Characterization of yeast mutant strains for starter culture in Arabica coffee fermentation.

Arabica coffee is the most popular and best-selling type of coffee. During coffee fermentation, microorganisms are essential for the production of metabolites and volatile compounds that affect coffee flavor quality. This work aimed to study the mutation, selection, and characterization of the Wickerhamomyces anomalus strain YWP1-3 as a starter culture to enhance the flavor quality of Arabica coffee. The results revealed that six mutants could produce relatively high levels of the pectinase enzyme on pectin agar media and exhibited high activity levels, ranging from 332.35 to 415.88 U/ml in mucilage broth. Strains UV22-2, UV22-3, UV41-1 and UV32-1 displayed higher levels of amylase activity than did the wild type. The UV22-2 and UV22-3 mutants exhibited the highest pectin degradation indices of 49.22% and 45.97%, respectively, and displayed significantly enhanced growth rates in nitrogen yeast base media supplemented with various sugars; thus, these mutants were evaluated for their ability to serve as a starter for fermentation of Arabica coffee. The cupping scores of coffees derived from UV22-2 and UV22-3 were 83.5 ± 1.5 and 82.0 ± 2.14, respectively. The volatile compounds in the roasted coffee fermented by UV22-2 were analyzed by GC‒MS, which revealed higher levels of furfuryl alcohol and furfuryl acetate than did the other samples. These findings suggested that UV22-2 could be an influential starter culture for Arabica coffee fermentation.

Towards a Miniaturized Culture Screening for Cellulolytic Fungi and Their Agricultural Lignocellulosic Degradation.

The substantial use of fungal enzymes to degrade lignocellulosic plant biomass has widely been attributed to the extensive requirement of powerful enzyme-producing fungal strains. In this study, a two-step screening procedure for finding cellulolytic fungi, involving a miniaturized culture method with shake-flask fermentation, was proposed and demonstrated. We isolated 297 fungal strains from several cellulose-containing samples found in two different locations in Thailand. By using this screening strategy, we then selected 9 fungal strains based on their potential for cellulase production. Through sequence-based identification of these fungal isolates, 4 species in 4 genera were identified: Aspergillus terreus (3 strains: AG466, AG438 and AG499), Penicillium oxalicum (4 strains: AG452, AG496, AG498 and AG559), Talaromyces siamensis (1 strain: AG548) and Trichoderma afroharzianum (1 strain: AG500). After examining their lignocellulose degradation capacity, our data showed that P. oxalicum AG452 exhibited the highest glucose yield after saccharification of pretreated sugarcane trash, cassava pulp and coffee silverskin. In addition, Ta. siamensis AG548 produced the highest glucose yield after hydrolysis of pretreated sugarcane bagasse. Our study demonstrated that the proposed two-step screening strategy can be further applied for discovering potential cellulolytic fungi isolated from various environmental samples. Meanwhile, the fungal strains isolated in this study will prove useful in the bioconversion of agricultural lignocellulosic residues into valuable biotechnological products.

Effects of acid and alkali promoters on compressed liquid hot water pretreatment of rice straw.

In this study, effects of homogeneous acid and alkali promoters on efficiency and selectivity of LHW pretreatment of rice straw were studied. The presences of acid (0.25%v/v H2SO4, HCl, H3PO4, and oxalic acid) and alkali (0.25 w/v NaOH) efficiently promoted hydrolysis of hemicellulose, improved enzymatic digestibility of the solids, and lower the required LHW temperature. Oxalic acid was a superior promoter under the optimal LHW conditions at 160 °C, leading to the highest glucose yield from enzymatic hydrolysis (84.2%) and the lowest formation of furans. Combined with hydrolyzed glucose in the liquid, this resulted in the maximal 91.6% glucose recovery from the native rice straw. This was related to changes in surface area and crystallinity of pretreated biomass. The results showed efficiency of external promoters on increasing sugar recovery and saving energy in LHW pretreatment.

Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, ß-glucosidase, xylanase, and ß-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of ß-glucosidase and core hemicellulases (xylanase and ß-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external ß-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.

Autohydrolysis of tropical agricultural residues by compressed liquid hot water pretreatment.

Pretreatment is an essential step in biorefineries for improving digestibility of recalcitrant agricultural feedstocks prior to enzymatic hydrolysis to composite sugars, which can be further converted to fuels and chemicals. In this study, autohydrolysis by compressed liquid hot water (LHW) pretreatment of various tropical agricultural residues including sugarcane bagasse (BG), rice straw (RS), corn stover (CS), and empty palm fruit bunch (EPFB) was investigated. It was found that LHW pretreatment at 200 °C for 5-20 min resulted in high levels of hemicellulose solubilization into the liquid phase and marked improvement on enzymatic digestibility of the solid cellulose-enriched residues. The maximal yields of glucose and pentose were 409.8-482.7 mg/g and 81.1-174.0 mg/g of pretreated substrates, respectively. Comparative analysis based on severity factor showed varying susceptibility of biomass to LHW in the order of BG> RS> CS> EPFB. Structural analysis revealed surface modification of the pretreated biomass along with an increase in crystallinity index. Overall, 75.7-82.3 % yield of glucose and 27.4-42.4 % yield of pentose from the dried native biomass was recovered in the pretreated solid residues, while 18.3-29.7 % of pentoses were recovered in the liquid phase with dehydration by-product concentration under the threshold for ethanologens. The results suggest the potential of LHW as an efficient pretreatment strategy for implementation in biorefineries operated using various seasonal agricultural feedstocks.

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation.