Thanh Yen Nguyen

Co‐solvent Pretreatment Reduces Costly Enzyme Requirements for High Sugar and Ethanol Yields from Lignocellulosic Biomass

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover.

CELF pretreatment of corn stover boosts ethanol titers and yields from high solids SSF with low enzyme loadings

A major challenge to economically produce ethanol from lignocellulosic biomass is to achieve industrially relevant ethanol titers (>50 g L−1) to control operating and capital costs for downstream ethanol operations while maintaining high ethanol yields. However, due to reduced fermentation effectiveness at high biomass solids loadings, excessive amounts of enzymes are typically required to obtain reasonable ethanol titers, thereby trading off reduced operating and capital costs with high enzyme costs. In this study, we applied our newly developed Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment to produce highly digestible glucan-rich solids from corn stover. Simultaneous saccharification and fermentation (SSF) was then applied to pretreated solids from CELF at 15.5 wt% solids loadings (corresponding to 11 wt% glucan loadings) in modified shake flasks to achieve an ethanol titer of 58.8 g L−1 at 89.2% yield with an enzyme loading of 15 mg-protein per g-glucan-in-raw-corn-stover (-RCS) in only 5 days. By comparison, SSF of corn stover solids from dilute acid pretreatment at 18.3 wt% solids loading (or 10 wt% glucan loading) only achieved an ethanol titer and a yield of 47.8 g L−1 and 73.0%, respectively, despite needing longer fermentation times (∼20 days) and an additional 18 h of prehydrolysis at 50 °C. Remarkably, although longer fermentation times were required at more economical enzyme loadings of 5 and 2 mg-protein per g-glucan-in-RCS, high solids SSF of CELF pretreated corn stover realized final ethanol titers consistently above 50 g L−1 and yields over 80%.

An In Vitro Mechanism Study on the Proliferation and Pluripotency of Human Embryonic Stems Cells in Response to Magnesium Degradation

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials, we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg, cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence, their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose, the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4–40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages, the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4, SSEA3, and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM, however, hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications, providing their degradation rate is moderate. Additionally, the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro, and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds.

Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol

Significance Future production of renewable transportation fuels such as ethanol must rely on abundant nonfood plant sources also known as lignocellulosic biomass. However, a major historical barrier to low-cost production of ethanol from biomass is the low ethanol yields and titers that result from fermentation of biomass solids at high solids when compared with simple sugar fermentations. Here, we show that combining a cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment process with subsequent simultaneous saccharification and fermentation (SSF) can achieve similar high ethanol yields and titers that match that of separate pure glucose fermentations. We demonstrate a strategy whereby direct fermentation of biomass to ethanol is now limited by the microbe rather than by the process. Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF. First, fed-batch glucose fermentations by Saccharomyces cerevisiae D5A revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g⋅L−1. Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3, and 85.6 g⋅L−1 in batch shake flask, corresponding to ethanol yields of 90.5%, 86.1%, and 80.8% at solids loadings of 20.0 wt %, 21.5 wt %, and 23.0 wt %, respectively. Ethanol yields remained at over 90% despite reducing enzyme loading to only 10 mg protein⋅g glucan−1 [∼6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by D5A and not loss of enzyme activity or mixing issues, thereby demonstrating an SSF-based process that was limited by a strain’s metabolic capabilities and tolerance to ethanol.

In vitro interactions of blood, platelet, and fibroblast with biodegradable magnesium-zinc-strontium alloys

Magnesium (Mg) alloy is an attractive class of metallic biomaterial for cardiovascular applications due to its biodegradability and mechanical properties. In this study, we investigated the degradation in blood, thrombogenicity, and cytocompatibility of Magnesium-Zinc-Strontium (Mg-Zn-Sr) alloys, specifically four Mg-4 wt % Zn-xSr (x = 0.15, 0.5, 1, and 1.5 wt %) alloys, together with pure Mg control and relevant reference materials for cardiovascular applications. Human whole blood and platelet rich plasma (PRP) were used as the incubation media to investigate the degradation behavior of the Mg-Zn-Sr alloys. The results showed that the PRP had a greater pH increase and greater concentration of Mg(2+) ions when compared with whole blood after 2 h of incubation with the same respective Mg alloys, suggesting that the Mg alloys degraded faster in PRP than in whole blood. The Mg alloy with 4 wt % Zn and 0.15 wt % Sr (named as ZSr41A) was identified as the most promising alloy for cardiovascular stent applications, because it showed slower degradation and less thrombogenicity, as indicated by the lower concentrations of Mg(2+) ions released and less deposition of platelets. Additionally, ZSr41 alloys were cytocompatible with fibroblasts in direct exposure culture in which the cells adhered and proliferated around the samples, with no statistical difference in cell adhesion density compared with the blank reference. Future studies on the ZSr41 alloys are necessary to investigate their direct interactions with other important cells in cardiovascular system, such as vascular endothelial cells and smooth muscle cells.

