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NC State Researchers Developing Ozonolysis Pretreatment for Energy Grass Feedstocks

Researchers at North Carolina State University are developing an ozone-based pre-treatment technique (ozonolysis) to release sugars from the energy grass miscanthus for production into renewable fuels or chemicals with minimal generation of chemical waste streams and degradation of the carbohydrate components.

Breaking down lignin—the woody, protective material that provides structural support. for plants—is an essential first step for processes that use the plant’s component carbohydrates. At present, pretreatment techniques include physical, chemical, physicochemical and/or biological methods such as steam explosion; hot water extraction; sulfuric acid; sodium hydroxide; hydrogen peroxide; peracetic acid; ammonia fiber explosion (AFEX); and wet oxidation in addition to an emerging body of work on ozonolysis.

Following pretreatment, enzymatic hydrolysis turns the carbohydrates into sugars that can be then used for fermentation or other production pathways. However, many of the chemical pretreatment techniques often result in a significant portion of the plant’s carbohydrates being siphoned off with the liquid waste stream, the NC State researchers note. Producers must either incorporate additional processes to retrieve those carbohydrates, or lose them altogether.

Other research has shown the efficacy of ozonolysis pretreatment in degrading the lignin polymer but also slightly solubilizing hemicellulose content of lignocellulosic biomass.

Ozone is highly reactive towards compounds incorporating conjugated double bonds and functional groups with high electron densities. Therefore, the moiety most likely to be oxidized in ozonization of lignocellulosic materials is lignin due to its high content of C=C bounds. Ozone attacks lignin releasing soluble compounds of less molecular weight, mainly organic acids such as formic and acetic acid which can result in a drop in pH from 6.5 to 2.

The main advantages linked to this process are the lack of any degradation products which might interfere with subsequent hydrolysis or fermentation, and the reactions occurring at ambient temperature.García-Cubero et al. (2008)

Exposing the miscanthus to gaseous ozone, with very little moisture, the NC State researchers are able to produce a carbohydrate-rich solid with no solid or liquid waste.

The NC State researchers are investigating the effect of ozonolysis as a pretreatment method under room temperature and pressure. Ozone concentrations up to 60 ppm at flow rates up to 0.5 L/min are being used to pretreat several varieties of miscanthus for varying times to enhance enzymatic hydrolysis. The efficiency of pretreatment will be determined by measuring the reducing sugars generated after hydrolysis.

They expect that the results of this study will help in the development of a pretreatment process that provides higher specificity towards lignin removal compared than other delignifying agents/pretreatments.

This technique makes the process more efficient and less expensive. The technique could open the door to making lignin-rich plant matter a commercially viable feedstock for biofuels, curtailing biofuel’s reliance on staple food crops. Our eventual goal is to use this technique for any type of feedstock, to produce any biofuel or biochemical that can use these sugars.

—Dr. Ratna Sharma-Shivappa, associate professor of biological and agricultural engineering at NC State

Sharma-Shivappa notes that the ozonolysis process is more expensive than using a bath of harsh chemicals to free the carbohydrates, but is ultimately more cost-effective because it makes more efficient use of the plant matter.

The researchers have recently received a grant from the Center for Bioenergy Research and Development to fine-tune the process for use with switchgrass and miscanthus grass.

The research is partially funded by the Biofuels Center of North Carolina and was presented June 23 at the 2010 Annual International Meeting of the American Society for Agricultural and Biological Engineers in Pittsburgh, PA.


  • Anushadevi Panneerselvam, Ratna Sharma-Shivappa, Praveen Kolar, Thomas Ranney (2010) Effect of ozonolysis on bioconversion of miscanthus to bioethanol. Presented: June 23, 2010, 2010 Annual International Meeting of the American Society for Agricultural and Biological Engineers in Pittsburgh, Penn.

  • M.T. García-Cubero et al. (2009) Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw. Bioresource Technology 100 1608–1613 doi: 10.1016/j.biortech.2008.09.012



Nice to see research continue on miscanthus...haven't heard anything about the plant in a while...for a period of time it seemed like miscanthus was the holy grail of all energy crops.



work is still in progress with miscanthus and other energy crops as well as all biorefineries approach being investigated, the problem is that it is a very long and tedious process to develop a new crop and refining process at industrial scale and especially when the profitability is far form certain. Scaling up of biofuels will be slow, and it's better be slow because it is risky, financially and ecologically.


Asides this Ozone pretreatment sounds pretty smart, attack the CC double bounds to make them convertible in sugars looks sensible to me. Future will tell...


Ozone does a number on all the rubber in your car, stands to reason it would attack cellulose fiber. Good work.


my concern is that I wonder if ozone pretreatment would be scalable, ozone is pretty toxic even at very low concentration, so when you treat hundreds of tons of biomass with ozone every day how can you avoid that some amount to escape in the atmosphere around due to the gazeous nature of ozone ?


Ozone has a very short half-life in the atmosphere. There is absolutely no bio-accumulation. Its toxicity is because of its high reactivity. It reacts with almost anything (and oxidises it), thereby destructing itself.
'ozone pollution' is actually pollution with organic solvents, which continuously generate ozone when exposed to sunlight. On sunny days, ozone levels indoors are much lower than outdoors, simply because the ozone that gets inside reacts immedately with your wallpaper (and becomes oxygen)
Because it is so reactive, it is very easy to neutralise it with almost anything. Ozone is now already used as green disinfectant for swimming pools and drinking water.

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