Study on Xylan Degradation System of New Isolated Thermophilic Facultatively Anaerobic Bacterium Paenibacillus sp. Strain DA-C8

概要

Xylan is an abundant and renewable resource from many biomass materials, including empty fruit, oil palm, corn hull/straw, rice straw, and birch wood. To expand and add value on this biomass resources, microbial xylanase production has been considered for bioconversion of the plant biomass and used in various biotechnological applications like biofuel production, pulp, and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper.

Currently, researchers revealed the xylanase produced from microbial used for commercial-level production of enzymes is limited due to the low yield and do not meet the demand of industries level as well as stability of pH value and temperature. Additionally, xylanase microbial from the thermophilic condition is less information. In this research, screening, identification, and study of xylanolytic bacteria's functional properties were conducted. To obtain a bacterium exhibiting efficient xylan-degradation ability - a thermophilic, facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) as a pure culture was isolated from compost. Strain DA-C8 was observed under light microscopy after gram staining and by SEM and TEM. The strain was Gram-negative, and cell morphology was straight or slightly straight curve rod shape, a non-motile bacterium with 0.4-0.5 µm in diameter and 2-7 µm in length. Physiological characteristic of strain DA-C8 was studied. In terms of optimum condition for cell growth, it was at 55ºC with pH value 9.0 under anaerobic condition, 1% of xylose or glucose was used as a carbon source in BM. Strain can utilize variety of carbon sources, including L-arabinose, D-fructose, D-mannose, raffinose, D-glucose, D- xylose, cellobiose, maltose, sucrose, ribose, L-galactose. Strain DA-C8 can hydrolyze xylan, aesculin, arabinoxylan, galactan, pectin but could not hydrolyze Cellulose, gelatin, casein, chitin, CMC. To confirm the ability of xylan degradation with strain DA-C8, strain can degrade completely 1% beechwood xylan in 4 days under anaerobic conditions. From 16S rRNA and ANI sequence analysis, strain DA-C8 was closely related to many Paenibacillus sp and to Xylanibacillus composti K-13, known to utilize xylan as carbon sources. The observation and parameters below were conducted to confirm that strain DA-C8 belongs to a member of the genus’s Paenibacillus sp or Xylanibacillus sp,
To understand the xylan degradation system, the genes related to the xylan degradation in DA-C8 and K-13 were compared; beechwood xylan and corn hull biomass degradation were investigated, cell mass and protein of cells of both strains were measured, and extracellular enzymes from anaerobic or aerobic growth conditions were analyzed. The result revealed that the predicted genes regarding β-1,4-xylanases and endo-β-1,4-glucanases belonging to GH family-5, 9, 10, and 11 were found in the genome sequence of both strains. Meanwhile, genes of β-glucosidases and β-xylosidases belonging to GH family-3 and 16 were abundantly confirmed in genome sequence of stain DA-C8. For debranching genes, called the accessory enzymes such as α-L-arabinofuranosidase, β-galactosidase, ɑ-glucosidase, and polygalacturonase were also confirmed to the genome of strain DA-C8. Strain DA-C8 was revealed to equip various genes regarding lignocellulose degradation more than X. composti K-13. Extracellular fraction of strain DA-C8 showed relatively high degradation activity of 33.00±0.03 U/mg toward xylan but low activity for crystalline cellulose. Optimum pHs and temperatures of the extracellular fractions of strain DA-C8 and X. composti K-13 were observed at pH 5.0-8.0 and pH 6.0-8.0, and 50-70°C and 50-55°C, respectively.

Extracellular fraction of X. composti K-13 was two times lower xylanase ability than that of strain DA-C8. Furthermore, extracellular fraction from strain DA-C8 possessed high β- xylosidase, β-glycosidase, and α-L-arabinofuranosidase activities compared with those of X. composti K-13. These results were an agreement to high xylan and plant biomass degradation abilities with strain DA-C8, indicating that high β-xylosidase, β-glycosidase and α-L- arabinofuranosidase activities of DA-C8 can enhance the degradation ability because these enzymes be able to avoid not only enzymatic feedback inhibition by xylobiose and cellobiose, but also debranching enzyme can remove arabinose substituents from arabinoxylan and arabinoxylooligomers, thereby, improving bioconversion of lignocellulosic biomass. When strain DA-C8 was grown on BM using xylose as the sole carbon source, the growth speed and cell densities under anaerobic growth conditions were slightly slower than aerobic growth conditions; however, both states were not a big difference and showed good growths. In contrast, growth of strain DA-C8 was drastically decreased on BM medium supplemented xylan as sole carbon sources under aerobic growth conditions. The cell density as seen from total proteins was two times lower than that of the anaerobic culture condition. These results indicate that strain DA-C8 especially prefers anaerobic conditions when xylan is used as carbon source and needs to shift the metabolisms from aerobic to anaerobic conditions to obtain an efficient xylan degradation ability. As a result, xylan degradation ability of strain DA-C8 decreased only 27% under aerobic culture conditions as well as its growth curves, but strain can degrade 100% until four days under anaerobic conditions. These results indicated that the xylan degradation system of strain DA-C8 might depressed by aerobically growth conditions. The xylanase activity in extracellular fraction prepared from a culture of strain DA-C8 under aerobic growth condition was observed 1.24±0.01 unit/mg protein as xylanase activity. Strain DA-C8 indicated significantly high xylan degradation ability and enzymatic activity under anaerobic culture conditions. However, the degradation ability was deficient in aerobic growth conditions. These results suggest that the xylan degradation activity of DA-C8 may be strictly regulated by aerobically growth conditions.
Based on the above result, a new isolated strain DA-C8 should be represented in
Paenibacillus sp.

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