Implementing time-domain 1 H-Nuclear Magnetic Resonance relaxometry to investigate 1 recovery and stability of vegetative oil in bioenergy crops following feedstock preprocessing

12 Background: The bioenergy crops energycane, miscanthus and, sorghum are being genetically 13 modified using state of the art synthetic biotechnology techniques to accumulate energy-rich 14 molecules such as triacylglycerides (TAGs) in their vegetative cells to enhance their utility for 15 biofuel production. Typically, measuring and analyzing vegetative lipid contents at each step of 16 feedstock preprocessing requires tedious sample preparation and extraction with an organic 17 solvent. In the present study, proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy was 18 successfully adapted for non-invasive and rapid quantification of vegetative oil in untreated and 19 pretreated cellulosic biomass. 20 Results: We show that the establishment of a precise and specific NMR calibration for each 21 biomass with a distinct oil composition is key for accurate quantification of vegetative oil. The 22 values obtained with 1 H-NMR were validated using a conventional solvent extraction method 23 and cross-referenced values were within 10 % deviation. 1 H-NMR relaxation time distribution 24 provided insight into the proton environment associated with the vegetative oil in the biomass. 25 T1T2 correlation spectra resolved two distinct populations of proton molecules based on their 26 ‘molecular tumbling’ rate. The population of protons with short and long relaxation times was 27 characterized as bound and free oil in the biomass sample, respectively. Besides, we show that 28 biomass pretreated with two-staged hydrothermal and mechanical pretreatment can be directly 29 used for NMR analysis unlike dilute acid and alkaline pretreated biomass which needs an 30 additional step for neutralization of sample. 31 Conclusion: Time-domain 1 H-NMR provides a chemical-free and one-step analysis of in situ 32 vegetative oil in transgenic cellulosic biomass. T1T2 correlation spectra facilitated the resolution 33 of the influence of various pretreatment procedures typical of cellulosic bioprocessing on the 34 chemical composition of molecular and local 1 H population in each sample, hence yield 35 information on the stability and oil recovery subsequent to each step of feedstock preprocessing.

values obtained with 1 H-NMR were validated using a conventional solvent extraction method 23 and cross-referenced values were within 10 % deviation. 1    Research to develop and learn to process energy crops with in situ oil requires rapid and 60 convenient analytical methods to characterize and quantify their oil contents. Sample processing 61 is particularly complicated because new, likely multistep, processes will be needed to recover the 62 oil and optimizing the process necessities measuring oil recoveries and losses at each step. 63 Traditional organic solvent-based methods are too slow and tedious.

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In cellulosic related studies, 1 H-NMR has been used to explore water mobility within untreated 73 and pretreated biomasses [15][16][17]. Therefore, it was of interest to see if it could be used for the 74 measurement of vegetative oil within non-seed biomass-the future of biodiesel production. In 75 this study, besides quantification, 1 H-NMR technology has been used to differentiate between 76 bound and free oil present in the biomass and investigate their fate after three distinct feedstock 77 preprocessing i.e., two-staged hydrothermal and mechanical, dilute acid and alkaline 78 pretreatment to confirm their suitability for bioenergy crops with in situ oil.

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The present study is proof of the above-mentioned concept using ground energycane bagasse 80 soaked in crude corn oil as model biomass. The model biomass was used to investigate the fate 81 of vegetative oil during pretreatment procedures. It helped in analyzing the accuracy of measured 82 values and the correlation between the NMR quantification and relaxometry spectra as the 83 concentration of oil per gram of dry biomass was known. A two-way validation that involved 84 two distinct biomasses (soyhull and transgenic lipidcane-having in situ oil) and the conventional 85 oil extraction method i.e., organic solvent extraction was performed to validate 1 H-NMR 86 spectroscopy for quantification of vegetative oil in cellulosic biomass. This study aims to 87 5 develop an analytical method based on td-1 H-NMR spectroscopy for non-invasive, chemical-88 free, and, rapid quantification of vegetative oil in cellulosic biomass. Moreover, we also 89 investigated proton relaxometry correlation spectra associated with oil to understand the fate of 90 oil in biomass during different pretreatment.    Table 1 show that increasing the concentration of externally 134 added oil from 0.096 g oil per g dry biomass to 0.501 g oil per g dry biomass increases the 135 magnitude of T2 (1) and T2 (2) from 46 ± 3 ms and 192 ± 4 ms to 60 ± 2 ms and 235 ± 3 ms, 136 respectively, while the magnitude of T1 (1) and T1 (2 increased from 19 ± 3 ms and 70 ± 10 ms 137 to 31 ± 6 ms and 120 ± 10 ms (after deducting control), respectively. The significant effect of  The oil contents measured using NMR spectroscopy were further compared with the values 162 obtained using the classical organic solvent extraction gravimetric method for further validation.

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Since feedstock preprocessing is indispensable for biofuel production using lignocellulosic 169 biomass, energycane test sample containing ~0.20 g crude corn oil per g dry biomass was    (Fig. 4). The decline can be ascribed to either extraction of the free oil or release of bound oil 190 during pretreatment and hence, was further investigated using NMR relaxometry study.

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However, as mentioned previously, biomass pretreated with dilute acid exhibit a higher proton 217 signal due to the increased concentration of H + ions, thus, the magnitudes of T1 and T2 might 218 vary for the neutralized sample.

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In contrast, pretreatment of biomass containing oil with alkali at high temperature had an adverse  The biomass pretreated with two-staged hydrothermal and mechanical pretreatment showed 225 promising results with 1 H-NMR analysis without sample preparation hence, it was studied in 226 detail. Analysis of total oil content (Fig. 5 a and b) and T1T2 relaxometry correlation spectra 227 ( Table   228 Table 3) of pretreated biomass provided insight into the stability and percent recovery of bound 229 and free oil during the pretreatment processes. The T2 relaxometry study presented in Table   230 Table 3 Table 2 and Additional 286 file, Table 1 show that acid pretreated samples exhibit a significantly higher magnitude on NMR

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The degradation of oil and inconsistency in relaxometry analysis ( Although the analysis of oil composition is critical, however, from the NMR relaxometry 301 analysis and corresponding quantification of oil, it can be inferred that hydrothermal 302 pretreatment at 180 ºC followed by disk milling maintains the stability and quality of vegetative 303 oil in biomass.

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Analyzing the effectiveness of feedstock preprocessing 305 The assessment of water-associated proton molecules for their mobility in biomass structure has 306 been successfully established using NMR relaxometry correlation spectra. The moisture content 307 of biomass is maintained ≥ 10% to study the water-associated relaxometry spectra.    Table   585 Table 3 1 H-NMR relaxometry spectra for the evaluation of percent loss of bound and free oil in biomass samples after three distinct 586 feedstock preprocessing i.e., two-staged hydrothermal and mechanical, dilute acid and alkaline procedures.