A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified ... more A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of the COVID-19 pandemic that originated in China in December 2019. Although extensive research has been performed on SARS-CoV-2, the binding behavior of spike (S) protein and receptor binding domain (RBD) of SARS-CoV-2 at different environmental conditions have yet to be studied. The objective of this study is to investigate the effect of temperature, fatty acids, ions, and protein concentration on the binding behavior and rates of association and dissociation between the S protein and RBD of SARS-CoV-2 and the hydrophobic aminopropylsilane (APS) biosensors using biolayer interferometry (BLI) validated with molecular dynamics simulation. Our results suggest three conditions—high ionic concentration, presence of hydrophobic fatty acids, and low temperature—favor the attachment of S protein and RBD to hydrophobic surfaces. Increasing the temperature within an hour from 0 to 2...
Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extrac... more Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extracted microalgae (LEA). The advantages of protein hydrolysates over other protein products encompass enhanced solubility, digestibility, and potential bioactivity. The development of an economically feasible process to produce protein hydrolysates depends on maximizing the recovery of hydrolyzed native protein from the lipid-extracted algal biomass and subsequent fractionation of hydrolyzed protein slurry. Previously, we reported a method for fractionation of enzymatically generated protein hydrolysates by acidic precipitation of algal cell debris and unhydrolyzed protein, precipitate wash, centrifugation, and depth filtration. The present study evaluates tangential flow ultrafiltration as a single-step alternative to centrifugation, precipitate wash, and depth filtration. The results demonstrate that the tangential flow ultrafiltration process has a potential that deserves further investi...
Microalgae have received a substantial amount of attention as an alternative fuel feedstock due t... more Microalgae have received a substantial amount of attention as an alternative fuel feedstock due to their ability to produce large quantities of lipids. The goal of this research was to determine the ideal operating parameters for electrocoagulation; a low cost, low energy method of dewatering cultures of microalgae. The objectives of this research focus on recognizing parameters that influence the overall efficiency of the process, effective electrode materials, and finally directional improvements in operating parameters contributing to a high reduction in optical density. Variables found to have a statistically significant effect on the efficiency of electrocoagulation were the electrode material, current, and duration. With no adjustment of the algae culture prior to electrocoagulation, iron and nickel were identified as the best performing electrode materials, in terms of optical density reduction. Of the materials tested, iron was found to achieve the greatest recovery of microalgae at the lowest power consumption, while staying below the threshold for animal feed tolerance. The most desirable operating parameters for electrocoagulation, within the confines of the experimental apparatus and using iron electrodes, were found to be a current of 0.3 amps and a 15 min reaction time. Increases in current and duration were found to provide the highest levels of optical density reduction. However, the average voltage, and therefore, power consumption are the highest when current and duration are maximized. Additional testing should be performed at higher currents and longer durations, in an attempt to find a peak in the optical density reduction. iii DEDICATION To my wife and son.
