Poly(l-lactide)-Degrading Enzyme Production by Laceyella sacchari LP175 Under Solid State Fermentation Using Low Cost Agricultural Crops and Its Hydrolysis of Poly(l-lactide) Film

  • Thanasak Lomthong
  • Rangrong Yoksan
  • Saisamorn Lumyong
  • Vichien KitpreechavanichEmail author
Original Paper



The aim of this study was to investigate the enhancement of poly(l-lactide) (PLLA)-degrading enzyme production by Laceyella sacchari LP175 under solid state fermentation (SSF) using low-cost agricultural crops as substrates and its hydrolysis of poly(l-lactide) film.


Cassava chip, soybean meal and corncob were used to investigate the enzyme production using a statistical mixture design method. The effect of various inducers and SSF growth conditions parameters affecting PLLA-degrading enzyme production were also investigated. The fermentation of solid substrates was up-scale in a static tray bioreactor. The crude enzyme extracted from the fermented solid substrate was used to evaluate the biological degradation of PLLA film at 50 °C for 24 h.


The results of substrate combination showed that 5 g of the substrate mixture, consisting of 4.3 g cassava chips and 0.7 g soybean meal, yielded 320 U/g dry solid. The addition of 0.1 g peptone to the mixture of solid materials increased enzyme production up to 456 U/g dry solid. The production of enzyme in a static tray bioreactor with optimized physical conditions yielded the maximum enzyme production, 472 U/g dry solid. The 20 mL reaction consisting of 8600 mg/L PLLA film was degraded by the crude enzyme extracted from the fermented solid substrate.


Agricultural crops and wastes contain significant amounts of nutrients for microbial growth and products. This is the first reported of PLLA degrading enzyme production under SSF using low-cost substrates showing the possibility for application in large-scale biological recycling of bio-plastic as a future sustainable process.


Poly(l-lactide)-degrading enzyme Laceyella sacchari LP175 Solid state fermentation Agricultural products Biodegradation 



A scholarship from the National Research Council of Thailand (NRCT) is gratefully acknowledged. This research has been supported by the Center of Excellence on Biodiversity (BDC) of the Higher Education Commission (Grant No. BDC-PG2-159012). Part of this research was financially supported by the Kasetsart University Research and Development Institute, and Center for Advanced Studies in Tropical Natural Resources, National Research University Kasetsart University (CASTNAR, NRU-KU). Part of this work was supported by the RGJ Advanced Programme (Grant No. RAP61K0008). Thanks to Carbios Company for kindly providing materials and substrates. The authors also thank ICEO, which is part of the PICT platform of Toulouse, for analytical facilities. Thanks to Campus France for management of financial support.

Compliance with Ethical Standards

Conflict of interest

The authors declared that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (DOCX 4269 KB)
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Supplementary material 2 (DOCX 31 KB)


