If biosurfactants are to achieve their promise in environmental oil‐spill remediation, their toxicity to marine life must be assessed. A killifish larvae assay is commonly used as a measure of toxicity but is difficult and nonlinear in response. Red blood cell (RBC) hemolysis has also been the basis for assays of some surfactant levels. Here we present a modified sheep RBC suspension assay and compare its response to that of the fish assay for surfactin and its genetically modified variant fatty‐acyl‐glutamate (FA‐Glu). This is the first report of hemolytic activity as a property of FA‐Glu. The method's potential for screening for toxicity against marine organisms is demonstrated. © 2016 American Institute of Chemical Engineers Environ Prog, 36: 505–510, 2017

Microscopic observation of sunflower meal before and after extraction indicated that extensive cellular disruption was achieved by extrusion, but that unextracted oil remained sequestered as coalesced oil within the void spaces of disrupted cotyledon cells. A full factorial design experiment was defined to develop aqueous extraction processing (AEP) with and without enzymes to improve vegetable oil extraction yields of extruded sunflower meal. This experimental design studied the influence of four parameters, agitation, liquid/solid (L/S) ratio, and cellulase and protease addition, on extraction yield of lipid and protein. Agitation and addition of cellulases increased oil extraction yield, indicating that emulsification of oil and alteration of the geometry of the confining cellular matrix were important mechanisms for improving yields. Protease and liquid-solid ratio of the extraction mixture did not have significant effects, indicating key differences with previously established soy oil extraction mechanisms. Maximum yields attained for oil and protein extraction were 39% and 90%, respectively, with the aid of a surfactant.

Host cell proteins (HCP) are a problematic set of impurities in downstream processing (DSP) as they behave most similarly to the target protein during separation. Approaching DSP with the knowledge of HCP separation behavior would be beneficial for the production of high purity recombinant biologics. Therefore, this work was aimed at characterizing the separation behavior of complex mixtures of HCP during a commonly used method: anion-exchange chromatography (AEX). An additional goal was to evaluate the performance of a statistical methodology, based on the characterization data, as a tool for predicting protein separation behavior. Aqueous two-phase partitioning followed by two-dimensional electrophoresis provided data on the three physicochemical properties most commonly exploited during DSP for each HCP: pI (isoelectric point), molecular weight, and surface hydrophobicity. The protein separation behaviors of two alternative expression host extracts (corn germ and E. coli) were characterized. A multivariate random forest (MVRF) statistical methodology was then applied to the database of characterized proteins creating a tool for predicting the AEX behavior of a mixture of proteins. The accuracy of the MVRF method was determined by calculating a root mean squared error value for each database. This measure never exceeded a value of 0.045 (fraction of protein populating each of the multiple separation fractions) for AEX. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1453-1463, 2016.

Because of safety concerns and product consistency issues with the use of animal-derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple-helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia-produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre- and post-treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13-fold purification with a 2.7-fold volume reduction which would reduce the pepsin requirement for post-recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016

Fatty-acyl-glutamate (FA-Glu), a surfactin variant has been successfully produced using a genetically modified strain of Bacillus subtilis grown on glucose. However, yields with soybean hulls (SBH) replacing glucose were lower. This work was undertaken to reduce the yield loss when using SBH as the carbon source and to evaluate two other soy by-products, namely fiber and skim from aqueous oil extraction as alternative carbon and nitrogen sources.Fermentation of soybean hulls, fibers and skim at various concentrations produced lower FA-Glu titers compared to S-7 medium. Neither increasing their amount nor supplementing with glucose increased the FA-Glu titer, suggesting the presence of an inhibitor in these feedstocks. By using a mixture of polysaccharide-degrading enzymes, over 65% of SBH solids were converted to soluble carbohydrates. FA-Glu titers obtained from SBH hydrolysates containing residual hull solids were still low; however, with the removal of the solids, cell growth improved and FA-Glu yield was 60% higher than with glucose. Thus, this low-cost material can be converted to a substrate for production of FA-Glu biosurfactant. Unmodified fiber and skim components of aqueous oil extraction were not beneficial. (C) 2014 Elsevier BAT. All rights reserved.

Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were similar to 30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (OUR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10 g/L, respectively. However, CMC values were higher, 0.02 and 0.40 g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per trillion (ppt) Instant Ocean (R) sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean (R) solutions containing 12 ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis, which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25 ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate. (C) 2013 Elsevier Inc. All rights reserved.

