Steven O. Salley

Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta

Production of biofuel from algae is dependent on the microalgal biomass production rate and lipid content. Both biomass production and lipid accumulation are limited by several factors, of which nutrients play a key role. In this research, the marine microalgae Dunaliella tertiolecta was used as a model organism and a profile of its nutritional requirements was determined. Inorganic phosphate PO4(3-) and trace elements: cobalt (Co2+), iron (Fe3+), molybdenum (Mo2+) and manganese (Mn2+) were identified as required for algae optimum growth. Inorganic nitrogen in the form of nitrate NO3- instead of ammonium (NH4+) was required for maximal biomass production. Lipids accumulated under nitrogen starvation growth condition and this was time-dependent. Results of this research can be applied to maximize production of microalgal lipids in optimally designed photobioreactors.

Influence of silicone surface roughness and hydrophobicity on adhesion and colonization of Staphylococcus epidermidis

Bacterial adhesion and colonization are complicated processes that depend on many factors, including surface chemistry, hydrophobicity, and surface roughness. The contribution of each of these factors has not been fully elucidated because most previous studies used different polymeric surfaces to achieve differences in properties. The objective of this study was to modify hydrophobicity and roughness on one polymeric surface, eliminating the confounding contribution of surface chemistry. Mechanically assembled monolayer (MAM) preparation methods (both one- and two-dimensional) were used to impart different degrees of hydrophobicity on fluoroalkylsilane (FAS)-coated silicone. Surface roughness was varied by casting the silicone to templates prepared with different abrasives. Surface hydrophobicity was determined by contact angle measurement, whereas surface roughness was determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Bacterial adhesion and colonization were analyzed using a direct colony-counting method and SEM images. Hydrophobicity increased as a function of stretched length or width (Deltax or Deltay); it reached a maximum at Deltax = 60% with one-dimensional MAM and decreased as Deltax further increased to 80 and 100%. The same trend was observed for the two-dimensional MAM. After 12-h incubation, all the FAS/silicone surfaces had significantly reduced adherence of Staphylococcus epidermidis by 42-89%, compared to untreated silicone, and the degree of which is inversely related to surface hydrophobicity. On the other hand, surface roughness had a significant effect on bacterial adhesion and colonization only when the root-mean-square roughness was higher than 200 nm.

Culture of microalgae Chlorella minutissima for biodiesel feedstock production

Microalgae are among the most promising of non‐food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light‐emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two‐phase strategy with an initial nutrient‐sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011;108: 2280–2287.

Continuous microalgae cultivation in a photobioreactor

New biomass sources for alternative fuels has become a subject of increasing importance as the nation strives to resolve the economic and strategic impacts of limited fossil fuel resources on our national security, environment, and global climate. Algae are among the most promising non‐food‐crop‐based biomass feedstocks. However, there are currently no commercially viable microalgae‐based production systems for biofuel production that have been developed, as limitations include less‐than optimal oil content, growth rates, and cultivation techniques. While batch studies are critical for determining basic growth phases and characteristics of the algal species, steady‐state studies are necessary to better understand and measure the specific growth parameters. This study evaluated the effects of dilution rate on microalgal biomass productivity, lipid content, and fatty acid profile under steady‐state conditions with continuous illumination and carbon dioxide supplemention for two types of algae. Continuous cultures were conducted for more that 3 months. Our results show that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) when the dilution rate varied from 0.08 to 0.64 day−1. The biomass productivity of C. minutissima reached a maximum value (137 mg/L/day) at a dilution rate of 0.33 day−1, while the productivity of Dunaliella tertiolecta varied from 46 to 91 mg/L/day at a dilution rate of 0.17 to 0.74 day−1. The biomass productivity of D. tertiolecta reached a maximum value of 91 mg/L/day at a dilution rate of 0.42 day−1. Moreover, the lipid content had no significant change with various dilution rates. Biotechnol. Bioeng. 2012; 109: 2468–2474.

Performance of heterogeneous ZrO2 supported metaloxide catalysts for brown grease esterification and sulfur removal

In order to achieve a viable biodiesel industry, new catalyst technology is needed which can process a variety of less expensive waste oils, such as yellow grease and brown grease. However, for these catalysts to be effective for biodiesel production using these feedstocks, they must be able to tolerate higher concentrations of free fatty acids (FFA), water, and sulfur. We have developed a class of zirconia supported metaloxide catalysts that achieve high FAME yields through esterification of FFAs while simultaneously performing desulfurization and de-metallization functions. In fact, methanolysis, with the zirconia supported catalysts, was more effective for desulfurization than an acid washing process. In addition, using zirconia supported catalysts to convert waste grease, high in sulfur content, resulted in a FAME product that could meet the in-use ASTM diesel fuel sulfur specification (<500 ppm). Possible mechanisms of desulfurization and de-metallization by methanolysis were proposed to explain this activity.

Performance of plasma-perfused, microencapsulated hepatocytes: Prospects for extracorporeal liver support

The growing success of liver transplantation and the shortage of donor livers has turned attention to the possibility of utilizing hepatocytes within artificial liver support systems to allow time for donor livers to become available and to improve the condition of patients with hepatic failure. This study evaluated encapsulated hepatocytes, a technology which might allow the possibility of using xenogenic or human hepatoma cells. Rabbit hepatocytes were encapsulated using the ionic polysaccharides carboxymethylcellulose, chondroitin sulfate A, chitosan, and polygalacturonic acid. Encapsulated cells were maintained in perfusion culture for at least 6 days in heparinized, normal human plasma or in a defined culture medium. Parallel cultures of plated hepatocytes were also conducted. The metabolic capability of the cells was evaluated by following the rates of urea, albumin, and transferrin synthesis and the transformation rate of the drug antipyrine. Protein synthesis and ureogenesis in plasma were depressed from the levels expressed in defined culture medium. Drug detoxification as measured by antipyrine metabolism appeared to be enhanced in plasma. We conclude that encapsulated rabbit hepatocytes retain significant levels of function for at least 6 days of perfusion with human plasma, suggesting the feasibility of this technology as a potential method of short-term liver support.

The effect of support material on the transesterification activity of CaO–La2O3 and CaO–CeO2 supported catalysts

The effects of support materials—lanthanum oxide, cerium oxide, zirconium oxide, titanium oxide, γ-alumina, and ZSM-5—on the transesterification activity of CaO–La2O3 and CaO–CeO2 catalysts were investigated. The metal composition and surface acidity (or basicity) of the supported catalysts played a significant role in determining the activity of the catalyst. Results showed that both catalytic activity and basicity of the supported catalysts decreased in the following order: CaO–La2O3/La2O3 ≥ CaO–La2O3/CeO2 > CaO–La2O3/ZrO2 > CaO–La2O3/γ-Al2O3 > CaO–La2O3/ZSM-5 > CaO–La2O3/TiO2. In addition, leaching of Ca species from the catalyst was more pronounced with basic supports. However, Ca leaching could be minimized by coupling with La2O3 or CeO2 on an appropriate support. This was verified in a flow reactor study of the CaO–CeO2/La2O3 catalyst, where, over 200 h of continuous operation, the transesterification yield held constant at 88∼90% while the initial Ca concentration in the product decreased from 194 ppm to below 5.0 ppm after 144 h. This further suggests that Ca leaching had little long-term effect on the overall FAME activity of the catalyst.

ECMO without heparin: Laboratory and clinical experience

To evaluate the feasibility of long-term extracorporeal membrane oxygenation (ECMO) without heparin, we placed six lambs on standard venoarterial ECMO for 71 to 96 hours. Group 1 (3 animals) was given doses of heparin to maintain activated clotting times (ACT) greater than 400 seconds. No form of anticoagulant was used for the three animals in group 2. Blood flow was maintained at 60 mL/kg/min. No histological evidence of thrombosis was noted at necropsy. ACT, prothrombin time, and partial thromboplastin time were higher in group 1, and much lower, although still above normal in group 2. Fibrinogen was significantly lower in group 2 (75 +/- 35 v 219 +/- 64 mg/dL group 1), and, although the platelet count was lower in group 2 (142 +/- 76 x 10(3)/mm3 v 225 +/- 167 x 10(3)/mm3), it was clinically acceptable. These results encouraged us to discontinue heparin when faced with severe hemorrhage in four patients on ECMO, rather than withdraw support at a time when there was little chance of survival. Heparin was discontinued for 10.5 +/- 6 hours. The mean ACT was reduced from 220 +/- 23 seconds to 144 +/- 22 seconds. One patient, who required repair of gastric necrosis while on ECMO following repair of a congenital diaphragmatic hernia, survived and had a decrease in blood loss from 2 to 0 mL/kg/h after the heparin was discontinued. One of the three patients who died had an autopsy with no evidence of thrombosis. We conclude that it may be reasonable to discontinue heparin in the face of life-threatening hemorrhage while on ECMO.

Competitive transesterification of soybean oil with mixed methanol/ethanol over heterogeneous catalysts.

Methylesters and ethylesters of fatty acids were synthesized using homogeneous CH(3)ONa and CH(3)CH(2)ONa, anion exchanged resin, and CaO-La(2)O(3) catalysts. Methanol, ethanol, and methanol/ethanol mixtures were used as the alcohol feed for transesterification of soybean oil. With a homogeneous catalyst (CH(3)ONa) there was essentially no difference in conversion rates between methanolysis and ethanolysis in batch reactions. However, with a heterogeneous resin and CaO-La(2)O(3) catalysts, significant differences in the conversion rates between the methanolysis and ethanolysis were observed. The formation rate of methylesters over a CaO-La(2)O(3) catalyst was higher than that of ethylesters, which may be attributable to a steric hindrance effect. Conversely, with a heterogeneous resin catalyst, the conversion rate of ethylester was higher than that of methylesters which may be attributable to the surface hydrophobicity of the anion exchanged resin. When the transesterification of soybean oil was carried out with an equimolar methanol/ethanol mixture, the yield ratio of methylester to ethylester formed within the first 30 min was 2.6 for the homogeneous catalyst (0.3% CH(3)ONa), and 3.4 for the heterogeneous CaO-La(2)O(3)catalyst. These differences in selectivity are likely due to both the higher reactivity of methoxide and to a steric hindrance effect of ethoxide on the catalyst surface. In addition, the transformation of methylester to ethylester was observed when a methanol/ethanol mixture was used.

Regional blood flow during extracorporeal membrane oxygenation in lambs

To determine changes in blood flow to different organs during extracorporeal membrane oxygenation (ECMO), the authors performed venoarterial ECMO in four young lambs for 71-96 hr (Group 1). Macroaggregated albumin microspheres labeled with technetium 99m were injected through the perfusion cannula before termination of ECMO to determine percent of blood flow by measuring radioactivity from the microspheres lodged in specific organs. The control group (Group 2) consisted of three animals not on bypass; injections were made through a catheter placed in the left ventricle. Relative coronary blood flow from the perfusion cannula was significantly less than relative coronary blood flow in the control group, possibly because of cannula location. Renal flow from the perfusion cannula also was decreased. Contrary to observations in rabbits, cerebral perfusion did not decrease in the bypass group despite ligation of the carotid artery and the external jugular vein. There were no statistically significant differences between the two groups in the relative blood flow to other organs. The authors conclude that ECMO may significantly alter myocardial and renal perfusion, with minimal effects to other organs.