  1. 1.
    Fan, Y., Nishida, H., Hoshihara, S., Shirai, Y., Tokiwa, Y., Endo, T.: Pyrolysis kinetics of poly(L-lactide) with carboxyl and calcium salt end structures. Polym. Degrad. Stab. 79, 547–562 (2003)CrossRefGoogle Scholar
  2. 2.
    Piemonte, V., Sabatini, S., Gironi, F.: Chemical recycling of PLA: a great opportunity towards the sustainable development. J. Polym. Environ. 21, 640–647 (2013)CrossRefGoogle Scholar
  3. 3.
    Jarerat, A., Tokiwa, Y., Tanaka, H.: Production of poly(L-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(L-lactide). Appl. Microbiol. Biotechnol. 72, 726–731 (2006)CrossRefGoogle Scholar
  4. 4.
    Youngpreda, A., Panyachanakul, T., Kitpreechavanich, V., Sirisansaneeyakul, S., Suksamrarn, S., Tokuyama, S., Krajangsang, S.: Optimization of poly(dl-Lactic acid) degradation and evaluation of biological re-polymerization. J. Polym. Environ. 25, 1–9 (2016)Google Scholar
  5. 5.
    Ambone, T., Joseph, S., Deenadayalan, E., Mishra, S., Jaisankar, S., Saravanan, P.: Polylactic acid (PLA) biocomposites filled with waste leather buff (WLB). J. Polym. Environ. 25, 1099–1109 (2017)CrossRefGoogle Scholar
  6. 6.
    Lomthong, T., Hanphakphoom, S., Kongsaeree, P., Srisuk, N., Guicherd, M., Cioci, G., Kitpreechavanich, V.: Enhancement of poly (L-lactide)-degrading enzyme production by Laceyella sacchari LP175 using agricultural crops as substrates and its degradation of poly (L-lactide) polymer. Polym. Degrad. Stab. 143,64–73 (2017)CrossRefGoogle Scholar
  7. 7.
    Sukkhum, S., Tokuyama, S., Kitpreechavanich, V.: Poly(L-lactide)-degrading enzyme production by Actinomadura keratinilytica T16-1 in 3 L airlift bioreactor and its degradation ability for biological recycle. J. Microbiol. Biotechnol. 22, 92–99 (2012)CrossRefGoogle Scholar
  8. 8.
    Hanphakphoom, S., Maneewong, N., Sukkhum, S., Tokuyama, S., Kitpreechavanich, V.: Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175. J. Gen. Appl. Microbiol. 60, 13–22 (2014)CrossRefGoogle Scholar
  9. 9.
    Robinson, T., Singh, D., Nigam, P.: Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl. Microbiol. Biotechnol. 55, 284–289 (2001)CrossRefGoogle Scholar
  10. 10.
    Thomas, L., Larroche, C., Pandey, A.: Current developments in solid-state fermentation. Biochem. Eng. J. 81, 146–161 (2013)CrossRefGoogle Scholar
  11. 11.
    Krishna, C.: Solid state fermentation systems—an overview. Crit. Rev. Biotechnol. 25, 1–30 (2005)CrossRefGoogle Scholar
  12. 12.
    John, R.P., Nampoothiri, K.M., Pandey, A.: Solid state fermentation for L-lactic acid production from agro wastes using Lactobacillus delbrueckii. Process Biochem. 41, 759–763 (2006)CrossRefGoogle Scholar
  13. 13.
    Zhang, B.B., Lu, L.P., Xu, G.R.: Why solid state fermentation is more advantageous over submerged fermentation for converting high concentration of glycerol into Monacolin K by Monascus purpureus 9901: a mechanistic study. J. Biotechnol. 206, 60–65 (2015)CrossRefGoogle Scholar
  14. 14.
    Raimbault, M.: General and microbiological aspects of solid substrate fermentation. Electron. J. Biotechnol. 1, 26–27 (1998)CrossRefGoogle Scholar
  15. 15.
    Kaur, P., Satyanarayana, T.: Production and starch saccharification by a thermostable and neutral glucoamylase of a thermophilic mould Thermomucorindicae-seudaticae. World J. Microbiol. Biotechnol. 20, 419–425 (2004)CrossRefGoogle Scholar
  16. 16.
    Lomthong, T., Hanphakphoom, S., Yoksan, R., Kitpreechavanich, V.: Co-production of poly(L-lactide)-degrading enzyme and raw starch-degrading enzyme by Laceyella sacchari LP175 using agricultural products as substrate, and their efficiency on biodegradation of poly(L-lactide)/thermoplastic starch blend film. Int. Biodeterior. Biodegrad. 104, 401–410 (2015)CrossRefGoogle Scholar
  17. 17.
    Sadaf, A., Khare, S.K.: Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis. Bioresour. Technol. 153, 126–130 (2014)CrossRefGoogle Scholar
  18. 18.
    de Castro, R.J.S., Ohara, A., Nishide, T.G., Bagagli, M.P., Dias, F.F.G., Sato, H.H.: A versatile system based on substrate formulation using agro industrial wastes for protease production by Aspergillus niger under solid state fermentation. Biocatal. Agric. Biotechnol. 4, 678–684 (2015)CrossRefGoogle Scholar
  19. 19.