During the spring 2012 semester, a task force of graduate students, staff, and faculty created a survey to gather information from women graduate students about their experiences at Iowa State University (ISU). The survey was adapted from similar surveys at MIT and University of Maryland. Our survey had four main sections: (1) Campus climate, (2) Professional development and academic services, (3) Student workload and student services, (4) Wellness, family and housing. Disciplines Educational Assessment, Evaluation, and Research | Higher Education | Women's Studies Authors Lorraine D. Acker, Elizabeth M. Asque, Glennda M. Bivens, Marilyn Ann Cornish, Charles E. Glatz, Julia A. Graden, Susana Hernandez, Bethany J. Juhnke, Kimberley LeVelle, Cynthia R. Maroney, Santos Nunez, Craig A. Ogilvie, Angelica Reina, Alissa Stoehr, Judith K. Strand, and Emma M. H. White This report is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/grad_reports/1 ISU Female Grad Student Survey Page 1 Results from Iowa State Female Graduate Student Needs Assessment Survey, July 2012 Task force members: Lorraine Acker, Elizabeth Asque, Glennda Bivens, Marilyn Cornish, Chuck Glatz, Julia Graden, Susana Hernandez, Bethany Juhnke, Kimberly LeVelle, Cindy Maroney, Santos Nunez, Craig Ogilvie, Angelica Reina, Alissa Stoehr, Judy Strand, and Emma White During the spring 2012 semester, a task force of graduate students, staff, and faculty created a survey to gather information from women graduate students about their experiences at Iowa State University (ISU). The survey was adapted from similar surveys at MIT and University of Maryland. Our survey had four main sections: 1. Campus climate 2. Professional development and academic services 3. Student workload and student services 4. Wellness, family and housing 1,922 female graduate students were sent the survey on March 27, 2012—49.4% clicked on the survey link. Reminder emails were sent on April 4—13.3% clicked on the survey link—and on April 11—11.2% clicked on the survey link. The survey was closed on April 16. In total 654 students started the survey and 438 completed every question. This corresponds to a response rate of 438/1922 = 22.8% The demographic breakdown of the responders is Asian Black/African American Hawaiian/Pacific Islander Latino/Hispanic White Native American Other Total 65 23 0 23 319 2 13 427 Master's degree Graduate Certificate Undeclared Graduate Student PhD Total

The most significant cost of recombinant protein production lies in the optimization of the downstream purification methods, mainly due to a lack of knowledge of the separation behavior of the host cell proteins (HCP). To reduce the effort required for purification process development, this work was aimed at modeling the separation behavior of a complex mixture of proteins in cation-exchange chromatography (CEX). With the emergence of molecular pharming as a viable option for the production of recombinant pharmaceutical proteins, the HCP mixture chosen was an extract of corn germ. Aqueous two phase system (ATPS) partitioning followed by two-dimensional electrophoresis (2DE) provided data on isoelectric point, molecular weight and surface hydrophobicity of the extract and step-elution fractions. A multivariate random forest (MVRF) method was then developed using the three characterization variables to predict the elution pattern of individual corn HCP. The MVRF method achieved an average root mean squared error (RMSE) value of 0.0406 (fraction of protein eluted in each CEX elution step) for all the proteins that were characterized, providing evidence for the effectiveness of both the characterization method and the analysis approach for protein purification applications.

Isoelectric precipitation and whey nanofiltration were evaluated in recovering protein from skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes. Countercurrent two-stage EAEP was performed at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h to extract oil and protein from soybeans. Two protein recovery strategies were applied to skim fractions produced by different extraction treatments: Treatment 1 using 0.5% protease (wt/g extruded flakes) in both extraction stages; Treatment 2 using 0.5% protease only in the 2nd extraction stage; and Treatment 3 using no enzyme in either extraction stage. Protein recovery by using isoelectric precipitation was inversely related to the extent of hydrolysis with recoveries of 27, 61, and 87% of skim proteins from Treatments 1, 2, and 3, respectively. Overall protein recoveries of 26, 54, and 57% of the original protein in the extruded full-fat flakes were achieved when combining extraction treatments and isoelectric precipitation. Nanofiltering isoelectric wheys (500-Da membrane) achieved protein retentate yields of 96.3, 94.5, and 91.8% (1.9–2.8 concentration factor) with permeate fluxes up to 1.35 kg/h m2. About 97, 98, and 99% of skim protein were recovered by isoelectric precipitation and whey nanofiltration for Treatments 1, 2, and 3, respectively. Overall protein recoveries of 93, 87, and 65% of the protein in the extruded flakes were achieved for Treatments 1, 2, and 3, respectively. Although high protein retentions were achieved, very low permeate fluxes were observed for whey nanofiltration.

