Research Article: Optimisation of enzymatic hydrolysis of apple pomace for production of biofuel and biorefinery chemicals using commercial enzymes

Date Published: June 20, 2015

Publisher: Springer Berlin Heidelberg

Author(s): Repson Gama, J. Susan Van Dyk, Brett I. Pletschke.


Apple pomace, a waste product from the apple juice industry is a potential feedstock for biofuel and biorefinery chemical production. Optimisation of hydrolysis conditions for apple pomace hydrolysis using Viscozyme L and Celluclast 1.5L was investigated using 1 L reaction volumes. The effects of temperature, pH, β-glucosidase supplementation and substrate feeding regimes were determined. Hydrolysis at room temperature using an unbuffered system gave optimal performance. Reactors in batch mode resulted in a better performance (4.2 g/L glucose and 16.8 g/L reducing sugar, 75 % yield for both) than fed-batch (3.2 g/L glucose and 14.6 g/L reducing sugar, 65.5 and 73.1 % yield respectively) in 72 h. The addition of Novozyme 188 to the core mixture of Viscozyme L and Celluclast 1.5L resulted in the doubling of glucose released. The main products (yield %) released from apple pomace hydrolysis were galacturonic acid (78 %), glucose (75 %), arabinose (90 %) and galactose (87 %). These products are potential raw materials for biofuel and biorefinery chemical production.

Partial Text

Apple pomace is the waste produced from the extraction of juice from apples. Large quantities of waste are produced worldwide every year, with 25–35 % of the 70 million metric tonnes produced ending up as waste (Food and Agricultural Organisation of the United Nations 2012; Gullon et al. 2008), an estimated 17–24 million metric tonnes per year (Van Dyk et al. 2013). In most cases, the pomace is disposed of, which poses major environmental and health problems due to its high moisture content (70–75 %), high chemical oxygen demand (COD, 10,000 mg/L) and biological oxygen demand (BOD) (Bhushan et al. 2008; Capek et al. 1995; Mahmood et al. 2010; Parmar and Rupasinghe 2013). Combining treatment of this waste with production of value-added products can solve disposal problems, while potentially creating additional revenue in the bio-economy.

Celluclast, Viscozyme and Biocip enzyme cocktails were tested in different combinations for activity on apple pomace. Selection was based on reports in the literature that indicated that Celluclast exhibited mainly cellulase activity; Viscozyme hemicellulase, arabinase, β-glucanase, cellulase and xylanase activities and Biocip polygalacturonase and cellulase activities. Combinations of Viscozyme and Biocip, or Celluclast and Biocip displayed a much lower yield of reducing sugars (glucose equivalents) than combinations of Viscozyme and Celluclast (See “Appendix”, Fig. 6). A combination of Viscozyme, Celluclast and Biocip displayed the same yields as the Viscozyme and Celluclast alone. These results indicated that a combination of Viscozyme and Celluclast was the best for the hydrolysis of apple pomace. Therefore, further experiments were performed using Celluclast and Viscozyme. Enzyme assays using different substrates were carried out to determine the enzyme activity profile for Celluclast and Viscozyme which are shown in Table 1.Table 1Activities of Viscozyme and Celluclast on different substratesSubstrateActivity measuredViscozymeCelluclastCarboxymethylcelluloseEndoglucanase263.6 (±1.6)385.1 (±2.4)Birchwood xylanEndoxylanase191.1 (±1.5)813.9 (±12.5)PectinPectinase1177.3 (±28.3)180.6 (±7.9)Locust bean gumEndomannanase406.5 (±10.7)124.4 (±0.8)Polygalacturonic acidPolygalacturonase1470.7 (15.6)149.6 (±2.7)Filter paperTotal cellulase33 (±0.3)95.2 (±0.9)4-Nitrophenyl-β-D-cellobiosideCellobiohydrolase0.004 (±0.0002)0.03 (±0.001)4-Nitrophenyl-β-D-glucopyranosideβ-D-glucosidase0.2 (±0.001)0.3 (±0.002)4-Nitrophenyl-β-D-mannopyranosideβ-D-mannosidase0.006 (±0.0002)0.0003 (±0.0001)4-Nitrophenyl-β-D-xylopyranosideβ-D-xylosidase0.005 (±0.0002)0.4 (±0.002)4-Nitrophenyl-α-L-arabinofuranosideα-L-Arabinofuranosidase0.4 (±0.002)0.06 (±0.001)Activities are expressed as reducing sugar equivalents released (µg/ml/min) per mg protein (endoglucanase, endoxylanase, endomannanase, pectinase, polygalacturonase and total cellulose) and 4-nitrophenol liberated (µmol/ml/min) per mg protein (cellobiohydrolase, β-D-glucosidase, β-D-xylosidase, β-D-mannosidase, α-L-arabinofuranosidase)Values are presented as mean values ± SD (n = 3)

The obtained results indicated that hydrolysis of apple pomace using Viscozyme L and Celluclast 1.5L can be successfully performed at room temperature without buffering, therefore lowering the operational costs involved in the treatment of this waste, while producing multiple products that can further contribute in value addition. β-glucosidase supplementation to Viscozyme L and Celluclast 1.5L was very important as it resulted in the doubling of the amount of glucose released over longer periods of incubation. The best results were obtained by operating the bioreactors in a batch mode producing 4.2 g/L glucose and 16.8 g/L reducing sugar (75 % yield for both glucose and reducing sugar) compared to the fed-batch bioreactors. Apple pomace hydrolysis products such as glucose, galacturonic acid, arabinose and galactose (75, 78, 90, and 87 % yield, respectively) can be further explored for value addition, making the treatment process more cost-effective. This study revealed that there were multiple and complementary enzymes present in both the Viscozyme and Celluclast cocktails. Employing similar hydrolysis conditions for the enzymes allow for the cost-effective application in industrial bioreactors. The inhibitory effect of various sugars on Viscozyme and Celluclast can be minimised using a SSF or a membrane bioreactor system.




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