The effects of poly(3,4-ethylenedioxythiophene) coating on magnesium degradation and cytocompatibility with human embryonic stem cells for potential neural applications

Magnesium (Mg) is a promising conductive metallic biomaterial due to its desirable mechanical properties for load bearing and biodegradability in human body. Controlling the rapid degradation of Mg in physiological environment continues to be the key challenge toward clinical translation. In this study, we investigated the effects of conductive poly(3,4-ethylenedioxythiophene) (PEDOT) coating on the degradation behavior of Mg substrates and their cytocompatibility. Human embryonic stem cells (hESCs) were used as the in vitro model system to study cellular responses to Mg degradation because they are sensitive and can potentially differentiate into many cell types of interest (e.g., neurons) for regenerative medicine. The PEDOT was deposited on Mg substrates using electrochemical deposition. The greater number of cyclic voltammetry (CV) cycles yielded thicker PEDOT coatings on Mg substrates. Specifically, the coatings produced by 2, 5, and 10 CV cycles (denoted as 2×-PEDOT-Mg, 5×-PEDOT-Mg, and 10×-PEDOT-Mg) had an average thickness of 31, 63, and 78 µm, respectively. Compared with non-coated Mg samples, all PEDOT coated Mg samples showed slower degradation rates, as indicated by Tafel test results and Mg ion concentrations in the post-culture media. The 5×-PEDOT-Mg showed the best coating adhesion and slowest Mg degradation among the tested samples. Moreover, hESCs survived for the longest period when cultured with the 5×-PEDOT-Mg samples compared with the non-coated Mg and 2×-PEDOT-Mg. Overall, the results of this study showed promise in using PEDOT coating on biodegradable Mg-based implants for potential neural recording, stimulation and tissue engineering applications, thus encouraging further research.

Effects of magnesium on growth and proliferation of human embryonic stem cells

The effects of magnesium on the growth and proliferation of human embryonic stem cells (hESCs) was explored to advance magnesium as an implant biomaterial. When magnesium ions from magnesium salt were added to the culture media at 10, 100, 250, 500, 750, and 1000 ppm (0.4, 4, 10, 20, 30, 40 mM) the rate of increase in viable cell coverage over time was higher for the larger doses of magnesium salt. Thus, the addition of magnesium ions exerted a positive effect on viable cell coverage. When hESCs were cultured with pure magnesium metal strips through transwell inserts, the cells underwent an initial increase in viable cell coverage, followed by rapid cell death within the first 24 hours. This initial increase in viable cell coverage corresponded to the colonies dispersing and losing their tightly packed morphologies. The cell death may be attributed to an increased alkalinity in the culture media incubated with the magnesium metal strips. In conclusion, since the degradation of magnesium results in both magnesium ions and OH- ions (an increase of pH), controlling the degradation of magnesium to obtain the perfect balance of ions is critical for advancing magnesium as an implant biomaterial.

Characterization of Magnesium Alloy Degradation in Whole Blood and Platelet Rich Plasma

Magnesium (Mg) is an attractive biomaterial due to its desirable biodegradable and mechanical properties. In this study, we compared the degradation behavior of Mg and a new Mg alloy incubated in both whole blood and platelet rich plasma (PRP) for two hours under standard cell culture conditions. To avoid settling of red blood cells, tubes with whole blood were under constant rotation during the incubation. Post-incubation solutions were collected, centrifuged, and analyzed for pH and Mg ion concentration. Mg and Mg alloy samples were fixed with a 3% glutaraldehyde solution, dehydrated using an ethanol series, critical point dried, sputter coated, and imaged with a field emission scanning electron microscope. Analysis of the post-incubation solutions showed PRP had greater concentrations of Mg ions and higher pH values when compared with whole blood. This indicated that the Mg and Mg alloy degraded faster when incubated in PRP than in whole blood. When comparing the surface of the materials after incubation with whole blood and PRP, the surfaces of Mg and Mg alloy that was incubated in PRP had larger cracks and grain boundaries than the samples incubated in whole blood. Additionally, more particulate microstructures were observed on the samples incubated in PRP as opposed to whole blood. Further studies are still needed to elucidate the differences in degradation of Mg alloys in whole blood and PRP.