Showing papers in "Biotechnology and Bioengineering in 2009"
TL;DR: The experiments showed that the eustigmatophyte Nannochloropsis sp.
Abstract: Thirty microalgal strains were screened in the laboratory for their biomass productivity and lipid content. Four strains (two marine and two freshwater), selected because robust, highly productive and with a relatively high lipid content, were cultivated under nitrogen deprivation in 0.6-L bubbled tubes. Only the two marine microalgae accumulated lipid under such conditions. One of them, the eustigmatophyte Nannochloropsis sp. FM102: 100–112. © 2008 Wiley Periodicals, Inc.
2,714 citations
TL;DR: The use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogel hybrids that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix are discussed.
Abstract: Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three-dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three-dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic-biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism.
2,298 citations
TL;DR: The ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([Emim][CH3COO]), was used as a pretreatment solvent to extract lignin from wood flour, resulting in a highly concentrated solution of chemically unmodified lign in, which may serve as a valuable source of a polyaromatic material as a value‐added product.
Abstract: Lignocellulose represents a key sustainable source of biomass for transformation into biofuels and bio-based products. Unfortunately, lignocellulosic biomass is highly recalcitrant to biotransformation, both microbial and enzymatic, which limits its use and prevents economically viable conversion into value-added products. As a result, effective pretreatment strategies are necessary, which invariably involves high energy processing or results in the degradation of key components of lignocellulose. In this work, the ionic liquid, 1-ethyl-3-methylimidazolium acetate ([Emim][CH3COO]), was used as a pretreatment solvent to extract lignin from wood flour. The cellulose in the pretreated wood flour becomes far less crystalline without undergoing solubilization. When 40% of the lignin was removed, the cellulose crystallinity index dropped below 45, resulting in > 90% of the cellulose in wood flour to be hydrolyzed by Trichoderma viride cellulase. [Emim] [CH3COO] was easily reused, thereby resulting in a highly concentrated solution of chemically unmodified lignin, which may serve as a valuable source of a polyaromatic material as a value-added product.
914 citations
TL;DR: This review presents a critical analysis of the current status of research in enzymatic biodiesel production and accentuates the main obstacles to the widespread use of enzymes for commercial biodiesel transesterification.
Abstract: Enzymatic biodiesel production has been investigated intensively, but is presently employed industrially only in a 20,000 tons/year pilot plant in China (Du et al. [2008] Appl Microbiol Technol 79(3):331-337). This review presents a critical analysis of the current status of research in this area and accentuates the main obstacles to the widespread use of enzymes for commercial biodiesel transesterification. Improved results for enzymatic catalysis are seen with respect to increased yield, reaction time and stability, but the performance and price of the enzymes need further advances for them to become attractive industrially for biodiesel production. Critical aspects such as mass transfer limitations, use of solvents and water activity are discussed together with process considerations and evaluation of possible reactor configurations, if industrial production with enzymes is to be carried out. Results of published studies on the productivity of enzymes are also presented and compared to the use of chemical catalysts.
702 citations
TL;DR: In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval.
Abstract: Auto-fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1-n-ethyl-3-methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter- and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti-solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval.
379 citations
TL;DR: The herein described extensive characterization studies could help understand the limitations to high‐level, stable recombinant protein production and find ways to improving and accelerating the process for high‐producer cell line generation and selection.
Abstract: Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.
293 citations
TL;DR: It is demonstrated that algae can in principle, be used as a renewable source of electricity production in MFCs and the fingerprints of microbial communities developed in reactors had only 11% similarity to inocula and clustered according to the type of bioprocess used.
