Browsing by Author "Wen, Zhiyou"

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    Bio-based composites that mimic the plant cell wall
    Li, Zhuo (Virginia Tech, 2009-04-22)
    Nature creates high performance materials under modest conditions, i.e., neutral pH and ambient temperature and pressure. One of the most significant materials is the plant cell wall. The plant cell wall is a composite of oriented cellulose microfibrils reinforcing a lignin/hemicellulose matrix. In principle, the plant cell wall composite is designed much like a synthetic fiber-reinforced polymer composite. Unlike synthetic composites, the plant cell wall has an excellent combination of high modulus, strength, toughness and low density that originates in the optimal interactions between the biopolymers. Therefore, to produce high performance composites, a unique route may be to mimic a biological system like the plant cell wall. The present work focuses on understanding the thermodynamics of biopolymer assembly to exploit the process in vitro. In our system, we use an already polymerized nanocellulose template and polymerize phenolic monomers on the template using a peroxidase enzyme. In the first part, we have polymerized phenol using horseradish peroxidase (HRP) in the presence of TEMPO-oxidized nanocellulose. Similar to native plant cell wall structures, the polyphenol-nanocellulose composite had intimate mixing of polyphenol and cellulose at the nanoscale with the presence of cellulose promoting a uniquely organized structure. The obtained composite material showed synergy that enhanced the thermal stability, hydrophobicity, and possibly mechanical properties. In the second part, monolignol coniferyl alcohol was polymerized in the presence of nanocellulose by the same procedure. A comparison between the polyphenol composite and poly(coniferyl alcohol) (PCA) composite revealed that the propanyl substitution imparted flexibility to the PCA molecules so that they could bend and form a hollow globule structure to envelope nanocellulose inside. Polyphenol could not do this because of its rigidity.
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    Biodiesel fuel
    Wen, Zhiyou; Grisso, Robert D.; Ogejo, Jactone Arogo; Vaughan, David H. (Virginia Cooperative Extension, 2009-05-01)
    The purpose of this publication is to introduce the basics of biodiesel fuel and address some myths and answer some questions about biodiesel fuel before farmers and fleet owners use this type of fuel.
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    Biomethane technology
    Ogejo, Jactone Arogo; Wen, Zhiyou; Ignosh, John; Bendfeldt, Eric S.; Collins, Eldridge R. Jr. (Virginia Cooperative Extension, 2009-05-01)
    This publication provides a general overview of anaerobic digestion and the current status of biomethane technology on livestock farms in the United States. It is part of the Bioenergy Engineering Education Program (BEEP) of the Biological Systems Engineering Department at Virginia Tech. Most of the discussion uses dairy manure as an example of feedstock for an anaerobic digester.
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    Comparison of Dairy Manure Anaerobic Digestion Performance in Gas-lift and Bubble Column Digesters
    Tang, Jing (Virginia Tech, 2009-12-04)
    Anaerobic digestion is one of the most promising management options for dairy manure treatment. Manure wastewater from dairy farms has been used for methane production for decades. However, performance failure due to inadequate mixing is routine. In general, the mixng of anaerobic digester is achived throguh mechnical stirring, liquid circulation, and gas circulation, among which the gas circulation proves to be the most effcient way. In this work, we studied the liquid flow pattern of two differetn type of gas-circualtion based anaerobic digesters, with the aim to understand the effects of hydrodynamic behavior of the digesting liquid on the anaerobic digestion performance, so a better mixing strategy can be provided. We used two 20-L gas circulation based anaerobic digesters with confined (gas-lift) and unconfined (bubble column) design. The anaerobic digestion performance and mixing behaviors were studied at different gas recirculation rate. It was found that the biogas production from the bubble column was constantly higher than that from gas-lift digester. However, the overall flow of the two digesters, which is indicated by residence time distribution (RTD), showed a similar pattern. Furhter investigation of local liquid flow behavior using Computational Fluid Dynamic (CFD) indicate that the bubble column accumulated higher portion of sludge in the bottom of the digester, which has a higher TS and VS, COD, and biomethane production potential than those from the gas-lift digester. This is the reason that the biogas production from the bubble column is higher than the gas-lift digester. Through this study, a thorough characterization of the flow behavior of the anaerobic digester were developed, and provided a deep insight of its influence on the anaerobic digestion performance.