GENERAL INTRODUCTION Glucoamylase (a-l,4-glucan glucohydrolase, EC 3.2.1.3) is a typical exo-gluc... more GENERAL INTRODUCTION Glucoamylase (a-l,4-glucan glucohydrolase, EC 3.2.1.3) is a typical exo-glucanase in its production of D-glucose from the nonreducing ends of amylose, amylopectin, and maltooligosaccharide molecules with inversion of configuration of the anomeric carbon (1). It is an important industrial enzyme used in the hydrolysis of liquefied starch to syrups of high glucose concentration. Although glucoamylase is capable of hydrolyzing a-1,6-as well as a-l,4-glucosidic linkages (2), glucose yields higher than 95% of theoretical are not achieved in concentrated starch solutions because of the occurrence of glucose condensation reactions, commonly called reversion reactions (3). Formation of reversion product, mainly di-and trisaccharides, was often attributed to the catalytic action of transglucosylase contamination present in the glucoamylase preparations. However, a number of studies published in the last twenty years indicate that glucoamylase itself is capable of synthesizing various a-linked di-and trisaccharides in concentrated glucose solutions (4-10), The reported reversion products include isomaltose (6-0-aD -glucopyranosyl-D-glucose), maltose (4-0-aD -glucopyranosyl-D-glucose), nigerose (3-0-aD -glucopyranosyl-D-glucose), kojibiose (2-0-aD -glucopyranosyl-D-glucose), a,3-trehalose (a-O-D-glucopyranosyl-3-D-glucopyranoside), isomaltotriose 2 2 (6-0-ct-D-glucopyranosyl-D-isomaltose), panose (6-0-aD -glucopyranosyl-D-2 maltose), isopanose (4-0-aD -glucopyranosyl-D-isomaltose), and maltotriose 2 (4-O-cx-D-glucopyranosyl-D-maltose). Two disaccharides, a,3-trehalose and kojibiose, were observed for the first time in our previous work (10). Recently research Interest has been directed towards the kinetics and modeling of reversion reactions (9, 11-13). The common features of these studies are that maltose and isomaltose were the only condensation products detected and modeled, except for Beschkov et al. (11) who also included maltotriose, and that high performance liquid chromatography (HPLC) was used as an analytical method. Critical examination of the literature data shows significant dis agreement in the number and the type of reversion products formed. Some of the discrepancies may be caused by differences in experimental condi tions, like pH, temperature, and initial substrate concentration, or use of glucoamylase preparations with a different degree of purity and from a different origin. In addition, the applied chromatographic methods were probably not efficient enough to separate and identify all reaction products. Although evidence exists that glucoamylase is capable of synthesizing a-glucosidic bonds other than a-1,4 and a-1,6, fundamental understanding of condensation reactions is still lacking. The objectives of the kinetic studies (Section IV) were to examine the effect of reaction conditions on the rates and equilibria of reversion reactions as well as to compare the catalytic activities of A. niger glucoamylase forms. Attention has been focused on testing the validity and generality of the derived model which describes the simultaneous formation of reversion products. Explanation of Dissertation Format The dissertation contains four sections written in a form suitable for publication. The first three sections, which deal with HPLC of 3 carbohydrates, have already been published in the Journal of Chromatography (14-16), The last section presents the work on glucose reversion reac tions. The purification procedure of glucoamylase forms is described in the Appendix. Each section, as well as the General Introduction and Appendix, contains a list of references cited within.
The degradation performance of 11 types of commercially produced degradable starch-polyethylene p... more The degradation performance of 11 types of commercially produced degradable starch-polyethylene plastic compost bags was evaluated in municipal yard waste compost sites at Iowa State University (Ames) and in Carroll, Dubuque, and Grinnell, Iowa. Masterbatches for plastic production were provided by Archer Daniels Midland Co. (Decatur, Ill.), St. Lawrence Starch Co. Ltd. (Mississauga, Ontario, Canada), and Fully Compounded Plastics (Decatur, Ill.). Bags differed in starch content (5 to 9%) and prooxidant additives (transition metals and a type of unsaturated vegetable oil). Chemical and photodegradation properties of each material were evaluated. Materials from St. Lawrence Starch Co. Ltd. and Fully Compounded Plastics photodegraded faster than did materials from Archer Daniels Midland Co., whereas all materials containing transition metals demonstrated rapid thermal oxidative degradation in 70°C-oven (dry) and high-temperature, high-humidity (steam chamber) treatments. Each compost ...
