A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

Background Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions.

Effect of sucrose on the fatty acid metabolism of adventitious root cultures in vitro of Stevia rebaudiana

In this study, the effect of sucrose on the neutral lipid profile of adventitious root cultures of Stevia rebaudiana was evaluated. The cultures were obtained employing a roller bottle system. In this system, Schott-type flasks were used, which contained Murashige and Skoog liquid medium at 33.3% strength (MS/3) supplemented with 30, 60, and 80 g L-1 of sucrose, respectively, and 10.7 mM 1-naphthaleneacetic acid (NAA). The spectroscopic analyzes showed that the portion of polyunsaturated fatty acids (PUFAs) was highest in roots treated with 30 g L-1 of sucrose. The spectrometric analyzes showed that the palmitic acid was found to be present in relatively higher amounts in the roots submitted to the MS/3-30 g L-1 (31.9%) and MS/3-60 g L-1 (29.5%) sucrose treatments, and lower in the treatment with MS/3-80 g L-1 (28.8%) of sucrose. Also, the treatment using 30 g L-1 of sucrose was the best for obtaining unsaturated fatty acids (UFAs) in the culture, with a relative percentage of 62.9%. Our results indicate that the MS medium that received 30 g L-1 of sucrose induced a lesser abiotic stress condition, which favored PUFAs production in the adventitious root cultures of S. rebaudiana.

Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

Potential Role of Marine Snow in the Fate of Spilled Oil in Cook Inlet, Alaska

ABSTRACT The objective of the research is to inform response decision-making and understanding of the potential association of spilled oil with marine snow in Cook Inlet, Alaska. While extensive research has been conducted on minerals aggregating with spilled oil, larger organic aggregates, such as marine snow, have only recently been studied as a transport mechanism. This knowledge gap in understanding the fate of oil was highlighted as part of the Gulf of Mexico Research Initiative (GoMRI) following the Deepwater Horizon (DWH) spill. It was determined that significant percentages of spilled oil reached the seafloor as a result of association with marine snow during both DWH and the Ixtoc 1 blowouts. As development of oil resources continues in Alaska and the Arctic, marine snow is a significant oil exposure pathway that must be considered during oil spill response. In parallel with a corresponding sedimentation study, input from local, federal and industry experts was used to develop laboratory scale oil exposure experiments to evaluate the potential for oil-marine snow aggregate formation in Cook Inlet. Roller-bottle experiments were conducted from May to July 2019 to assess the interactions between a 5 μm sheen of Alaska North Slope crude oil and Cook Inlet surface water. Aggregate formation was documented and sinking flocs were observed and analyzed with fluorescence microscopy to estimate oil content. The total oil volumes estimated in aggregates were between 0.01 to 0.4 μl. Estimates of total oil volume associated with the aggregates ranged from 0.6 to 9.3 % ± 1.4% of the total oil volume (80 μl) that was added to the bottles. The incorporation of spilled oil in surface forming aggregates will contribute to understanding fate and response implications in Cook Inlet and other northern regions at risk of spilled oil entering the benthic food web via association with sinking marine snow.

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Large-Scale Expansion of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent stem cells with strong immunosuppressive property that renders them an attractive source of cells for cell therapy. MSCs have been studied in multiple clinical trials to treat liver diseases, peripheral nerve damage, graft-versus-host disease, autoimmune diseases, diabetes mellitus, and cardiovascular damage. Millions to hundred millions of MSCs are required per patient depending on the disease, route of administration, frequency of administration, and patient body weight. Multiple large-scale cell expansion strategies have been described in the literature to fetch the cell quantity required for the therapy. In this review, bioprocessing strategies for large-scale expansion of MSCs were systematically reviewed and discussed. The literature search in Medline and Scopus databases identified 26 articles that met the inclusion criteria and were included in this review. These articles described the large-scale expansion of 7 different sources of MSCs using 4 different bioprocessing strategies, i.e., bioreactor, spinner flask, roller bottle, and multilayered flask. The bioreactor, spinner flask, and multilayered flask were more commonly used to upscale the MSCs compared to the roller bottle. Generally, a higher expansion ratio was achieved with the bioreactor and multilayered flask. Importantly, regardless of the bioprocessing strategies, the expanded MSCs were able to maintain its phenotype and potency. In summary, the bioreactor, spinner flask, roller bottle, and multilayered flask can be used for large-scale expansion of MSCs without compromising the cell quality.