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    Facile protein and amino acid substitution reactions and their characterization using thermal, mechanical and optical techniques
    Budhavaram, Naresh Kumar (Virginia Tech, 2010-11-01)
    The work focused on addressing four main objectives. The first objective was to quantify protein and amino acid substitution reactions. Michael addition reactions were used to modify the amino acids and protein. Amino acids alanine, cysteine, and lysine, and protein ovalbumin (OA) were substituted with different concentrations of ethyl vinyl sulfone (EVS). The substituted products were analyzed using Raman spectroscopy and UV-spectroscopy based ninhydrin assay. In case of alanine, Raman and UV results correlated with each other. With cysteine at lower EVS substitutions amine on the main chain was the preferred site while the substitution shifted to thiols at higher substitutions. This could only be discerned using Raman spectroscopy. Lysine has amines on the main chain and side chain while main chain amine was the most reactive site at lower concentrations of EVS while at higher concentrations side chain amines were also substituted. This information could be discerned using Raman spectroscopy only and not UV spectroscopy. In case of protein as observed by Raman and UV spectroscopy the reaction continued at higher concentrations of EVS indicating the participation of glutamine and asparagines at higher substitutions. However, the reaction considerably slowed down at higher EVS substitutions. The second objective of the study was to decrease the glass transition temperature (Tg) of OA through internal plasticization and also study the effects of the substituents on the thermal stability of OA. The hypothesis was by covalently attaching substituents to OA, number of hydrogen bonds can be reduced while increasing the free volume and this would reduce Tg. EVS, acrylic acid (AA), butadiene sulfone (BS) and maleimide (MA) were the four groups used. EVS was the most efficient plasticizer of all the four substituents. The Tg decreased with the increasing concentration of EVS until all of the reactive of groups on OA were used up. Tg decreased slightly with AA and BS while no change was observed with MA. However, the substituents showed exact opposite trend in thermal stability as measured using thermogravimetric analysis (TGA). The thermal stability of MA substituted OA was the highest and that of EVS substituted OA was least. FT-IR spectroscopy results indicated that all four substituents caused structural changes in OA. This implied that there were intermolecular interactions between substituted protein chains in case of AA, BS, and MA. This caused an increase in the thermal stability. EVS on the other hand is a linear chain monomer with a hydrophobic end group and hence could not participate in the intermolecular interactions and hence caused a decrease in Tg. As mentioned above the limitation to this technique is the number of available reactive groups on the protein. However, we successfully demonstrated the feasibility of this method in decreasing Tg of protein. The third objective was to create hydrogels by crosslinking OA with divinyl sulfone (DVS). Protein hydrogels due to their biocompatible nature find applications in drug delivery and tissue engineering. For tissue engineering applications the hydrogels need to be mechanically stable. In this study the protein was substituted with EVS or AA and then crosslinked with DVS. The swelling ratio was measured as a function of pH. All the hydrogels showed the same trend and swelled the least at pH 4.5 which is the isoelectric point of the protein. At basic pH conditions EVS substituted hydrogels swelled the most while AA substituted hydrogels showed least swelling. The static and dynamic moduli of the hydrogels were determined using tensile tester and rheometer respectively. The static modulus values were three times the dynamic modulus. The modulus of the control which is crosslinked OA was least and that of AA substituted OA was highest. The stress relaxation test also showed similar results in which AA substituted OA relaxed the most and the control relaxed the least. FT-IR of the dry hydrogels showed that the amount of hydrogen bonding increased with AA substitution. The hydrophilic AA end groups interacted with each other forming hydrogen bonds. These hydrogen bonds served as additional crosslinks there by increasing the modulus of the hydrogels. EVS on the other hand was incapable of interactions due to the lack of hydrophilic end groups. We were successfully able to create protein hydrogels and control the swelling and mechanical properties by varying the amount of substituted group. The final objective of the study was to create and characterize microstructures from substituted alanine and lysine. Alanine and lysine were substituted with different concentrations of EVS. Bars and fibers were observed for alanine at moderate substitutions while at higher concentrations random structures were observed using scanning electron microscopy (SEM). Lysine formed tubes at moderate EVS substitutions and rosettes at high concentrations of EVS as evidenced by SEM. FT-IR results suggested that instead of carbonyl one of sulfonyl bonded to the available amine in modified amino acids. And only in this case fibers, tubes and rosettes were observed. X-ray diffraction (XRD) results supported this observation. Using these results we hypothesized that the self assembled structures very much depended on the amount of EVS present in the substituted product and sulfonyl forming β-sheet analogs with amine.