Extraction of multiple high-value products is recommended for sustainability of the microalgal pr... more Extraction of multiple high-value products is recommended for sustainability of the microalgal production platform. This study proposes a process for selective extraction of carotenoids and chlorophylls with ethanol, followed by alkaline pH extraction of proteins from wet, freeze-thawed Chlorella vulgaris biomass. A biomass-tosolvent ratio of 1:5 and 3 extraction stages were required to achieve maximum extraction yield of chlorophylls and carotenoids. The main compounds in the ethanol extract were identified as lutein, chlorophyll a and chlorophyll b. The lutein and total chlorophyll yield in the extracts were 5.4 mg/g and 15.4 mg/g dry biomass respectively. Effective protein release from freeze-thawed biomass was contact-time dependent and > 76% of total protein could be extracted in 15 min via bead-milling, and in 6 min by high-pressure homogenization at 15000 psi. Ethanol extraction of pigments affected protein solubility, and an alkaline pH was required to release the same total protein. Concentration and fractionation of proteins was carried out using a two-stage membrane filtration process and 78-80% of proteins remained in the 300 kDa retentate. Ethanol treatment and higher pH conditions did not negatively impact membrane filtration, nutritive value, or the emulsification properties of protein concentrates.
Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic appl... more Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point (pI), and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant E. coli lysates. Reported processes for OPN recovery from recombinant sources use non-orthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixedmodal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes.
Energy-efficient and scalable harvesting and lipid extraction processes must be developed in orde... more Energy-efficient and scalable harvesting and lipid extraction processes must be developed in order for the algal biofuels and bioproducts industry to thrive. The major challenge for harvesting is the handling of large volumes of cultivation water to concentrate low amounts of biomass. For lipid extraction, the major energy and cost drivers are associated with disrupting the algae cell wall and drying the biomass before solvent extraction of the lipids. Here we review the research and development conducted by the Harvesting and Extraction Team during the 3-year National Alliance for Advanced Biofuels and Bioproducts (NAABB) algal consortium project. The harvesting and extraction team investigated five harvesting and three wet extraction technologies at lab bench scale for effectiveness, and conducted a techoeconomic study to evaluate their costs and energy efficiency compared to available baseline technologies. Based on this study, three harvesting technologies were selected for further study at larger scale. The selected harvesting technologies: electrocoagulation, membrane filtration, and ultrasonic harvesting, were evaluated in a field study at minimum scale of 100 L/h. None of the extraction technologies were determined to be ready for scale-up; therefore, an emerging extraction technology (wet solvent extraction) was selected from industry to provide scale-up data and capabilities to produce lipid and lipidextracted materials for the NAABB program. One specialized extraction/adsorption technology was developed that showed promise for recovering high value co-products from lipid extracts. Overall, the NAABB Harvesting and Extraction Team improved the readiness level of several innovative, energy efficient technologies to integrate with algae production processes and captured valuable lessons learned about scale-up challenges. 1. Introduction 1.1. Preface Harvesting algae and extracting the lipids are significant cost drivers in the biofuel production process [1-6]. Therefore, the goal of the Harvesting and Extraction task within the National Alliance for Biofuels and Bioproducts (NAABB) was to develop low-energy, low-cost harvesting and extraction technologies that could feed lipids into highly efficient fuel-conversion processes [7]. At the outset of the project, the NAABB leadership team determined that the harvesting and extraction technologies developed in this task must have: • Low capital expense (CAPEX) and operating expense (OPEX) • Ease of operation and low maintenance requirements
The main objective of NAABB was to combine science, technology, and engineering expertise from ac... more The main objective of NAABB was to combine science, technology, and engineering expertise from across the nation to break down critical technical barriers to commercialization of algae-based biofuels. The approach was to address technology development across the entire value chain of algal biofuels production, from selection of strains to cultivation, harvesting, extraction, fuel conversion, and agricultural coproduct production. Sustainable practices and financial feasibility assessments ununderscored the approach and drove the technology development
The present invention includes a system and method for making a biofuel comprising: providing a n... more The present invention includes a system and method for making a biofuel comprising: providing a nitrogen-limiting, minimal growth media comprising glycerol, sugars generated from cellulosic biomass or both, under conditions in which an oleaginous microbe converts the growth media into at least one of triacylglycerol, neutral lipids, fatty acids, long-chain fatty acids, and hydrocarbons that is secreted by the microbe.U
The present invention provides a method for controlling the partitioning of a recombinant protein... more The present invention provides a method for controlling the partitioning of a recombinant protein between the supernatant and the periplasm in E.coli host cell cultures wherein expression of the recombinant protein by said cells is under the control of an inducible system, which method comprises: a) providing an E. coli host cell culture b) changing the growth rate of theE.coli host cells c) inducing expression of the recombinant protein wherein steps (b) and (c) can be performed in any order or simultaneously; and subsequently d) determining the yield of recombinant protein in the culture supernatant and the E.coli host cell periplasm e) comparing the yield determined in step (d) with the yield determined when at least one other growth rate has been used in step (b) f) selecting a growth rate from the comparison made in step (e) in which the partitioning of the recombinant protein between the supernatant and the periplasm is most suited to the primary recovery of the recombinant protein.