    Trakarnpaiboon, S., Srisuk, N., Piyachomkwan, K., Sakai, K., Kitpreechavanich, V.: Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production. Prep. Biochem. Biotechnol. 47, 813–823 (2017)CrossRefGoogle Scholar
  20. 20.
    de Castro, R.J.S., Sato, H.H.: Synergistic effects of agroindustrial wastes on simultaneous production of protease and α-amylase under solid state fermentation using a simplex centroid mixture design. Ind. Crop. Prod. 49, 813–821 (2013)CrossRefGoogle Scholar
  21. 21.
    Thirunavukarasu, K., Purushothaman, S., Sridevi, J., Aarthy, M., Gowthaman, M.K., Nakajima-Kambe, T., Kamini, N.R.: Degradation of poly (butylene succinate) and poly (butylene succinate-co-butylene adipate) by a lipase from yeast Cryptococcus sp. grown on agro-industrial residues. Int. Biodeterior. Biodegrad. 110, 99–107 (2016)CrossRefGoogle Scholar
  22. 22.
    Lu, Y., Warner, R., Sedlak, M., Ho, N., Mosier, N.S.: Comparison of glucose/xylose cofermentation of poplar hydrolysates processed by different pretreatment technologies. Biotechnol. Prog. 25, 349–356 (2009)CrossRefGoogle Scholar
  23. 23.
    Sukkhum, S., Tokuyama, S., Kitpreechavanich, V.: Development of fermentation process for PLA- degrading enzyme production by a new thermophilic Actinomadura sp. T16-1. Biotechnol. Bioprocess. Eng. 14, 302–306 (2009)CrossRefGoogle Scholar
  24. 24.
    Sana, B., Ghosh, D., Saha, M., Mukherjee, J.: Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans. Process Biochem. 41, 208–215 (2006)CrossRefGoogle Scholar
  25. 25.
    Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)CrossRefGoogle Scholar
  26. 26.
    Lomthong, T., Lertwattanasakul, N., Kitpreechavanich, V.: Production of raw starch degrading enzyme by the thermophilic filamentous bacterium Laceyella sacchari LP175 and its application for ethanol production from dried cassava chips. Starch-Stärke 68, 1264–1274 (2016)CrossRefGoogle Scholar
  27. 27.
    Soni, S.K., Goyal, N., Gupta, J.K., Soni, R.: Enhanced production of α-amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in the hydrolysis of raw potato starch. Starch/Stärke 64, 64–77 (2012)CrossRefGoogle Scholar
  28. 28.
    Adinarayana, K., Ellaiah, P.: Response surface optimization of the critical medium components for the production of alkaline protease by a newly isolated Bacillus sp. J. Pharm. Pharm. Sci. 5, 272–278 (2002)Google Scholar
  29. 29.
    Mahanta, N., Gupta, A., Khare, S.K.: Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate. Bioresour. Technol. 99, 1729–1735 (2008)CrossRefGoogle Scholar
  30. 30.
    Patel, R.K., Dodia, M.S., Joshi, R.H., Singh, S.P.: Production of extracellular halo-alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. isolated from seawater in Western India. World J. Microbiol. Biotechnol. 22, 375–382 (2006)CrossRefGoogle Scholar
  31. 31.
    Pitol, L.O., Biz, A., Mallmann, E., Krieger, N., Mitchell, D.A.: Production of pectinases by solid-state fermentation in a pilot-scale packed-bed bioreactor. Chem. Eng. J. 283, 1009–1018 (2016)CrossRefGoogle Scholar
  32. 32.
    Apinya, T., Sombatsompop, N., Prapagdee, B.: Selection of a Pseudonocardia sp. RM423 that accelerates the biodegradation of poly(lactic) acid in submerged cultures and in soil microcosms. Int. Biodeterior. Biodegrad. 99, 23–30 (2015)CrossRefGoogle Scholar
  33. 33.
    Tokiwa, Y., Jarerat, A.: Biodegradation of poly(L-lactide). Biotechnol. Lett. 26, 771–777 (2004)CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Thanasak Lomthong
    • 1
  • Rangrong Yoksan
    • 2
  • Saisamorn Lumyong
    • 3
  • Vichien Kitpreechavanich
    • 4
    • 5
    Email author
  1. 1.Division of Biology, Faculty of Science and TechnologyRajamangala University of Technology ThanyaburiPathumthaniThailand
  2. 2.Department of Packaging and Materials Technology, Faculty of Agro-IndustryKasetsart UniversityBangkokThailand
  3. 3.Department of Biology, Faculty of ScienceChiang Mai UniversityChiang MaiThailand
  4. 4.Department of Microbiology, Faculty of ScienceKasetsart UniversityBangkokThailand
  5. 5.Center for Advanced Study in Tropical Natural ResourcesNational Research University-Kasetsart University (CASTNAR, NRU-KU), Kasetsart UniversityBangkokThailand

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