Quinoa Stalks Glucuronoarabinoxylan: Biorefinery,xylooligosaccharides production and potential applications

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

Abstract

Quinoa stalks were used as a source of hemicellulose for XOs production and further as prebiotic source for probiotic bacteria. Three methods for extraction of hemicellulose were used, of which alkaline extraction (NaOH [0.5 M]) was the optimum method. This methodology allowed the establishment of a sequential extraction platform, including saponins obtention via PHWE, hemicellulose via alkaline extraction and cellulose via acid purification. Maximum yields of 15.4, 120 and 296 mg/g raw material of saponins, hemicellulose and cellulose yield were obtained, respectively. Saponins and hemicellulose extractions were dependent on PHWE conditions used, while cellulose extraction was not dependent on conditions in previous steps and resulted in purest fraction. The purified hemicellulose fraction consisted of glucuronoarabinoxylan (GAX) (HPAEC-PAD, FT-IR) with an estimated Mw of 1758 ± 31 kDa (SEC). The xylopyranosyl backbone was linked via β-(1,4) bonds, and substituted with 4-O-Methyl glucuronosyl and arabinofuranosyl residues. The later was linked as oligomers via α-(1-5) or as monomers α-(1-3) or α-(1-2) to the xylose skeleton (NMR). The monosaccharides composition of the GAX included xylose, glucuronic acid, arabinose and galactose in a molar ratio of 114:23:5:1, respectively. The GAX purified from quinoa stalks was treated by two methodologies to produce XOs: an enzymatic method (using xylanases) and a dilute acid method using H2SO4 [0.25 M]. In the enzymatic method, in house produced thermostable endoxylanases from GH10 (from R. marinus and B. halodurans) and the commercial GH11 PENTOPAN® , were used. The maximum yield of linear XOs obtained from the hydrolysis of GAX was 1.217 g XOs/100 g of GAX. Dilute acid treatment resulted in a maximum yield of 0.564 g XOs/100 g GAX. However, the DP in dilute acid treatment was more widely distributed, ranging from xylobiose to xylohexaose compared to the enzymatic method. B. adolescentis ATCC15703 and Weissella sp. strain 92 were cultivated using XOs from quinoa stalks GAX as carbon source. After 48 hours of cultivation, B. adolescentis grew to a maximum OD600 of 0.326, producing the followig amounts of SCFA and lactate (g/L): Acetate (1.243); lactate (1.013); propionate (0.812); formate (0.179) and; butyrate (0.124). Weissella sp. 92 grew to an OD600 of 0.626, producing Acetate (0.379 g/L) and lactate (0.259 g/L). The consumption of XOs by Weissella sp. 92 was mainly consuming monosaccharides (xylose and arabinose), and XOs: xylobiose, xylotriose and xylotetraose, while B. adolescentis was able to consume all linear XOs at different ratios, and was, moreover, able to consume substituted XOs (according to HPAEC-PAD). A draft genome sequencing of Clostridium boliviense strain E-1 was made, and a number of genes encoding hemicellulose-active enzymes were identified. Two enzymes were fully characterized; one two domain GH43-like endo-β-xylanase (CbE1Xyn43, MW 52.9 kDa) and one bifunctional acetyl esterase/endo-β-xylanase (CbE1Est1XynX, MW 44.2 kDa), also consisting of two domains (one carbohydrate esterase and one potential glycoside hydrolase). Both enzymes were thermostable and most active at neutral pH. Both enzymes also were active on birchwood glucuronoxylan, wheat bran arabinoxylan and quinoa stalks GAX. No xylosidase activity was determined. CbE1Xyn43 kinetic parameters was determined to Kcat (1.587 min-1) and Km (0.126 mM) using p-nitrophenyl xylobioside (pNPX2) as substrate. For CbE1Est1XynX the Kcat (6.645 min-1) and Km (0.233mM) was determined using pNPX2 and, using p-nitrophenyl acetate (pNPAc) Kcat was 243.363 min-1 and Km 2.25 mM. Despite the sequence similarity of CbE1Xyn43 to enzymes in GH43, the conserved catalytic residues of GH43 could not be identified, making classification of the enzyme difficult. In conclusion, the potential of quinoa stalks as a raw material for valorization has been demonstrated (separating saponins, GAX and cellulose) for further biorefinery applications. GAX in particular, resulted in successfully XOs production by enzymatic and acid methodologies for prebiotic usage. Also additional xylanases were explored, and were demonstrated as potential tools to diversify and increase linear and substituted XOs production from different material.

Detaljer

Författare
  • Daniel Salas Veizaga
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Teknik och teknologier

Nyckelord

Originalspråkengelska
KvalifikationDoktor
Tilldelande institution
Handledare/Biträdande handledare
Sponsorer för avhandling
  • Swedish International Development Cooperation Agency (SIDA)
Tilldelningsdatum2019 apr 25
Förlag
  • Department of Biotechnology, Lund University
Tryckta ISBN978-91-7422-647-8
StatusPublished - 2019 apr 25
PublikationskategoriForskning

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