Abstract: Bioelectricity production from a phytoplankton, Chlorella vulgaris, and a macrophyte, Ulva lactuca was examined in single chamber microbial fuel cells (MFCs). MFCs were fed with the two algae (as powders), obtaining differences in energy recovery, degradation efficiency, and power densities. C. vulgaris produced more energy generation per substrate mass (2.5 kWh/kg), but U. lactuca was degraded more completely over a batch cycle (73 +/- 1% COD). Maximum power densities obtained using either single cycle or multiple cycle methods were 0.98 W/m(2) (277 W/m(3)) using C. vulgaris, and 0.76 W/m(2) (215 W/m(3)) using U. lactuca. Polarization curves obtained using a common method of linear sweep voltammetry (LSV) overestimated maximum power densities at a scan rate of 1 mV/s. At 0.1 mV/s, however, the LSV polarization data was in better agreement with single- and multiple-cycle polarization curves. The fingerprints of microbial communities developed in reactors had only 11% similarity to inocula and clustered according to the type of bioprocess used. These results demonstrate that algae can in principle, be used as a renewable source of electricity production in MFCs.
289 citations
TL;DR: Development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium is reported, which should be useful for the bio-based production of putresCine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals.
Abstract: A four carbon linear chain diamine, putrescine (1,4-diaminobutane), is an important platform chemical having a wide range of applications in chemical industry. Biotechnological production of putrescine from renewable feedstock is a promising alternative to the chemical synthesis that originates from non-renewable petroleum. Here we report development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and deleting the ornithine carbamoyltransferase chain I gene argI to make more precursors available for putrescine synthesis. Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid-based and chromosome-based overexpression of the coding genes under the strong tac or trc promoter. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the trc promoter, which replaced the native promoter in the genome, to increase the ornithine pool. Finally, strain performance was further improved by the deletion of the stress responsive RNA polymerase sigma factor RpoS, a well-known global transcription regulator that controls the expression of ca. 10% of the E. coli genes. The final engineered E. coli strain was able to produce 1.68 g L(-1) of putrescine with a yield of 0.168 g g(-1) glucose. Furthermore, high cell density cultivation allowed production of 24.2 g L(-1) of putrescine with a productivity of 0.75 g L(-1) h(-1). The strategy reported here should be useful for the bio-based production of putrescine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals.
271 citations
TL;DR: This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells by demonstrating the beneficial effects of stirred bioreactor culture, aggregate size‐control and hypoxia on both cell growth and cell differentiation towards cardiomyocytes.
Abstract: The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called “embryoid bodies” (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493–507. © 2008 Wiley Periodicals, Inc.
251 citations
TL;DR: While identifiability of both parameters in the BMP tests was generally good, only f(d) could be well identified using continuous data, and this translated to very poor model performance when BMP-estimated values were used in the continuous model.
Abstract: In hydrolysis-limited anerobic systems, the key parameters describing degradation are degradability extent ( fd), and the lumped apparent first order coefficient (khyd). These are often measured in biological methane potential (BMP) tests. Using modern techniques, it should also be possible to estimate these parameters in full-scale systems, especially where inputs are dynamic. In this study, we evaluated fd and khyd values and uncertainty based on nonlinear parameter estimation from (i) BMP tests and (ii) effluent gas and solids from two full-scale digesters fed with highly variable feed flows and concentrations (up to 6 kgCODm3 day1). The substrate was thermally hydrolyzed activated sludge, and the inoculum for BMP tests was from the full-scale digesters. While identifiability of both parameters in the BMP tests was generally good, only fd could be well identified using continuous data. For khyd using continuous data, normally only a lower limit could be found (upper was unbounded). In addition, parameters as estimated on different outputs (VS and gasflow) and two different digesters were consistent, with an fd value of 0.45– 0.55, and a khyd value of >5 day1. Gradual changes in fd over the 450 days could be related to upstream changes. fd values as estimated in BMP tests were consistent (if conservative)with continuous estimates, with a fd in BMP of 0.4–0.5. khyd values were an order of magnitude lower (0.15– 0.25 day1 vs. >5 day1), and this translated to very poor model performance when BMP-estimated values were used in the continuous model. This means that while BMP testing may be used for project feasibility analysis, values obtained should not be used for dynamic modeling. The parameter confidence regions found were highly nonlinear, especially for continuous systems, indicating that iterative or sampling techniques are required for an estimate of real parameter uncertainty.
