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of its AX-AFH activity (pH 4) it had relatively low xylanase activity, hence this enzyme preparation was selected to investigate selective arabinose solubilisation from destarched ground corn fibre. Soaking in aqueous ammonia pretreatment was found to be necessary to make the hemicellulose structure accessible for the hemicellulolytic enzymes. During the enzymatic hydrolyses at pH 4 and 6 using Hemicellulase NS22002, high amounts of hemicellulosic oligomers, considerable amount of monomer arabinose and negligible amounts of other monomer sugars were solubilised. Enzymatic hydrolysis at pH 6 resulted in the solubilisation of more than 80% of the hemicellulose fraction and only 13% of the cellulose content within 2 days. Therefore, enzymatic hydrolysis of corn fibre using Hemicellulase NS22002 is a promising method to hydrolyse the hemicellulose fraction under mild reaction conditions, but it is not suitable for selective arabinose release.
Sulphuric acid treatments of corn fibre
Arabinose moieties are more sensitive against acid catalysed hydrolysis compared to the xylose units building up the hemicellulose backbone. Hence, in order to selectively release arabinose acid hydrolysis of destarched ground corn fibre and corn fibre was investigated at different temperatures, acid concentrations and reaction times according to experimental designs. Temperatures of 140°C and 120°C were found to be extremely high in terms of the selectivity of arabinose release, however, high arabinose yields were achieved. Acidic hydrolysis of destarched ground corn fibre at 5% (w/w) sulphuric acid concentration, 90°C and 5 min reaction time resulted in a total arabinose yield of 82.3%
with sufficient selectivity, hence an arabinose-rich liquid fraction was produced. During the investigations of acidic treatments of non-ground corn fibre at 90°C, the previous destarching step was omitted. The most favourable hydrolysis condition was determined by desirability function optimisation. At 1.1% (w/w) sulphuric acid concentration and 51 min reaction time, a total arabinose yield of 75.9% was achieved and the starch fraction was completely solubilised. Acidic hydrolysis of corn fibre under these conditions was referred to as first-hydrolysis in the two-step acidic fractionation process. After the first hydrolysis, considerable amount of oligosaccharides was obtained in the supernatant, thus an oligomer hydrolysis step (120°C, 1.1% (w/w) sulphuric acid, 30 min) was required to recover the sugars in monomeric form, which enabled to produce a glucose- and arabinose-rich supernatant. The solid residue of the first hydrolysis was utilised in a second acidic hydrolysis (120°C, 1.1% (w/w) sulphuric acid, 30 min,10% (w/w) dry matter), which resulted in a xylose-rich supernatant and a cellulose-rich solid fraction.
Arabinose biopurification and xylitol fermentation on semidefined media
Biopurification of hemicellulosic hydrolysate is an interesting and inexpensive strategy to produce pure arabinose solution through the depletion of other sugars using the adequate microorganisms. The capability of four yeast strains (C. boidinii, C. guilliermondii, C.
parapsilosis and H. anomala) for arabinose biopurification was tested on semidefined medium containing xylose, arabinose and galactose under aerobic conditions. C.
guilliermondii, C. parapsilosis and H. anomala utilised xylose, galactose and arabinose simultaneously, while C. boidiniidid not consume the arabinose, even if the other carbon sources had been depleted. Biopurification of semidefined media using C. boidinii resulted in an arabinose solution with an arabinose purity of 97%.
of its AX-AFH activity (pH 4) it had relatively low xylanase activity, hence this enzyme preparation was selected to investigate selective arabinose solubilisation from destarched ground corn fibre. Soaking in aqueous ammonia pretreatment was found to be necessary to make the hemicellulose structure accessible for the hemicellulolytic enzymes. During the enzymatic hydrolyses at pH 4 and 6 using Hemicellulase NS22002, high amounts of hemicellulosic oligomers, considerable amount of monomer arabinose and negligible amounts of other monomer sugars were solubilised. Enzymatic hydrolysis at pH 6 resulted in the solubilisation of more than 80% of the hemicellulose fraction and only 13% of the cellulose content within 2 days. Therefore, enzymatic hydrolysis of corn fibre using Hemicellulase NS22002 is a promising method to hydrolyse the hemicellulose fraction under mild reaction conditions, but it is not suitable for selective arabinose release.
Sulphuric acid treatments of corn fibre
Arabinose moieties are more sensitive against acid catalysed hydrolysis compared to the xylose units building up the hemicellulose backbone. Hence, in order to selectively release arabinose acid hydrolysis of destarched ground corn fibre and corn fibre was investigated at different temperatures, acid concentrations and reaction times according to experimental designs. Temperatures of 140°C and 120°C were found to be extremely high in terms of the selectivity of arabinose release, however, high arabinose yields were achieved. Acidic hydrolysis of destarched ground corn fibre at 5% (w/w) sulphuric acid concentration, 90°C and 5 min reaction time resulted in a total arabinose yield of 82.3%
with sufficient selectivity, hence an arabinose-rich liquid fraction was produced. During the investigations of acidic treatments of non-ground corn fibre at 90°C, the previous destarching step was omitted. The most favourable hydrolysis condition was determined by desirability function optimisation. At 1.1% (w/w) sulphuric acid concentration and 51 min reaction time, a total arabinose yield of 75.9% was achieved and the starch fraction was completely solubilised. Acidic hydrolysis of corn fibre under these conditions was referred to as first-hydrolysis in the two-step acidic fractionation process. After the first hydrolysis, considerable amount of oligosaccharides was obtained in the supernatant, thus an oligomer hydrolysis step (120°C, 1.1% (w/w) sulphuric acid, 30 min) was required to recover the sugars in monomeric form, which enabled to produce a glucose- and arabinose-rich supernatant. The solid residue of the first hydrolysis was utilised in a second acidic hydrolysis (120°C, 1.1% (w/w) sulphuric acid, 30 min,10% (w/w) dry matter), which resulted in a xylose-rich supernatant and a cellulose-rich solid fraction.
