Background
The bioenergy crops energycane, miscanthus and, sorghum are being genetically modified using state of the art synthetic biotechnology techniques to accumulate energy-rich molecules such as triacylglycerides (TAGs) in their vegetative cells to enhance their utility for biofuel production. Typically, measuring and analyzing vegetative lipid contents at each step of feedstock preprocessing requires tedious sample preparation and extraction with an organic solvent. In the present study, proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy was successfully adapted for non-invasive and rapid quantification of vegetative oil in untreated and pretreated cellulosic biomass.
Results
We show that the establishment of a precise and specific NMR calibration for each biomass with a distinct oil composition is key for accurate quantification of vegetative oil. The values obtained with 1 H-NMR were validated using a conventional solvent extraction method and cross-referenced values were within 10% deviation. 1 H-NMR relaxation time distribution provided insight into the proton environment associated with the vegetative oil in the biomass. T1T2 correlation spectra resolved two distinct populations of proton molecules based on their ‘molecular tumbling’ rate. The population of protons with short and long relaxation times was characterized as bound and free oil in the biomass sample, respectively. Besides, we show that biomass pretreated with two-staged hydrothermal and mechanical pretreatment can be directly used for NMR analysis unlike dilute acid and alkaline pretreated biomass which needs an additional step for neutralization of sample.
Conclusion
Time-domain 1 H-NMR provides a chemical-free and one-step analysis of in situ vegetative oil in transgenic cellulosic biomass. T1T2 correlation spectra facilitated the resolution of the influence of various pretreatment procedures typical of cellulosic bioprocessing on the chemical composition of molecular and local 1 H population in each sample, hence yield information on the stability and oil recovery subsequent to each step of feedstock preprocessing.