A recombinant cyclodextrin glucanotransferase mutant is provided, which mutant produces lowered a... more A recombinant cyclodextrin glucanotransferase mutant is provided, which mutant produces lowered amounts of β-cyclodextrins and γ-cyclodextrins compared to wild-type cyclodextrin glucanotransferase
The retention parameters of a series of carbohydrates (eg, digitoxose, sucrose, glucose and myo-i... more The retention parameters of a series of carbohydrates (eg, digitoxose, sucrose, glucose and myo-inositol) have been investigated on columns (25 cm × 4.6 mm) of Zorbax-NH2, Supelcosil LC-NH2 and Supelcosil LC-Si. Mobile phases comprised aq. methanol and aq. acetonitrile ...
Abstract The relative retention times of seventeen trimethylsilyl disaccharides (cellobiose, gent... more Abstract The relative retention times of seventeen trimethylsilyl disaccharides (cellobiose, gentiobiose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose, maltulose, melibiose, nigerose, palatinose, sophorose, sucrose, αα-trehalose, turanose and xylobiose) on a fused-silica capillary column coated with SE-54 are reported. Equilibrium anomeric compositions of fourteen of these disaccharides in pyridine are also given. Isothermal operation at 240°C allowed complete quantitation of the mixture.
Abstract Granular starch was used as a biospecific adsorbent to investigate the possible applicat... more Abstract Granular starch was used as a biospecific adsorbent to investigate the possible application of the starch-binding domain (SBD) as an affinity tail for a one-step purification of target proteins from crude cell extracts. A β-galactosidase (β-gal) fusion protein containing the C-terminal 119 amino-acids from GA-I (BSB119) was used as a model system to study the starch binding and elution. Because of proteolysis, approximately 40% of initial β-gal activity lacked the SBD, and the remaining fusion protein contained from to one to four SBDs per molecule of β-gal tetramer. The fusion protein forms containing at least one intact SBD adsorbed to starch. The bound fusion protein was eluted by using 10 m m solutions of various maltooligosaccharides and cyclodextrins. The best eluants were 10 m m maltodextrin with an average degree of polymerization ( DP ) of 10 and 10 m m β-cyclodextrin. The elution of BSB119 with maltooligosaccharides of increasing DP suggested that the starch-binding site of the SBD consists of at least five glucosyl binding sites. SDS-PAGE gels and Western blots showed that the purity of the fusion protein eluted from starch was as good as or better than that obtained by conventional affinity chromatography.
Abstract Five α-linked diglucosyl disaccharides (isomaltose, nigerose, kojibiose, maltose, and α,... more Abstract Five α-linked diglucosyl disaccharides (isomaltose, nigerose, kojibiose, maltose, and α, β-trehalose) and two trisaccharides (isomaltotriose and panose) were produced in 90% aqueous solutions of 2-methoxyethyl ether, 2-ethoxyethyl ether, or triethylene glycol ...