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    Fate of Omega-3 Fatty Acids from Algae in Mozzarella Cheese
    Orders, Margaret (Virginia Tech, 2008-08-14)
    Increased consumer interest in omega-3 fatty acids (FA) has led to novel foods with added omega-3 FA. Additional information regarding omega-3 FA fate within foods is needed for improving quality and stability. This research modeled DHA, an omega-3 FA, fate and explored means of preventing degradation and oxidation of FA in algal oil and mozzarella cheese. In algal oil, TBHQ (synthetic antioxidant) at 0.0175g/g algal oil prevented DHA degradation for at least 6 weeks, and mixed tocopherols (natural antioxidant) at 400ppm prevented DHA degradation and oxidation for about 4 weeks. DHA degradation in algal oil was modeled by an autocatalytic equation. The fate of DHA from algal oil in mozzarella cheese was also modeled by an autocatalytic equation. In an effort to prevent DHA degradation and oxidation, mixed tocopherols were added. The optimum combination of those tested was found, using a response surface design, to be 3% algal oil with 110ppm mixed tocopherols for maximum DHA and minimum oxidation over 2 weeks. This algal oil/antioxidant combination in mozzarella cheese successfully prevented oxidation and DHA degradation over 3 weeks of storage. Approximately 0.1g DHA may be consumed from a 28g serving of this cheese. Approximately 0.5-18 servings of this cheese are equivalent to DHA consumed from a 3oz serving of fish, depending on fish type. Sensory evaluation tests found consumers could distinguish between mozzarella cheese with/without algal oil. Results from this study improve understanding of omega-3 FA behavior in mozzarella cheese and provide a means for preserving quality and nutrition.
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    Fuel ethanol
    Wen, Zhiyou; Ignosh, John; Ogejo, Jactone Arogo (Virginia Cooperative Extension, 2009-05-01)
    This publication introduces the basics and answers questions regarding fuel ethanol.
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    Influence of wood on the pyrolysis of poultry litter
    Mante, Nii Ofei Daku (Virginia Tech, 2008-08-25)
    Pyrolytic oils produced from poultry litter differ in physico-chemical properties and the chemical composition. The litter is composed of manure and bedding material with traces of spilled feed and feathers. The type and amount of bedding material was varied to investigate its influence on the pyrolysis of layer manure. 400g of each feedstock: manure, wood (pine and oak), and mixtures of manure and wood in proportions (75:25 50:50, and 25:75 w/w %) respectively were subjected to fast pyrolysis at 450oC in a fluidized bed reactor. The total pyrolytic oil yield ranged from 43.3% to 64.5 wt%. The highest bio oil yield and the lowest char yield were obtained from oak wood. The manure oil had the highest HHV of 29.7 MJ/kg, the highest pH (5.89), the lowest density (1.14 g/cm3) and a relatively low viscosity of 130cSt. The oils had relatively high nitrogen content ranging from 5.88wt% to 1.36 wt%; low ash content (approximately <0.07wt %) and low sulfur content (<0.28wt %). FT-IR, 13CNMR, and 1HNMR analysis showed that manure oil was rich in aliphatic hydrocarbon and primary and secondary amides and the addition of wood introduced oxygenated compounds like aliphatic alcohols, phenols, aromatic ethers, and carbonyl/carboxylic groups into the oil. TG/DTG analysis also showed that the thermal decomposition of the oils were different depending on the amount and the type of wood in the manure/wood mixture. The parametric variables used for the mixture of 50% manure and 50% pine wood shavings study were; temperature (400-550°C), nitrogen gas flow rate (12-24 L/min), and feed rate (160-480 g/h). The results showed that the pyrolysis product yields, physical properties and the chemical composition of the oil were influenced by all parameters. Temperature was the most influential factor and its effect on the liquid, char and gas yields were significant. It was evident that depending on the gas flow rate and the feed rate, a maximum oil yield (51.1wt.%) can be achieved between 400-500 oC. Also an increase in temperature significantly increased the oil viscosity and decreased the carbonyl/carboxylic and the primary aliphatic alcohol functional groups in the oil. The study on the influence of wood on the stability of the oils when stored at ambient conditions for 8 months in a 30ml glass bottle showed that the viscosity of the oils increases when stored, however the manure oil was relatively more stable and the oil from the 50/50 mixture for both pine and oak was the least stable. It was found that the stability of the oils from the manure and wood mixtures were dependent on the amount and the type of wood (pine or oak) added to the manure. Also the addition of 10% solvent (methanol/ethanol) to the oil from 50% manure and 50% pine reduced the initial viscosity of the oil and was also beneficial in slowing down the increase in viscosity during storage.
