Aditya Bhalla

Conversion of corn stover alkaline pre-treatment waste streams into biodiesel via Rhodococci

The bioconversion of second-generation cellulosic ethanol waste streams into biodiesel via oleaginous bacteria is a novel optimization strategy for biorefineries with substantial potential for rapid development. In this study, one- and two-stage alkali/alkali-peroxide pretreatment waste streams of corn stover were separately implemented as feedstocks in 96 h batch reactor fermentations with wild-type Rhodococcus opacus PD 630, R. opacus DSM 1069, and R. jostii DSM 44719T. Here we show using 31P-NMR, HPAEC-PAD, and SEC analyses, that the more rigorous and chemically-efficient two-stage chemical pretreatment effluent provided higher concentrations of solubilized glucose and lower molecular weight (∼70–300 g mol−1) lignin degradation products thereby enabling improved cellular density, viability, and oleaginicity in each respective strain. The most significant yields were by R. opacus PD 630, which converted 6.2% of organic content with a maximal total lipid production of 1.3 g L−1 and accumulated 42.1% in oils based on cell dry weight after 48 h.

Predicting lignin depolymerization yields from quantifiable properties using fractionated biorefinery lignins

Lignin depolymerization to aromatic monomers with high yields and selectivity is essential for the economic feasibility of many lignin-valorization strategies within integrated biorefining processes. Importantly, the quality and properties of the lignin source play an essential role in impacting the conversion chemistry, yet this relationship between lignin properties and lignin susceptibility to depolymerization is not well established. In this study, we quantitatively demonstrate how the detrimental effect of a pretreatment process on the properties of lignins, particularly β-O-4 content, limit high yields of aromatic monomers using three lignin depolymerization approaches: thioacidolysis, hydrogenolysis, and oxidation. Through pH-based fractionation of alkali-solubilized lignin from hybrid poplar, this study demonstrates that the properties of lignin, namely β-O-4 linkages, phenolic hydroxyl groups, molecular weight, and S/G ratios exhibit strong correlations with each other even after pretreatment. Furthermore, the differences in these properties lead to discernible trends in aromatic monomer yields using the three depolymerization techniques. Based on the interdependency of alkali lignin properties and its susceptibility to depolymerization, a model for the prediction of monomer yields was developed and validated for depolymerization by quantitative thioacidolysis. These results highlight the importance of the lignin properties for their suitability for an ether-cleaving depolymerization process, since the theoretical monomer yields grows as a second order function of the β-O-4 content. Therefore, this research encourages and provides a reference tool for future studies to identify new methods for lignin-first biomass pretreatment and lignin valorization that emphasize preservation of lignin qualities, apart from focusing on optimization of reaction conditions and catalyst selection.

Cell wall-associated transition metals improve alkaline-oxidative pretreatment in diverse hardwoods

The responses of four diverse hardwoods (hybrid poplar, silver birch, hybrid aspen, and sugar maple) to alkaline hydrogen peroxide (AHP) pretreated at ambient temperature and pressure were analyzed to gain a deeper understanding of the cell wall properties that contribute to differences in enzymatic hydrolysis efficacy following alkaline-oxidative pretreatment. The enzymatic hydrolysis yields of these diverse hardwoods increased significantly with increasing the cell wall-associated, redox-active transition metal content. These increases in hydrolysis yields were directly correlated with improved delignification. Furthermore, we demonstrated that these improvements in hydrolysis yields could be achieved either through elevated levels of naturally-occurring metals, namely Cu, Fe, and Mn, or by the addition of a homogeneous transition metal catalyst (e.g. Cu 2,2′-bipyridine complexes) capable of penetrating into the cell wall matrix. Removal of naturally-occurring cell wall-associated transition metals by chelation resulted in substantial decreases in the hydrolysis yields following AHP pretreatment, while re-addition of metals in the form of Cu 2,2′-bipyridine complexes and to a limited extent Fe 2,2′-bipyridine complexes prior to pretreatment restored the improved hydrolysis yields. Glycome profiles showed improved extractability of xylan, xyloglucan, and pectin epitopes with increasing hydrolysis yields for the diverse hardwoods subjected to the alkaline-oxidative pretreatment, demonstrating that the strength of association between cell wall matrix polymers decreased as a consequence of improved delignification.

Production of single cell protein from agro-waste using Rhodococcus opacus

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.