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified ... more A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of the COVID-19 pandemic that originated in China in December 2019. Although extensive research has been performed on SARS-CoV-2, the binding behavior of spike (S) protein and receptor binding domain (RBD) of SARS-CoV-2 at different environmental conditions have yet to be studied. The objective of this study is to investigate the effect of temperature, fatty acids, ions, and protein concentration on the binding behavior and rates of association and dissociation between the S protein and RBD of SARS-CoV-2 and the hydrophobic aminopropylsilane (APS) biosensors using biolayer interferometry (BLI) validated with molecular dynamics simulation. Our results suggest three conditions—high ionic concentration, presence of hydrophobic fatty acids, and low temperature—favor the attachment of S protein and RBD to hydrophobic surfaces. Increasing the temperature within an hour from 0 to 2...
Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extrac... more Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extracted microalgae (LEA). The advantages of protein hydrolysates over other protein products encompass enhanced solubility, digestibility, and potential bioactivity. The development of an economically feasible process to produce protein hydrolysates depends on maximizing the recovery of hydrolyzed native protein from the lipid-extracted algal biomass and subsequent fractionation of hydrolyzed protein slurry. Previously, we reported a method for fractionation of enzymatically generated protein hydrolysates by acidic precipitation of algal cell debris and unhydrolyzed protein, precipitate wash, centrifugation, and depth filtration. The present study evaluates tangential flow ultrafiltration as a single-step alternative to centrifugation, precipitate wash, and depth filtration. The results demonstrate that the tangential flow ultrafiltration process has a potential that deserves further investi...
Microalgae have received a substantial amount of attention as an alternative fuel feedstock due t... more Microalgae have received a substantial amount of attention as an alternative fuel feedstock due to their ability to produce large quantities of lipids. The goal of this research was to determine the ideal operating parameters for electrocoagulation; a low cost, low energy method of dewatering cultures of microalgae. The objectives of this research focus on recognizing parameters that influence the overall efficiency of the process, effective electrode materials, and finally directional improvements in operating parameters contributing to a high reduction in optical density. Variables found to have a statistically significant effect on the efficiency of electrocoagulation were the electrode material, current, and duration. With no adjustment of the algae culture prior to electrocoagulation, iron and nickel were identified as the best performing electrode materials, in terms of optical density reduction. Of the materials tested, iron was found to achieve the greatest recovery of microalgae at the lowest power consumption, while staying below the threshold for animal feed tolerance. The most desirable operating parameters for electrocoagulation, within the confines of the experimental apparatus and using iron electrodes, were found to be a current of 0.3 amps and a 15 min reaction time. Increases in current and duration were found to provide the highest levels of optical density reduction. However, the average voltage, and therefore, power consumption are the highest when current and duration are maximized. Additional testing should be performed at higher currents and longer durations, in an attempt to find a peak in the optical density reduction. iii DEDICATION To my wife and son.