Arabinose biopurification and xylitol fermentation on semidefined media
Biopurification of hemicellulosic hydrolysate is an interesting and inexpensive strategy to produce pure arabinose solution through the depletion of other sugars using the adequate microorganisms. The capability of four yeast strains (C. boidinii, C. guilliermondii, C.
parapsilosis and H. anomala) for arabinose biopurification was tested on semidefined medium containing xylose, arabinose and galactose under aerobic conditions. C.
guilliermondii, C. parapsilosis and H. anomala utilised xylose, galactose and arabinose simultaneously, while C. boidiniidid not consume the arabinose, even if the other carbon sources had been depleted. Biopurification of semidefined media using C. boidinii resulted in an arabinose solution with an arabinose purity of 97%.
Xylitol fermentations on semidefined media containing xylose as carbon source were performed by using C. boidinii in order to determine the most favourable conditions in terms of xylitol yield. The effects of oxygen transfer rate, initial cell density, initial xylose concentration and cofactor (methanol) addition were investigated in shake flask experiments. A xylitol yield of 58% of theoretical was achieved by using 5 g/L initial cell concentration, 30 g/L initial xylose concentration at an oxygen transfer rate of 2.8 mmol/(L×h). The maximum xylitol concentration was obtained in one day, resulting in a xylitol volumetric productivity of 0.73 g/(L×h).
Integrated process of arabinose biopurification on the glucose- and arabinose-rich hydrolysate and xylitol fermentation on the xylose-rich hydrolysate
During aerobic biopurification, the undesired sugars are utilised mainly to produce cell mass, which occurs as by-product of the process. In the xylitol fermentation high initial cell concentration is required to achieve high xylitol yield and volumetric productivity. As C. boidinii was found to be suitable for both arabinose biopurification and xylitol fermentation, it seems to be reasonable to utilise the cell mass produced in the biopurification step as an inoculum in the xylitol fermentation. This kind of integration of the xylitol fermentation and the arabinose biopurification enables the utilization of the by-product cell mass of biopurification and results in a more effective carbon utilization, as the cell propagation of xylitol fermentation does not require additional carbon source or it does not consume xylose convertible into xylitol in the fermentation step. Utilization of on-site by-products and integration of different production routes are thought to be crucial to develop a viable biorefinery process. Considering those conceptions and the results of the acidic treatments of corn fibre, an integrated biorefinery process was invented, which is based on the two-step acidic fractionation of corn fibre and the diverse action of C.
boidinii(Figure 25). The two-step acidic fractionation of corn fibre resulted in a glucose-and arabinose-rich hydrolysate, a xylose-rich hydrolysate glucose-and a cellulose-rich solid fraction. The glucose- and arabinose-rich hydrolysate was utilised in arabinose biopurification after pH adjustment. After three days of biopurification the medium contained 9.2 g/L arabinose and 1 g/L galactose, hence the purity of arabinose was 90% of total sugars. The xylose-rich hydrolysate was utilised in xylitol fermentation after detoxification by activated carbon and pH adjustment. After the third day of xylitol fermentation the broth contained 10.4 g/L xylitol, 6.1 g/L arabinose, 4.1 g/L xylose (+galactose) and 2.7 g/L ethanol. As the xylitol concentration was approximately the same as the concentration of the residual sugars, further purification steps might be required to enable xylitol crystallization from the broth. The xylitol volumetric productivity was 0.14 g/(L×h).
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Figure 25: Process scheme of the proposed biorefinery process of corn fibre Process steps and material streams are indicated in capital letters and in italics,
respectively.
Future investigations
Utilisation of the cellulose-rich solid fraction is one of the main issues of the further development of the proposed biorefinery process. Preliminary studies have shown that the cellulose-rich solid fraction can be easily hydrolysed by commercial cellulase enzyme preparations using relatively high solid loading (15 (w/w) dry matter). Hence a glucose-rich liquid fraction can be obtained, which might be utilised to produce bioethanol or other value-added products via fermentation.
Investigation of purification techniques and recovery processes of arabinose and xylitol from the fermented broths are also crucial in terms of the development of a viable process.
Connected to this issue, it is important to increase the achievable arabinose and xylitol concentrations.
Figure 25: Process scheme of the proposed biorefinery process of corn fibre Process steps and material streams are indicated in capital letters and in italics,
respectively.
Future investigations
Utilisation of the cellulose-rich solid fraction is one of the main issues of the further development of the proposed biorefinery process. Preliminary studies have shown that the cellulose-rich solid fraction can be easily hydrolysed by commercial cellulase enzyme preparations using relatively high solid loading (15 (w/w) dry matter). Hence a glucose-rich liquid fraction can be obtained, which might be utilised to produce bioethanol or other value-added products via fermentation.
Investigation of purification techniques and recovery processes of arabinose and xylitol from the fermented broths are also crucial in terms of the development of a viable process.
Connected to this issue, it is important to increase the achievable arabinose and xylitol concentrations.