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Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

Rodrigues MA, Teixeira RS, Ferreira-Leitão VS, da Silva Bon EP - Biotechnol Biofuels (2015)

Bottom Line: The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes.High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity.These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Applied Photosynthesis Laboratory, Athos Avenida da Silveria Ramos, 149-Technology Centre, Block A, Room 532, University City, Rio de Janeiro, RJ 21941-909 Brazil.

ABSTRACT

Background: Chlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iβ polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus β-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low β-glucosidase; Aspergillus awamori, which secretes endoglucanases and β-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori β-glucosidase were evaluated.

Results: The highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high β-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and β-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme β-glucosidase load, indicating that the endoglucanase load was not rate-limiting.

Conclusions: High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

No MeSH data available.


Diffractograms ofC. homosphaerabiomass (continuous line, right axis) and Avicel Fluka (split line, left axis). X-ray diffraction was performed using a Rigaku MiniFlex diffractometer and filtered copper Kα radiation.
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Fig5: Diffractograms ofC. homosphaerabiomass (continuous line, right axis) and Avicel Fluka (split line, left axis). X-ray diffraction was performed using a Rigaku MiniFlex diffractometer and filtered copper Kα radiation.

Mentions: The fact that the A. awamori enzymes, which are devoid of exoglucanase activity, hydrolyzed C. homosphaera biomass at high rates and that the β-glucosidase activity load influences the hydrolysis yields and sometimes the initial rate, raised a question regarding the nature of the microalgal cell wall glucan. Chlorophytes are known to be enriched with cellulose type Iα, which is more susceptible to degradation than cellulose type Iβ, which is predominant in Charophyta and land plants [22,25,26,49]. Although the XRD of microalgae cellulose is generally similar to that of land plants, and except for some peaks at 14° and 16° that are not found in land plants [27], our results showed otherwise. Indeed, Figure 5 shows the X-ray diffractometry of C. homosphaera biomass and, for comparison, that of pure crystalline cellulose. The figure shows that, contrary to the data for crystalline cellulose, C. homosphaera biomass has a very low crystallinity, such that it was not possible to determine its degree of crystallinity. However, it was possible to identify a clear peak near 15°, which is typical for microalgae [27]. Remarkably, the XRD of C. homosphaera closely resembles that of the ball-milled XRD of microcrystalline cellulose after enzymatic hydrolysis, as found by Teixeira et al. [50], who also showed that cellobiohydrolase activity is not required to efficiently hydrolyze amorphous cellulose. These results suggest that the cell wall polysaccharides of C. homosphaera is mostly amorphous and, as such, C. homosphaera would be categorized as belonging to group 2, as established by Nicolai and Preston [17]. However, the low crystallinity may also be an artifact of biomass preparation; under stress, chlorophytes secrete a mucilaginous substance that was observed during the centrifugation process. In addition, the presence of algaenan in the cell wall may create structural changes and amorphous zones during the drying process, as suggested by Mihranyan [27]. However, regardless of whether the centrifugation and drying processes altered the cell wall polysaccharide structure, the dried C. homosphaera biomass showed such a low crystallinity that the enzyme mixtures devoid of exoglucanase activity were able to efficiently hydrolyze 40.5% of the algal glucan. In terms of biomass processing and enzyme use, this feature poses a great advantage for the biorefinery concept because it simplifies raw material processing and enzyme blend complexity, thereby reducing processing costs.Figure 5


Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

Rodrigues MA, Teixeira RS, Ferreira-Leitão VS, da Silva Bon EP - Biotechnol Biofuels (2015)

Diffractograms ofC. homosphaerabiomass (continuous line, right axis) and Avicel Fluka (split line, left axis). X-ray diffraction was performed using a Rigaku MiniFlex diffractometer and filtered copper Kα radiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356055&req=5

Fig5: Diffractograms ofC. homosphaerabiomass (continuous line, right axis) and Avicel Fluka (split line, left axis). X-ray diffraction was performed using a Rigaku MiniFlex diffractometer and filtered copper Kα radiation.
Mentions: The fact that the A. awamori enzymes, which are devoid of exoglucanase activity, hydrolyzed C. homosphaera biomass at high rates and that the β-glucosidase activity load influences the hydrolysis yields and sometimes the initial rate, raised a question regarding the nature of the microalgal cell wall glucan. Chlorophytes are known to be enriched with cellulose type Iα, which is more susceptible to degradation than cellulose type Iβ, which is predominant in Charophyta and land plants [22,25,26,49]. Although the XRD of microalgae cellulose is generally similar to that of land plants, and except for some peaks at 14° and 16° that are not found in land plants [27], our results showed otherwise. Indeed, Figure 5 shows the X-ray diffractometry of C. homosphaera biomass and, for comparison, that of pure crystalline cellulose. The figure shows that, contrary to the data for crystalline cellulose, C. homosphaera biomass has a very low crystallinity, such that it was not possible to determine its degree of crystallinity. However, it was possible to identify a clear peak near 15°, which is typical for microalgae [27]. Remarkably, the XRD of C. homosphaera closely resembles that of the ball-milled XRD of microcrystalline cellulose after enzymatic hydrolysis, as found by Teixeira et al. [50], who also showed that cellobiohydrolase activity is not required to efficiently hydrolyze amorphous cellulose. These results suggest that the cell wall polysaccharides of C. homosphaera is mostly amorphous and, as such, C. homosphaera would be categorized as belonging to group 2, as established by Nicolai and Preston [17]. However, the low crystallinity may also be an artifact of biomass preparation; under stress, chlorophytes secrete a mucilaginous substance that was observed during the centrifugation process. In addition, the presence of algaenan in the cell wall may create structural changes and amorphous zones during the drying process, as suggested by Mihranyan [27]. However, regardless of whether the centrifugation and drying processes altered the cell wall polysaccharide structure, the dried C. homosphaera biomass showed such a low crystallinity that the enzyme mixtures devoid of exoglucanase activity were able to efficiently hydrolyze 40.5% of the algal glucan. In terms of biomass processing and enzyme use, this feature poses a great advantage for the biorefinery concept because it simplifies raw material processing and enzyme blend complexity, thereby reducing processing costs.Figure 5

Bottom Line: The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes.High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity.These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Applied Photosynthesis Laboratory, Athos Avenida da Silveria Ramos, 149-Technology Centre, Block A, Room 532, University City, Rio de Janeiro, RJ 21941-909 Brazil.

ABSTRACT

Background: Chlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iβ polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus β-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low β-glucosidase; Aspergillus awamori, which secretes endoglucanases and β-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori β-glucosidase were evaluated.

Results: The highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high β-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and β-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme β-glucosidase load, indicating that the endoglucanase load was not rate-limiting.

Conclusions: High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

No MeSH data available.