected and showed their highest point of detection alongside the highest ß-glucosidase activity. No apparent changes in cellulose were observed, while hemicellulose content decreased, which could be related to production and activity of ß-glucosidase. This study shows that leaves of F. cernua are an important raw material for ß-glucosidase production and give a source of compounds of added value which also may have an important role for ß-glucosidase production.

Effect of growth conditions on ß-glucosidase production using Flourensia cernua leaves in a solid-state fungal bioprocess.

Flourensia cernua foliage was used in a solid-state fungal bioprocess to identify factors that could affect ß-glucosidase production such as growth medium components and partial identification of molecules from the plant material. Under an exploratory experimental design, each variable had their distinctive result on conditions, which affects and could further improve ß-glucosidase production. Under the experimental design, 1482 U/L of ß-glucosidase were detected, which marks an improvement in production compared to levels obtained in a control treatment with an activity of 1092 U/L. It was shown that inoculum, water content and pH were the factors with the greater effect on ß-glucosidase production. Polyphenolic content and cellulosic fiber in the form of raw fiber were measured to assess compound degradation of the plant material. Although fiber content was apparently unaffected, polyphenolic content decreased; ß-glucosidase was produced by A. niger GH1. This behavior could be associated with fiber level and polyphenolic content because molecules of this type can be hydrolyzed by ß-glucosidase. According to our results, F. cernua biomass can be used as a carbon source for ß-glucosidase production in a short culture time.

Study of enzymatic saccharification of Agave leaves biomass to yield fermentable sugars.

Agave is a good source of polysaccharides for the production of fermentable sugars as sustainable bioenergy feedstock solutions for semi-arid and arid lands. This plant grows in arid areas, which correspond to a large territory in northern Mexico. Having lignocellulose as the polysaccharide of interest, the information for the enzymatic saccharification of this kind of material is limited. Agave cell walls have a unique recalcitrant nature, but having a high cellulose content, makes this plant material an interesting research subject. In this work, acidic, alkaline and aqueous pretreatments were evaluated to generate a biomass rich in cellulose. The saccharification of pretreated Agave leaves-residue was evaluated under experimental designs to identify the most suitable conditions for enzymatic hydrolysis. Maximum value obtained was 31% glucose, which further increased to 41.4% at extended hydrolysis time of 96 h. The highest cellulose-saccharification reached was up to 61.81%, making Agave atrovirens an alternative for bioethanol production in its geographical area of cultivation.