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    Investigating biomass saccharification for the production of cellulosic ethanol
    Zhu, Zhiguang (Virginia Tech, 2009-04-28)
    The production of second generation biofuels -- cellulosic ethanol from renewable lignocellulosic biomass has the potential to lead the bioindustrial revolution necessary to the transition from a fossil fuel-based economy to a sustainable carbohydrate economy. Effective release of fermentable sugars through biomass pretreatment followed by enzymatic hydrolysis is among the most costly steps for emerging cellulosic ethanol biorefineries. In this project, two pretreatment methods (dilute acid, DA, and cellulose solvent- and organic solvent-lignocellulose fractionation, COSLIF) for corn stover were compared. It was found that glucan digestibility of the corn stover pretreated by COSLIF was much higher, along with faster hydrolysis rate, than that by DA- pretreated. This difference was more significant at a low enzyme loading. Quantitative measurements of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were established to find out the cause. The COSLIF-pretreated corn stover had a CAC nearly twice that of the DA-pretreated biomass. Further supported by qualitative scanning electron microscopy images, these results suggested that COSLIF treatment disrupted microfibrillar structures within biomass while DA treatment mainly removed hemicelluloses, resulting in a much less substrate accessibility of the latter than of the former. It also concluded that enhancing substrate accessibility was the key to an efficient bioconversion of lignocellulose. A simple method for determining the adsorbed cellulase on cellulosic materials or pretreated lignocellulose was established for better understanding of cellulase adsorption and desorption. This method involved hydrolysis of adsorbed cellulase in the presence of 10 M of NaOH at 121oC for 20 min, followed by the ninhydrin assay for the amino acids released from the hydrolyzed cellulase. The major lignocellulosic components (i.e. cellulose, hemicellulose, and lignin) did not interfere with the ninhydrin assay. A number of cellulase desorption methods were investigated, including pH adjustment, detergents, high salt solution, and polyhydric alcohols. The pH adjustment to 13.0 and the elution by 72% ethylene glycol at a neutral pH were among the most efficient approaches for desorbing the adsorbed cellulase. For the recycling of active cellulase, a modest pH adjustment to 10.0 may be a low-cost method to desorb active cellulase. More than 90% of cellulase for hydrolysis of the pretreated corn stover could be recycled by washing at pH 10.0. This study provided an in-depth understanding of biomass saccharification for the production of cellulosic ethanol for cellulose hydrolysis and cellulase adsorption and desorption. It will be of great importance for developing better lignocellulose pretreatment technologies and improving cellulose hydrolysis by engineered cellulases.
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    Investigating pretreatment methods for struvite precipitation in liquid dairy manure
    Shen, Yanwen (Virginia Tech, 2010-06-17)
    Phosphorus (P) recovery and re-use is very important today for sustainable nutrient cycling and water quality protection due to the declining global P reserves and increasingly stringent wastewater treatment regulations. P recovery as struvite (MgNH4PO4·6H2O) is a promising technology because it can be used as a slow-release fertilizer. The objective of this study was to investigate different pretreatment methods to enhance struvite precipitation in dairy manure. Generally there are two challenges that need to be overcome to precipitate struvite in liquid dairy manure. The first is the relatively high calcium (Ca) concentration. The present study investigated the effectiveness of two calcium binding reagents to reduce the calcium inhibitory effects to enhance the struvite precipitation. A chemical equilibrium model (Visual MINTEQ 2.60) was used to determine the pH to acidify manure and to precipitate struvite. Then, bench-scale experiments were conducted to validate the model results using synthetic and untreated manure. First, the manure was acidified (pH 4.5) to liberate the particulate-bounded Mg2+, Ca2+ and inorganic P. Second, EDTA and/or oxalate compounds were added to the manure. Third, pH was increased to 7.5 for struvite precipitation from the liquid filtrate. Results showed that struvite-containing crystals were obtained from the samples treated with calcium removal compounds, with a total suspended solids (TSS) concentration less than 4,000 mg/L. The second challenge is the low dissolved reactive P (DRP, i.e. PO4-P) to total P (TP) ratio. This study investigated different pretreatment methods for P release to enhance struvite precipitation in liquid dairy manure: enhanced biological phosphorus removal (EBPR), microwave heating (MW) and anaerobic digestion (AD). All of the pretreatment methods resulted in P release but struvite crystals were observed only in precipitates obtained in manure pretreated with MW+H2SO4 acidification. Without oxalic acid to reduce the Ca2+ effects, all the other pretreatment methods were not effective in enhancing struvite precipitation in liquid dairy manure. Hardly any struvite or crystalline-like solids were found in the non-centrifuged samples, regardless of any pretreatment, indicating the effects of suspended solids and organic matter on struvite precipitation. A partial economic analysis was conducted to assess the chemical costs of P recovery from liquid dairy manure with different pretreatment methods; P recovery amounts as struvite were modeled by Visual Minteq 2.61.Three pretreatment conditions (untreated manure + oxalic acid, MW + H2SO4 acidification, and AD) were selected based on the precipitation results. MW + H2SO4 acidification produced the highest struvite quantities among the pretreatment methods to enhance struvite recovery, also with the lowest chemical addition costs.
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    Microalgae as a Feedstock for Biofuel Production
    Wen, Zhiyou; Johnson, Michael Ben (Virginia Cooperative Extension, 2009-05-01)
    Introduces the basics of algal-biofuel production and the current status of this emerging biodiesel source.
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    Microalgal Biodiesel Production through a Novel Attached Culture System and Conversion Parameters
    Johnson, Michael Ben (Virginia Tech, 2009-04-29)
    Due to a number of factors, the biodiesel industry in the United States is surging in growth. Traditionally, oil seed crops such as soybean are used as the feedstock to create biodiesel. However, the crop production can no longer safely keep up with the demand for the growing biodiesel industry. Using algae as a feedstock has been considered for a number of years, but it has always had limitations. These limitations were mainly due to the production methods used to grow and harvest the algae, rather than the reaction methods of creating the biodiesel, which are the same as when using traditional crops. Algae is a promising alternative to other crops for a number of reasons: it can be grown on non arable land, is not a food crop, and produces much more oil than other crops. In this project, we propose a novel attached growth method to produce the algae while recycling dairy farm wastewater using the microalga Chlorella sp. The first part of the study provided a feasibility study as the attachment of the alga onto the supporting substrate as well as determining the pretreatment options necessary for the alga to grow on wastewater. The results showed that wastewater filtered through cheesecloth to remove large particles was feasible for production of Chlorella sp, with pure wastewater producing the highest biomass yield. Most importantly, the attached culture system largely exceeded suspended culture systems as a potentially feasible and practical method to produce microalgae. The algae grew quickly and were able to produce more than 3.2 g/m2-day with lipid contents of about 9% dry weight, while treating dairy farm wastewater and removing upwards of 90% of the total phosphorus and 79% of the nitrogen contained within the wastewater. Once the "proof-of-concept" work was completed, we investigated the effects of repeat harvests and intervals on the biomass and lipid production of the microalgae. The alga, once established, was harvested every 6, 10, or 15 days, with the remaining algae on the substrate material functioning as inoculums for repeated growth. Using this method, a single alga colony produced biomass and lipids for well over six months time in a laboratory setting. The second part of this study investigated another aspect of biodiesel production from algae. Rather than focus solely on biomass production, we looked into biodiesel creation methods as well. Biodiesel is created through a chemical reaction known as transesterification, alcoholysis, or commonly, methylation, when methanol is the alcohol used. There are several different transesterification methods. By simplifying the reaction conditions and examining the effects in terms of maximum fatty acid methyl esters (FAME) produced, we were able to determine that a direct transesterification with chloroform solvent was more effective than the traditional extraction-transesterification method first popularized by Bligh & Dyer in 1959 and widely used. This synergistic research helps to create a more complete picture of where algal biodiesel research and development is going in the future.
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    Microwave-based Pretreatment, Pathogen Fate and Microbial Population in a Dairy Manure Treatment System
    Jin, Ying (Virginia Tech, 2010-11-29)
    Anaerobic digestion and struvite precipitation are two effective ways of treating dairy manure for recovering biogas and phosphorus. Anaerobic digestion of dairy manure is commonly limited by slow fiber degradation, while one of the limitations to struvite precipitation is the availability of orthophosphate. The aim of this work was to study the use of microwave-based thermochemical pretreatment to simultaneously enhance manure anaerobic digestibility (through fiber degradation) and struvite precipitation (through phosphorus solubilization). Microwave heating combined with different chemicals (NaOH, CaO, H₂SO₄, or HCl) enhanced solubilization of manure and degradation of glucan/xylan in dairy manure. However, sulfuric acid-based pretreatment resulted in a low anaerobic digestibility, probably due to the sulfur inhibition and side reactions. The pretreatments released 20-40% soluble phosphorus and 9-14% ammonium. However, CaO-based pretreatment resulted in lower orthophosphate releases and struvite precipitation efficiency as calcium reacts with phosphate to form calcium phosphate. Collectively, microwave heating combined with NaOH or HCl led to a high anaerobic digestibility and phosphorus recovery. Using these two chemicals, the performance of microwave- and conventional-heating in thermochemical pretreatment was further compared. The microwave heating resulted in a better performance in terms of COD solubilization, glucan/xylan reduction, phosphorus solubilization and anaerobic digestibility. Lastly, temperature and heating time used in microwave treatment were optimized. The optimal values of temperature and heating time were 147°C and 25.3 min for methane production, and 135°C and 26 min for orthophosphate release, respectively. Applying manure or slurry directly to the land can contribute to pathogen contamination of land, freshwater and groundwater. Thus it is important to study the fate of pathogens in diary manure anaerobic digestion systems. The goal of the project was to establish a molecular based quantitative method for pathogen identification and quantification, compare the molecular based method with culture based method and study pathogen fate in dairy manure and different anaerobic digesters. Result showed that molecular based method detected more E.coli than the culture based method with less variability. Thermophilic anaerobic digestion can achieve more than 95% pathogen removal rate while mesophilic anaerobic digester had increased E.coli number than fresh manure, indicating temperature is a key factor for pathogen removal. In general, the overall goal of the study is to develop an integrated dairy manure treatment system. The microwave based pretreatment enhanced the subsequent biogas production and struvite precipitation, and the molecular tool based method provided a more precise and faster way to study the pathogen fate in various anaerobic digestions.
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    Mixotrophic Production of Omega-3 Fatty Acid-rich Alga Phaeodactylum tricornutum on Biodiesel-derived Crude Glycerol
    Woisard, Kevin Keith (Virginia Tech, 2010-11-29)
    Crude glycerol is the major byproduct of the biodiesel industry. There is an abundance of this byproduct and purifying it for use in industries such as food, pharmaceutical, or cosmetic is prohibitively expensive. Developing an alternative use for crude glycerol is needed. Utilizing it as a carbon source in the fermentation of algae is one potential method for using this under-utilized byproduct. In this research, crude glycerol is used in the mixotrophic production of the alga, Phaeodactylum tricornutum, which is an eicosapentaenoic acid (EPA) producing diatom. Mixotrophic growth is when cells perform autotrophic and heterotrophic modes of growth concurrently. EPA is an omega-3 polyunsaturated fatty acid that has been demonstrated to have a multitude of beneficial health effects, including maintaining human cardiovascular health, treating cancer and human depression diseases, and an anti-obesity effect. In this study, the potential of using crude glycerol in batch mode mixotrophic culture of P. tricornutum was investigated. Once the mixotrophic culture was established, parameters involved in increasing the biomass and EPA production were optimized. These included nitrogen source, level of supplemental carbon dioxide, and concentration of crude glycerol. Using nitrate, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide led to the highest biomass productivity of 0.446 g L?? day?? and the highest EPA productivity of 16.9 mg L?? day?? in batch mode culture. The continuous culture of the mixotrophic culture was then performed following the batch culture optimization. The effects of dilution rate were observed in continuous culture with the parameters of nitrate as the nitrogen source, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide held constant. The highest biomass productivity of 0.612 g L?? day?? was obtained at D = 0.24 day??. The highest EPA productivity of 16.5 mg L?? day?? was achieved at both D = 0.15 day?? and D = 0.24 day??. The maximum specific growth rate was estimated from the washing out dilution rate and was determined to be around 0.677 day??. Overall, it was found that crude glycerol increases the biomass and EPA productivity of Phaeodactylum tricornutum. Continuous culture with the use of crude glycerol can further increase these measurements. The potential for scaling up studies is demonstrated by these results and can help lead to a market for this abundant, little-used byproduct of the biodiesel industry.
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    Modeling and Production of Bioethanol from Mixtures of Cotton Gin Waste and Recycled Paper Sludge
    Shen, Jiacheng (Virginia Tech, 2008-12-19)
    In this study, the hydrolytic kinetics of mixtures of cotton gin waste (CGW) and recycled paper sludge (RPS) at various initial enzyme concentrations of Spezyme AO3117 and Novozymes NS50052 was investigated. The experiments showed that the concentrations of reducing sugars and the conversions of the mixtures increased with increasing initial enzyme concentration. The reducing sugar concentration and conversion of the mixture of 75% CGW and 25% RPS were higher than those of the mixture of 80% CGW and 20% RPS. The conversion of the former could reach 73.8% after a 72-hour hydrolysis at the initial enzyme loading of 17.4 Filter Paper Unit (FPU)/g substrate. A three-parameter kinetic model with convergent property based on enzyme deactivation and its analytical expression were derived. Using nonlinear regression, the parameters of the model were determined from the experimental data of hydrolytic kinetics of the mixtures. Based on this kinetic model of hydrolysis, two profit rate models, representing two kinds of operating modes with and without substrate recycling, were developed. Using the profit rate models, the optimal enzyme loading and hydrolytic time could be predicted for the maximum profit rate in ethanol production according to the costs of enzyme and operation, enzyme loading, and ethanol market price. Simulated results from the models based on the experimental data of hydrolysis of the mixture of 75% CGW and 25% RPS showed that use of a high substrate concentration and an operating mode with feedstock recycle could greatly increase the profit rate of ethanol production. The results also demonstrated that the hydrolysis at a low enzyme loading was economically required for systematic optimization of ethanol production. The development of profit rate model points out a way to optimize a monotonic function with variables, such as enzyme loading and hydrolytic time for the maximum profit rate. The study also investigated the ethanol production from the steam-exploded mixture of 75 wt% cotton gin waste and 25 wt% recycled paper sludge at various influencing factors, such as enzyme concentration, substrate concentration, and severity factor, by a novel operating mode: semi-simultaneous saccharification and fermentation (SSSF) consisting of a pre-hydrolysis and a simultaneous saccharification and fermentation (SSF). Four cases were studied: 24-hour pre-hydrolysis + 48-hour SSF (SSSF 24), 12-hour pre-hydrolysis + 60-hour SSF (SSSF 12), 72-hour SSF, and 48-hour hydrolysis + 12-hour fermentation (SHF). SSSF 24 produced higher ethanol concentration, yield, and productivity than the other operating modes. The higher temperature of steam explosion favored of ethanol production, but the higher initial enzyme concentration could not increase the final ethanol concentration though the hydrolytic rate of the substrate was increased. A mathematical model of SSSF, which consisted of an enzymatic hydrolysis model and a SSF model including four ordinary differential equations that describe the changes of cellobiose, glucose, microorganism, and ethanol concentrations with respect to residence time, was developed, and was used to simulate the data for the four components in the SSSF processes of ethanol production from the mixture. The model parameters were determined by a MATLAB program based on the batch experimental data of the SSSF. The analysis to the reaction rates of cellobiose, glucose, cell, and ethanol using the model and the parameters from the experiments showed that the conversion of cellulose to cellobiose was a rate-controlling step in the SSSF process of ethanol production from cellulose.
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    Producing eicosapentaenoic acid (EPA) from biodiesel-derived crude glycerol
    (United States Patent and Trademark Office, 2013-09-24)
    Pythium, when cultured with crude glycerol as a carbon source, produces useful polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). The crude glycerol is pretreated to remove soaps and methanol. An exemplary Pythium species for use in the production of EPA is Pythium irregulare.
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    Producing Omega-3 Polyunsaturated Fatty Acids from Biodiesel Waste Glycerol by Microalgae Fermentation
    Ethier, Shannon Elizabeth (Virginia Tech, 2010-05-04)
    Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae Schizochytrium limacinum and Pythium irregulare which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of S. limacinum, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day⁻¹, and a maintenance coefficient of 0.2216 day⁻¹. The second part of this study focused on morphology issues with P. irregulare, a prolific producer of eicosapentaenoic acid (EPA). P. irregulare has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of S. limacinum with high DHA productivity and the morphology of P. irregulare could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species.
