Xanthobacter-dominated biofilm as a novel source for high-value rhamnose.

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition-based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.

Opportunities to improve the conversion of food waste to lactate: Fine-tuning secondary factors.

Extensive research has demonstrated the potential for bioconversion of food waste to lactate, with major emphasis on adjusting temperature, pH, and loading rate of the fermentation. Each of these factors has a significant effect on lactate production; however, additional secondary factors have received little attention. Here we investigate three additional factors where opportunities exist for process improvement: freezing of samples during storage, discontinuous pH control, and holdover of fermentation broth between fermentations. Freezing samples prior to fermentation was shown to reduce the production rate of lactate by 8%, indicating freeze-thaw should be avoided in experiments. Prior work indicated a trade-off in pH control strategies, where discontinuous pH control correlated with higher lactate accumulation while continuous pH control correlated with higher production rate. Here we demonstrate that continuous pH control can achieve both higher lactate accumulation and higher production rate. Finally, holding over fermentation broth was shown to be a simple method to improve production rate (by 18%) at high food waste loading rates (>140 g volatile solids L-1) but resulted in lower lactate accumulation (by 17%). The results inform continued process improvements within the waste treatment of food waste through fermentation to lactic acid.

Enhancing silymarin fractionation using the conductor-like screening model for real solvents.

A significant hurdle for discovery of plant-derived products is the numerous trial-and-error experiments required to develop an effective purification strategy. To overcome the experimental burden, a quantum mechanics-based molecular modeling approach - known as the COnductor-like Screening Model for Real Solvents (COSMO-RS) - was used to predict a suitable two-phase solvent system to purify six silymarins from an aqueous mixture. Silymarins, a class of flavonolignans found in milk thistle (Silybum marianum L.), are well suited for assessing the use of a molecular modeling approach to predict partitioning in a countercurrent chromatography (CCC) separation because they are well characterized and previous studies report low purity fractionation in liquid-liquid solvent systems. They also present an opportunity to evaluate the use of COSMO-RS in predicting the partitioning of structurally similar isomeric compounds that are present together in an aqueous solution upon extraction from their native source. The COSMO-RS model results predicted the partition coefficients in: three traditional ARIZONA solvent systems (composed of heptane, ethyl acetate, methanol, and water), nine additional variations of this quaternary solvent system, and two chloroform, methanol, and water solvent systems. Predicted results were concise but not accurate when compared to experimental results determined by the shake flask method. The 1:4:3:5 n-heptane:ethyl acetate:methanol:water (v/v/v/v) system was identified to be an improvement on the 1:4:3:4 system previously reported. The present study verified the ability of COSMO-RS to hone in on one or two solvent systems that will yield the best fractionation using CCC.

Techno-economic analysis for incorporating a liquid-liquid extraction system to remove acetic acid into a proposed commercial scale biorefinery.

Mitigating the effect of fermentation inhibitors in bioethanol plants can have a great positive impact on the economy of this industry. Liquid-liquid extraction (LLE) using ethyl acetate is able to remove fermentation inhibitors-chiefly, acetic acid-from an aqueous solution used to produce bioethanol. The fermentation broth resulting from LLE has higher performance for ethanol yield and its production rate. Previous techno-economic analyses focused on second-generation biofuel production did not address the impact of removing the fermentation inhibitors on the economic performance of the biorefinery. A comprehensive analysis of applying a separation system to mitigate the fermentation inhibition effect and to provide an analysis on the economic impact of removal of acetic acid from corn stover hydrolysate on the overall revenue of the biorefinery is necessary. This study examines the pros and cons associated with implementing LLE column along with the solvent recovery system into a commercial scale bioethanol plant. Using details from the NREL-developed model of corn stover biorefinery, the capital costs associated with the equipment and the operating cost for the use of solvent were estimated and the results were compared with the profit gain due to higher ethanol production. Results indicate that the additional capital will add 1% to the total capital and manufacturing cost will increase by 5.9%. The benefit arises from the higher ethanol production rate and yield as a consequence of inhibitor extraction and results in a $0.35 per gallon reduction in the minimum ethanol selling price (MESP). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:971-977, 2016.

Acetic acid removal from corn stover hydrolysate using ethyl acetate and the impact on Saccharomyces cerevisiae bioethanol fermentation.

Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild-type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid-liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus™, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L(-1) . The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y-1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:929-937, 2016.

Purification of resveratrol, arachidin-1, and arachidin-3 from hairy root cultures of peanut (Arachis hypogaea) and determination of their antioxidant activity and cytotoxicity.

Antioxidant stilbenoids, such as resveratrol, arachidin-1, and arachidin-3, have demonstrated beneficial effects on human health. Although resveratrol is commercially available, arachidin-1 and arachidin-3 are not, resulting in an opportunity to explore purification methods and to confirm biological activity. Recently, Arachis hypogaea hairy root cultures (produced via Agrobacterium rhizogenes-mediated transformation) were reported to secrete stilbenoids into liquid growth media upon elicitation in quantities sufficient for commercial production. The purpose of this study was to purify substantial quantities of resveratrol, arachidin-1, and arachidin-3 from A. hypogaea hairy root cultures using centrifugal partition chromatography (CPC), determine the antioxidant activity of these compounds using the thiobarbituric acid reactive substances (TBARS) assay, and determine the cytotoxicity of the compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In a single run of CPC, resveratrol, arachidin-1, and arachidin-3 were separated to a purity of 97.1%, 97.0%, and 91.8%, respectively. Lipid oxidation was inhibited by a 27 and 7 µM dose for reference standards of resveratrol and arachidin-1, respectively, while oxidation was not inhibited up to a 27 µM dose for reference standard of arachidin-3. Oxidation was inhibited at a 14, 7, and 14 µM doses for CPC-purified resveratrol, arachidin-1, and arachidin-3, respectively. Arachidin-1 and arachidin-3 demonstrated cytotoxicity at 27 and 55 µM in RAW 264.7 and HeLa cell lines, respectively; while resveratrol exhibited no cytotoxicity to either cell line. These results demonstrate the integration of a production and purification system for the manufacturing of A. hypogaea-derived stilbenoids.