245 citations
TL;DR: To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current.
Abstract: We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro-organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real-time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation ( approximately 30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate-oxidizing ARB out-competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate-consuming ARB. When we inhibited methanogenesis with 50 mM 2-bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo-acetogenesis. This illustrates the key role of competition among the various H(2) scavengers and that, when the hydrogen-consuming methanogens were present, they out-competed the other groups. These findings also demonstrate the importance of a three-way syntrophic relationship among fermenters, acetate-consuming ARB, and a H(2) consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current.
TL;DR: A bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages and showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring.
Abstract: The higher ethanol titer inevitably requires higher solids loading during the simultaneous enzymatic saccharification and fermentation (SSF) using lignocellulose as the feedstock. The mixing between the solid lignocellulose and the liquid enzyme is crucially important. In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost between the increased mixing energy cost and the reduced distillation energy cost at the high solids loading should be made. The potentials of the new bioreactor were tested under various SSF conditions for obtaining optimal ethanol yield and titer.
TL;DR: Because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose.
Abstract: Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) pretreatments were measured at 4 degrees C. Langmuir adsorption parameters were then estimated by non-linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4 degrees C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO(2) pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness.
TL;DR: For the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylonase and β‐xylosidase improved performance significantly.
Abstract: Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).
TL;DR: This research investigated the use of algae for energy generation in a stand‐alone, closed‐loop system that continuously transforms solar energy into energy‐rich biogas and electricity and resulted in a power plant with a potential capacity of about 9 kW of solar algal panel.
Abstract: In the quest for renewable resources, algae are increasingly receiving attention. Their high growth rate, high CO2 fixation and their lack of requirement for fertile soil surface represent several advantages as compared to conventional (energy) crops. Through their ability to store large amounts of oils, they qualify as a source for biodiesel. Algal biomass, however, can also be used as such, namely as a substrate for anaerobic digestion. In the present research, we investigated the use of algae for energy generation in a stand-alone, closed-loop system. The system encompasses an algal growth unit for biomass production, an anaerobic digestion unit to convert the biomass to biogas and a microbial fuel cell to polish the effluent of the digester. Nutrients set free during digestion can accordingly be returned to the algal growth unit for a sustained algal growth. Hence, a system is presented that continuously transforms solar energy into energy-rich biogas and electricity. Algal productivities of 24–30 ton VS ha−1 year−1 were reached, while 0.5 N m3 biogas could be produced kg−1 algal VS. The system described resulted in a power plant with a potential capacity of about 9 kW ha−1 of solar algal panel, with prospects of 23 kW ha−1. Biotechnol. Bioeng. 2009;103: 296–304. © 2009 Wiley Periodicals, Inc.
TL;DR: An in vitro microscale cell culture analog (µCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations is developed and showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithelium and is further metabolizing by liver cells, resulting in liver cell toxicity in a dose‐dependent manner.
Abstract: The lining of the gastrointestinal (GI) tract is the largest surface exposed to the external environment in the human body. One of the main functions of the small intestine is absorption, and intestinal absorption is a route used by essential nutrients, chemicals, and pharmaceuticals to enter the systemic circulation. Understanding the effects of digestion on a drug or chemical, how compounds interact with and are absorbed through the small intestinal epithe- lium, and how these compounds affect the rest of the body is critical for toxicological evaluation. Our goal is to create physiologically realistic in vitro models of the human GI tract that provide rapid, inexpensive, and accurate predic- tions of the body's response to orally delivered drugs and chemicals. Our group has developed an in vitro microscale cell culture analog (mCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations. The GI tract mCCA, coupled with a multi- chamber silicon mCCA representing the systemic circula- tion, is described and challenged with acetaminophen. Proof of concept experiments showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithe- lium and is further metabolized by liver cells, resulting in liver cell toxicity in a dose-dependent manner. The mCCA response is also consistent with in vivo measurements in mice. The system should be broadly useful for studies on orally delivered drugs or ingestion of chemicals with potential toxicity. Biotechnol. Bioeng. 2009;104: 193-205.