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    Production of Eicosapentaenoic acid from biodiesel derived crude glycerol using fungal culture
    Athalye, Sneha Kishor (Virginia Tech, 2008-07-07)
    Omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA, C20:5, n-3) and docosahexaenoic acid (DHA, C22:6, n-3), have many medically established benefits against cardiovascular diseases, cancers, schizophrenia, and Alzheimer's. Currently, fish oil is the main source of omega-3 fatty acids, but there are many problems associated with it such as undesirable taste and odor, and heavy metal contamination. As a result, it is necessary to seek alternative production sources based on various microorganisms. In this thesis we have developed a novel microfungal culture process to produce EPA from the crude glycerol byproduct generated in biodiesel industry. This process provides both an alternative source of omega-3 fatty acids and a benefit to the biodiesel industry. Indeed, as oil prices reach historical highs, biodiesel has attracted increasing interest throughout the United States. The disposal of the crude glycerol byproduct has been a challenge faced by the biodiesel producers. Crude glycerol presents a cheap carbon source for growth of many microorganisms. In this thesis, we tested the feasibility of using crude glycerol for producing eicosapentaenoic acid (EPA, 20:5, n-3) by one algal species, Phaeodactylum tricornutum and two fungal species, Mortierella alpina and Pythium irregulare. We observed that the algal growth is inhibited in the crude glycerol while the fungi can grow very well in crude glycerol-containing medium. The fungus M. alpina produced significant amount of ARA but negligible amount of EPA. P. irregulare produced significant amount of biomass as well as a relatively high level of EPA. The maximum dry biomass for the P. irregulare culture was 2.9 g/L with an EPA productivity of 7.99 mg/L-day. Based on these results, we concluded that P. irregulare was a promising candidate for EPA production from biodiesel derived crude glycerol. Further optimization work showed that P. irregulare grown 30 g/L crude glycerol and 10g/L yeast extract results in the highest level of EPA production. A temperature of 20o C is optimal for high fungal biomass and EPA levels. Addition of vegetable oil (at 1%) enhanced the EPA production and almost doubled the amount of biomass reached. Soap inhibits growth as well as EPA production severely even in small amounts. Methanol completely inhibits growth. The final optimized growth conditions for the fungus P.irregulare were a medium with 30g/L of crude glycerol, 10 g/L of yeast extract at a pH of 6 with 1% supplementation of oil, at a temperature of 20o C for a period of 7 days.Thus we have established that the fungus P.irregulare can be used successfully to produce high mounts of EPA from crude glycerol.
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    Small-scale biodiesel production: safety, fuel quality, and waste disposal considerations
    Wen, Zhiyou; Bantz, Steve A.; Bachmann, Christopher G.; Brodrick, Christie-Joy; Schweitzer, Lisa A. (Virginia Cooperative Extension, 2009-05-01)
    This publication explains how explains how to produce biodiesel fuel on a small scale from waste oil, fats, and oilseed crops. It addresses safety concerns, fuel quality, and by-product disposal.
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    Sulfate and Hydroxide Supported on Zirconium Oxide Catalysts for Biodiesel Production
    Abdoulmoumine, Nourredine (Virginia Tech, 2010-06-18)
    Biodiesel is currently produced by homogeneous catalysis. More recently however, heterogeneous catalysis is being considered as a cheaper alternative to the homogeneous process. In this research project, heterogeneous catalysts of zirconium oxide were produced by impregnation. Zirconium oxide impregnation with sulfuric acid produced acidic solid catalysts. It was determined that impregnation and calcination at 550oC (SO₄/ZrO₂-550oC) produced the best catalyst for palmitic acid esterification with 10 wt % as the optimum concentration in esterification of palmitic acid. SO₄/ZrO₂-550oC was successfully recycled for eight consecutive runs before permanent deactivation. Its sulfur content was 1.04 wt % using SEM-EDS and 2.05 wt % using XPS for characterization. BET surface area was 90.89 m2/g. The reaction mechanism over Brønsted acid (SO₄/ZrO₂-550oC) and Lewis acid (Al₂O₃) catalysts obeyed Eley-Rideal kinetics with palmitic acid and methanol adsorbed on the active site respectively. Zirconium oxide was also impregnated with sodium hydroxide to produce basic catalysts. The best catalyst was produced when zirconium oxide was impregnated with 1.5 M NaOH and calcined at 600oC. Soybean oil was completely converted to biodiesel with 10 wt % catalyst and 1:6 oil to methanol. A mixture of the base catalyst with 30 wt % SO₄/ZrO₂-550oC effectively converted soybean oil containing 5% oleic acid indicating that this mixture could be used for waste oils. The reaction was first order with respect to triglyceride and second order with respect to methanol. The activation energy was 49.35 kJ/mol and the reaction mechanism obeyed Langmuir-Hinshelwood kinetics.