GENERAL INTRODUCTION Glucoamylase (a-l,4-glucan glucohydrolase, EC 3.2.1.3) is a typical exo-gluc... more GENERAL INTRODUCTION Glucoamylase (a-l,4-glucan glucohydrolase, EC 3.2.1.3) is a typical exo-glucanase in its production of D-glucose from the nonreducing ends of amylose, amylopectin, and maltooligosaccharide molecules with inversion of configuration of the anomeric carbon (1). It is an important industrial enzyme used in the hydrolysis of liquefied starch to syrups of high glucose concentration. Although glucoamylase is capable of hydrolyzing a-1,6-as well as a-l,4-glucosidic linkages (2), glucose yields higher than 95% of theoretical are not achieved in concentrated starch solutions because of the occurrence of glucose condensation reactions, commonly called reversion reactions (3). Formation of reversion product, mainly di-and trisaccharides, was often attributed to the catalytic action of transglucosylase contamination present in the glucoamylase preparations. However, a number of studies published in the last twenty years indicate that glucoamylase itself is capable of synthesizing various a-linked di-and trisaccharides in concentrated glucose solutions (4-10), The reported reversion products include isomaltose (6-0-aD -glucopyranosyl-D-glucose), maltose (4-0-aD -glucopyranosyl-D-glucose), nigerose (3-0-aD -glucopyranosyl-D-glucose), kojibiose (2-0-aD -glucopyranosyl-D-glucose), a,3-trehalose (a-O-D-glucopyranosyl-3-D-glucopyranoside), isomaltotriose 2 2 (6-0-ct-D-glucopyranosyl-D-isomaltose), panose (6-0-aD -glucopyranosyl-D-2 maltose), isopanose (4-0-aD -glucopyranosyl-D-isomaltose), and maltotriose 2 (4-O-cx-D-glucopyranosyl-D-maltose). Two disaccharides, a,3-trehalose and kojibiose, were observed for the first time in our previous work (10). Recently research Interest has been directed towards the kinetics and modeling of reversion reactions (9, 11-13). The common features of these studies are that maltose and isomaltose were the only condensation products detected and modeled, except for Beschkov et al. (11) who also included maltotriose, and that high performance liquid chromatography (HPLC) was used as an analytical method. Critical examination of the literature data shows significant dis agreement in the number and the type of reversion products formed. Some of the discrepancies may be caused by differences in experimental condi tions, like pH, temperature, and initial substrate concentration, or use of glucoamylase preparations with a different degree of purity and from a different origin. In addition, the applied chromatographic methods were probably not efficient enough to separate and identify all reaction products. Although evidence exists that glucoamylase is capable of synthesizing a-glucosidic bonds other than a-1,4 and a-1,6, fundamental understanding of condensation reactions is still lacking. The objectives of the kinetic studies (Section IV) were to examine the effect of reaction conditions on the rates and equilibria of reversion reactions as well as to compare the catalytic activities of A. niger glucoamylase forms. Attention has been focused on testing the validity and generality of the derived model which describes the simultaneous formation of reversion products. Explanation of Dissertation Format The dissertation contains four sections written in a form suitable for publication. The first three sections, which deal with HPLC of 3 carbohydrates, have already been published in the Journal of Chromatography (14-16), The last section presents the work on glucose reversion reac tions. The purification procedure of glucoamylase forms is described in the Appendix. Each section, as well as the General Introduction and Appendix, contains a list of references cited within.
The degradation performance of 11 types of commercially produced degradable starch-polyethylene p... more The degradation performance of 11 types of commercially produced degradable starch-polyethylene plastic compost bags was evaluated in municipal yard waste compost sites at Iowa State University (Ames) and in Carroll, Dubuque, and Grinnell, Iowa. Masterbatches for plastic production were provided by Archer Daniels Midland Co. (Decatur, Ill.), St. Lawrence Starch Co. Ltd. (Mississauga, Ontario, Canada), and Fully Compounded Plastics (Decatur, Ill.). Bags differed in starch content (5 to 9%) and prooxidant additives (transition metals and a type of unsaturated vegetable oil). Chemical and photodegradation properties of each material were evaluated. Materials from St. Lawrence Starch Co. Ltd. and Fully Compounded Plastics photodegraded faster than did materials from Archer Daniels Midland Co., whereas all materials containing transition metals demonstrated rapid thermal oxidative degradation in 70°C-oven (dry) and high-temperature, high-humidity (steam chamber) treatments. Each compost ...
