Impact of Ammonia Pretreatment Conditions on the Cellulose III Allomorph Ultrastructure and Its Enzymatic Digestibility

Leonardo Da Costa Sousa, James Humpula, Venkatesh Balan, Bruce E. Dale, Shishir P.S. Chundawat

Research output: Contribution to journalArticle

4 Scopus citations


Plant biomasses enriched in crystalline cellulose allomorphs, such as native cellulose I (CI), can be structurally altered using anhydrous liquid ammonia to form an enzymatically less recalcitrant cellulose III (CIII) allomorph. Here, we designed and implemented an advanced ammonia pretreatment reactor/sampler setup that allowed us to systematically study the impact of several process parameters on ammonia-cellulose complex and ultimately CIII formation, including ammonia-to-cellulose concentration, ammonia/cosolvent concentration, pretreatment time, and temperature. Pretreated cellulose ultrastructural characterization was performed using complementary X-ray diffraction-, FTIR-, and FT-Raman spectroscopy-based techniques. We found that the amorphous content of cellulose increased initially when the intermediate ammonia-cellulose complex was formed within the first 30 s of pretreatment. However, a reduction in the amorphous content was observed if the complex was annealed for longer periods of time and/or at high temperatures, resulting in a highly crystalline and well-ordered CIII allomorph. However, depending on the exact pretreatment conditions tested, CIII-"like" cellulosic substrates with varying allomorphic ultrastructures and crystalline order were formed. Interestingly, CIII-like allomorphs with higher crystallinity were more easily hydrolyzed by cellulase enzymes compared to native CI and lower crystallinity CIII-like substrates. This work highlights the challenges associated with systematically conducting ammonia treatments, interpretation of the CIII ultrastructure using appropriate analytical techniques, and the influence of cellulose ultrastructure on enzymatic digestibility in light of previous work. Finally, we explored reduced severity and more cost-effective pretreatment conditions that could lower recalcitrance of cellulose by producing crystalline CIII and enable adoption of more advanced ammonia-based pretreatments in cellulosic biorefineries.

Original languageEnglish (US)
Pages (from-to)14411-14424
Number of pages14
JournalACS Sustainable Chemistry and Engineering
Issue number17
StatePublished - Sep 3 2019

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Environmental Chemistry
  • Chemical Engineering(all)
  • Renewable Energy, Sustainability and the Environment


  • ammonia pretreatment
  • biofuels
  • cellulases
  • cellulose allomorphs
  • cellulose crystallinity

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