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New ammonia biomass pretreatment process improves yield with lower enzyme loading; improving cellulosic biofuel economics

A team from the US, China and India, led by researchers from Michigan State University, has developed a new liquid ammonia biomass pretreatment methodology called Extractive Ammonia (EA). EA-pretreated corn stover delivers a higher fermentable sugar yield compared to the older Ammonia Fiber Expansion (AFEX) process while using 60% lower enzyme loading.

As described in a paper in the RSC journal Energy & Environmental Science, the single-stage EA process achieves high biofuel yields (18.2 kg ethanol per 100 kg untreated corn stover, dry weight basis), comparable to those achieved using ionic liquid pretreatments. The EA process achieves these ethanol yields at industrially-relevant conditions using low enzyme loading (7.5 mg protein per g glucan) and high solids loading (8% glucan, w/v).

The EA-pretreated biomass hydrolysates are readily fermentable due to removal of lignin-derived inhibitors while preserving the microbial nutrient availability.

The EA process also preserves extracted lignin functionalities, offering the potential to co-produce lignin-derived fuels and chemicals in the biorefinery.

Process design differences between Extractive Ammonia (EA) and AFEX pretreatment. (a) EA laboratory equipment set up and mass balances for pretreatment performed at 120 ˚C, 6:1 ammonia:biomass weight ratio (NH3:BM) for 30 min residence time on corn stover with 10% (w/w) moisture (dry weight basis). (b) AFEX laboratory equipment setup and mass balances for optimal AFEX conditions to pretreat corn stover. Source: Sousa et al. Click to enlarge.

EA pretreatment involves a three-stage process: reaction; extraction; and product/solvent recovery. In the reaction process, liquid ammonia and biomass are combined in a reactor at a sufficiently high loading to fully immerse the biomass at a defined temperature and residence time. Unlike the older AFEX process (earlier post), external heat is required during EA pretreatment to increase reaction temperature due to the absence of high moisture levels.

As temperature increases, ammonia pressure builds up until a new vapor–liquid equilibrium is established. During this stage, the cellulose–ammonia complex is formed, ester bonds are cleaved, and lignin is partly solubilized in the liquid ammonia phase. Similarly to the AFEX process, EA pretreatment promotes ammonolysis of cell wall ester crosslinks that are particularly abundant in monocots. These key reactions disrupt lignin–polysaccharide crosslinks, thereby enabling biomass deconstruction by improving access of enzymes to embedded structural carbohydrates.

In the extraction stage, the EA-pretreated biomass is filtered to separate the ammonia-soluble components from residual solids. During this stage, lignin is extracted, and CIII—a highly digestible cellulose allomorph—is formed from the cellulose–ammonia complex as ammonia is continuously removed from the biomass into an extract-collection vessel.

Nitrogen overpressure is used to maintain ammonia in the liquid state at constant temperature.

During the recovery stage, the ammonia is evaporated from the extractives, which are subsequently recovered as a dark brown viscous liquid. About 0.022 g ammonia per 100 g biomass input cannot be recycled due to reactions between ammonia and the biomass. The remaining ammonia is recoverable and can be recycled.

EA simultaneously converts native crystalline cellulose to a highly digestible cellulose allomorph and selectively extracts up to ∼45% of the lignin from lignocellulosic biomass with near-quantitative retention of all polysaccharides.

Although CIII can be produced at room temperature, the EA pretreatment is more effective at higher temperatures, which are required to maximize ester bond cleavage, lignin solubilization, and thereby improve enzyme accessibility to CIII.


  • Leonardo da Costa Sousa, Mingjie Jin, Shishir P. S. Chundawat, Vijay Bokade, Xiaoyu Tang, Ali Azarpira, Fachuang Lu, Utku Avci, James Humpula,a Nirmal Uppugundla, Christa Gunawan, Sivakumar Pattathil, Albert M. Cheh, Ninad Kothari, Rajeev Kumar, John Ralph, Michael G. Hahn, Charles E. Wyman, Seema Singh, Blake A. Simmons, Bruce E. Dale and Venkatesh Balan (2016) “Next-generation ammonia pretreatment enhances cellulosic biofuel production” Energy Environ. Sci. doi: 10.1039/C5EE03051J


Henry Gibson

Bio-fuel production starves people and is not economic and does not substantially reduce and may even increase greenhouse gas release. ..HG..

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