Extraction of multiple high-value products is recommended for sustainability of the microalgal pr... more Extraction of multiple high-value products is recommended for sustainability of the microalgal production platform. This study proposes a process for selective extraction of carotenoids and chlorophylls with ethanol, followed by alkaline pH extraction of proteins from wet, freeze-thawed Chlorella vulgaris biomass. A biomass-tosolvent ratio of 1:5 and 3 extraction stages were required to achieve maximum extraction yield of chlorophylls and carotenoids. The main compounds in the ethanol extract were identified as lutein, chlorophyll a and chlorophyll b. The lutein and total chlorophyll yield in the extracts were 5.4 mg/g and 15.4 mg/g dry biomass respectively. Effective protein release from freeze-thawed biomass was contact-time dependent and > 76% of total protein could be extracted in 15 min via bead-milling, and in 6 min by high-pressure homogenization at 15000 psi. Ethanol extraction of pigments affected protein solubility, and an alkaline pH was required to release the same total protein. Concentration and fractionation of proteins was carried out using a two-stage membrane filtration process and 78-80% of proteins remained in the 300 kDa retentate. Ethanol treatment and higher pH conditions did not negatively impact membrane filtration, nutritive value, or the emulsification properties of protein concentrates.
Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic appl... more Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point (pI), and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant E. coli lysates. Reported processes for OPN recovery from recombinant sources use non-orthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixedmodal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes.
Energy-efficient and scalable harvesting and lipid extraction processes must be developed in orde... more Energy-efficient and scalable harvesting and lipid extraction processes must be developed in order for the algal biofuels and bioproducts industry to thrive. The major challenge for harvesting is the handling of large volumes of cultivation water to concentrate low amounts of biomass. For lipid extraction, the major energy and cost drivers are associated with disrupting the algae cell wall and drying the biomass before solvent extraction of the lipids. Here we review the research and development conducted by the Harvesting and Extraction Team during the 3-year National Alliance for Advanced Biofuels and Bioproducts (NAABB) algal consortium project. The harvesting and extraction team investigated five harvesting and three wet extraction technologies at lab bench scale for effectiveness, and conducted a techoeconomic study to evaluate their costs and energy efficiency compared to available baseline technologies. Based on this study, three harvesting technologies were selected for further study at larger scale. The selected harvesting technologies: electrocoagulation, membrane filtration, and ultrasonic harvesting, were evaluated in a field study at minimum scale of 100 L/h. None of the extraction technologies were determined to be ready for scale-up; therefore, an emerging extraction technology (wet solvent extraction) was selected from industry to provide scale-up data and capabilities to produce lipid and lipidextracted materials for the NAABB program. One specialized extraction/adsorption technology was developed that showed promise for recovering high value co-products from lipid extracts. Overall, the NAABB Harvesting and Extraction Team improved the readiness level of several innovative, energy efficient technologies to integrate with algae production processes and captured valuable lessons learned about scale-up challenges. 1. Introduction 1.1. Preface Harvesting algae and extracting the lipids are significant cost drivers in the biofuel production process [1-6]. Therefore, the goal of the Harvesting and Extraction task within the National Alliance for Biofuels and Bioproducts (NAABB) was to develop low-energy, low-cost harvesting and extraction technologies that could feed lipids into highly efficient fuel-conversion processes [7]. At the outset of the project, the NAABB leadership team determined that the harvesting and extraction technologies developed in this task must have: • Low capital expense (CAPEX) and operating expense (OPEX) • Ease of operation and low maintenance requirements
The main objective of NAABB was to combine science, technology, and engineering expertise from ac... more The main objective of NAABB was to combine science, technology, and engineering expertise from across the nation to break down critical technical barriers to commercialization of algae-based biofuels. The approach was to address technology development across the entire value chain of algal biofuels production, from selection of strains to cultivation, harvesting, extraction, fuel conversion, and agricultural coproduct production. Sustainable practices and financial feasibility assessments ununderscored the approach and drove the technology development
The present invention includes a system and method for making a biofuel comprising: providing a n... more The present invention includes a system and method for making a biofuel comprising: providing a nitrogen-limiting, minimal growth media comprising glycerol, sugars generated from cellulosic biomass or both, under conditions in which an oleaginous microbe converts the growth media into at least one of triacylglycerol, neutral lipids, fatty acids, long-chain fatty acids, and hydrocarbons that is secreted by the microbe.U
The present invention provides a method for controlling the partitioning of a recombinant protein... more The present invention provides a method for controlling the partitioning of a recombinant protein between the supernatant and the periplasm in E.coli host cell cultures wherein expression of the recombinant protein by said cells is under the control of an inducible system, which method comprises: a) providing an E. coli host cell culture b) changing the growth rate of theE.coli host cells c) inducing expression of the recombinant protein wherein steps (b) and (c) can be performed in any order or simultaneously; and subsequently d) determining the yield of recombinant protein in the culture supernatant and the E.coli host cell periplasm e) comparing the yield determined in step (d) with the yield determined when at least one other growth rate has been used in step (b) f) selecting a growth rate from the comparison made in step (e) in which the partitioning of the recombinant protein between the supernatant and the periplasm is most suited to the primary recovery of the recombinant protein.