Aqueous extraction processing technologies, having advanced in recent years, may be a viable alternative to hexane extraction to separate oil and protein from soybeans. Different extraction strategies incorporating various modes of comminution, extraction buffers, and enzymes allow production of a range of oil and protein products, but also create different processing challenges. Processes capable of achieving high free oil yields often result in a soluble protein fraction difficult to isolate and dilute oil emulsions difficult to break. Other processes can achieve high yields and purities of native soy protein, but with reduced free oil yield or require a high osmotic and ionic strength extraction buffer. This review article discusses these various advanced processes and their relative advantages and disadvantages. In addition, the current understanding of the underlying fundamental concepts of aqueous extraction is discussed in order to help direct future investigations to improve these technologies.

Enzyme‐assisted aqueous oil extraction from soybean is a “green” alternative to hexane extraction that must realize potential revenues from a value‐added protein co‐product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m 2 hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran‐grafted agarose quaternary‐amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010

A novel combination of 2-D electrophoresis with hydrophobic partitioning in aqueous two-phase systems (ATPS) was extended to an alternative ATPS and both systems used for the three-dimensional characterization of the proteins extracted from soybeans. The 3-D plots of molecular weight, isoelectric point, and surface hydrophobicity were obtained using two different phase-forming salts: Na2SO4 and potassium phosphate. Six proteins with known hydrophobicities were used to validate the ATPS-based method. Molecular properties obtained using the PEG-sulfate system resulted in a wider range of proteins characterized. The wide range of concentration and strongly hydrophilic character of the soy extracts limited the coverage obtained; reduction of the storage protein content aided detection. The number of proteins simultaneously and accurately characterized by this method as currently implemented is limited by the dynamic range of staining, the ability to quantify strongly partitioning proteins in both phases, and loss of proteins of limited solubility in the ATPS or during the removal of phase-forming components.

The effectiveness of ultrafiltration for the purification of recombinant proteins from aqueous corn endosperm and germ extracts was examined using model proteins of two different sizes, recombinant type I human collagen (rCollagen, 265kDa) and green fluorescent protein (GFP, 27kDa), to evaluate the effects of membrane pore size, transmembrane pressure (TMP), crossflow rate, and filtration pH on permeate flux and protein sieving. Using a 300kDa MWCO membrane resulted in a significant loss of rCollagen, whereas a 100kDa MWCO membrane completely retained rCollagen. Increasing the filtration crossflow rate and TMP resulted in a higher permeate flux without significantly altering the sieving of the host cell proteins (HCP) or GFP. The greatest HCP sieving was observed in the endosperm extract filtration at low pH and, compared to endosperm, the filtration of germ extracts had lower HCP sieving. GFP exhibited similar sieving as the average HCP for all filtration conditions. rCollagen purity of 89% was achieved with only diafiltration of endosperm extracts and, when preceded by precipitation, a purity of >99% was attained. Thus, ultrafiltration is a valuable method to separate and purify corn-hosted recombinant proteins >100kDa, particularly when the expression is targeted to the endosperm.

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

The effectiveness of tangential flow microfiltration for the solid/liquid clarification of aqueous corn endosperm and germ extracts was examined using recombinant type I human collagen (rCollagen) and green fluorescent protein (GFP) as model proteins. We identified the effects of transmembrane pressure (TMP), crossflow rate, protein molecular weight, and membrane chemistry on permeate flux, protein rejection, and internal membrane fouling in microfiltration. In dead-end filtrations, both endosperm and germ extracts formed highly compressible cakes that provided the dominant hydraulic resistance. For tangential flow filtration using a ceramic membrane, increasing the crossflow rate had a significant beneficial effect on the permeate flux for all TMPs examined. High fouling of the ceramic membrane occurred during the filtration and this was likely due to the presence of soluble corn starch in the feed. Filtration with a ceramic membrane resulted in low rejection (<10%) of both the host cell proteins (HCP) and GFP and very high rejection (∼90%) of rCollagen. In contrast to the ceramic membrane, tangential flow filtration using a poly(vinylidene fluoride) membrane resulted in much less internal fouling and no measurable rejection of HCP, GFP, or rCollagen. Microfiltration was an effective method for the solid/liquid clarification of corn protein extracts, except in cases where high internal membrane fouling resulted in increased rejection of large molecular weight proteins.