A recombinant cyclodextrin glucanotransferase mutant is provided, which mutant produces lowered a... more A recombinant cyclodextrin glucanotransferase mutant is provided, which mutant produces lowered amounts of β-cyclodextrins and γ-cyclodextrins compared to wild-type cyclodextrin glucanotransferase
The retention parameters of a series of carbohydrates (eg, digitoxose, sucrose, glucose and myo-i... more The retention parameters of a series of carbohydrates (eg, digitoxose, sucrose, glucose and myo-inositol) have been investigated on columns (25 cm × 4.6 mm) of Zorbax-NH2, Supelcosil LC-NH2 and Supelcosil LC-Si. Mobile phases comprised aq. methanol and aq. acetonitrile ...
Abstract The relative retention times of seventeen trimethylsilyl disaccharides (cellobiose, gent... more Abstract The relative retention times of seventeen trimethylsilyl disaccharides (cellobiose, gentiobiose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose, maltulose, melibiose, nigerose, palatinose, sophorose, sucrose, αα-trehalose, turanose and xylobiose) on a fused-silica capillary column coated with SE-54 are reported. Equilibrium anomeric compositions of fourteen of these disaccharides in pyridine are also given. Isothermal operation at 240°C allowed complete quantitation of the mixture.
Abstract Granular starch was used as a biospecific adsorbent to investigate the possible applicat... more Abstract Granular starch was used as a biospecific adsorbent to investigate the possible application of the starch-binding domain (SBD) as an affinity tail for a one-step purification of target proteins from crude cell extracts. A β-galactosidase (β-gal) fusion protein containing the C-terminal 119 amino-acids from GA-I (BSB119) was used as a model system to study the starch binding and elution. Because of proteolysis, approximately 40% of initial β-gal activity lacked the SBD, and the remaining fusion protein contained from to one to four SBDs per molecule of β-gal tetramer. The fusion protein forms containing at least one intact SBD adsorbed to starch. The bound fusion protein was eluted by using 10 m m solutions of various maltooligosaccharides and cyclodextrins. The best eluants were 10 m m maltodextrin with an average degree of polymerization ( DP ) of 10 and 10 m m β-cyclodextrin. The elution of BSB119 with maltooligosaccharides of increasing DP suggested that the starch-binding site of the SBD consists of at least five glucosyl binding sites. SDS-PAGE gels and Western blots showed that the purity of the fusion protein eluted from starch was as good as or better than that obtained by conventional affinity chromatography.
Abstract Five α-linked diglucosyl disaccharides (isomaltose, nigerose, kojibiose, maltose, and α,... more Abstract Five α-linked diglucosyl disaccharides (isomaltose, nigerose, kojibiose, maltose, and α, β-trehalose) and two trisaccharides (isomaltotriose and panose) were produced in 90% aqueous solutions of 2-methoxyethyl ether, 2-ethoxyethyl ether, or triethylene glycol ...
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Papers by Zivko Nikolov