TL;DR: This study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons.
Abstract: The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 x 10(9) to 8.04 x 10(3) cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log(10)) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominantly epithelial or mesenchymal characteristics?).
TL;DR: Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.
Abstract: Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations 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 measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.
TL;DR: The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing.
Abstract: Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications.
TL;DR: The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitotoxicity assays showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils.
Abstract: Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X-ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol-grafted chitosan nanoparticles (CHCA NPs) and the eugenol-grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram-negative bacterium, Escherichia coli (E. coli) and a gram-positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component-grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils.
TL;DR: Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds and microaerobic conditions were used as a means of eliminating the need for rich nutrients.
Abstract: Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed approximately 95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by-products succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.
TL;DR: Due to its modular nature, the proposed ISPR design exhibits strong potential for compatibility with future n‐ butanol fermentation efforts, and retrieval of n‐butanol from resins via thermal treatment was demonstrated with high efficiency and predicted to be economically favorable.
Abstract: Polymeric resins with high n-butanol adsorption affinities were identified from a candidate pool of commercially available materials representing a wide array of physical and chemical properties. Resin hydrophobicity, which was dictated by the chemical structure of its constituent monomer units, most greatly influenced the resin-aqueous equilibrium partitioning of n-butanol whereas ionic functionalization appeared to have no effect. In general, those materials derived from poly(styrene-co-divinylbenzene) possessed the greatest n-butanol affinity, while the adsorption potential of these resins was limited by their specific surface area. Resins were tested for their ability to serve as effective in situ product recovery (ISPR) devices in the n-butanol fermentation by Clostridium acetobutylicum ATCC 824. In small-scale batch fermentations, the addition of 0.05 kg/L Dowex Optipore SD-2 facilitated achievement of effective n-butanol titers as high as 2.22% (w/v), well above the inhibitory threshold of C. acetobutylicum ATCC 824, and nearly twice that of traditional, single-phase fermentations. Retrieval of n-butanol from resins via thermal treatment was demonstrated with high efficiency and predicted to be economically favorable. Due to its modular nature, the proposed ISPR design exhibits strong potential for compatibility with future n-butanol fermentation efforts.
TL;DR: The optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs and the advantages of fast and high‐levelproduction of VLP‐based vaccines using the BeYDV‐derivedDNA replicon system for transient expression in plants are described.
Abstract: Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants.
TL;DR: The results demonstrate that the microfluidic system can be used to study durotactic behavior of cells and neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.
Abstract: We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.
TL;DR: An environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods.
Abstract: Phanerochaete chrysosporium is a wood-rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin-degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett-Burman design and the Box-Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X-ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal-pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield.
TL;DR: Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors and indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer andcarbon dioxide removal.
Abstract: Bioprocess scale-up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell-free mixing studies were performed in production scale 5,000-L bioreactors to evaluate scale-up issues. Using the current bioreactor configuration, the 5,000-L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5- and 20-L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000-L configuration can only support a maximum viable cell density of 7 x 10(6) cells mL(-1). Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000-L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing-related engineering parameters in the 5,000-L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed-batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000-L bioreactors may need optimizing to mitigate the risk of different performance upon process scale-up.
TL;DR: Improved leader sequences allow for a 180‐fold improvement over previous reports in the secretion of full‐length, functional, glycosylated human IgG1, and will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs.
Abstract: Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three- to tenfold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFalpha1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16-fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180-fold improvement over previous reports in the secretion of full-length, functional, glycosylated human IgG(1). The production of full-length IgG(1) at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs.
TL;DR: The maximal productivity of a 14 mm light‐path panel photobioreactor under high irradiance was determined and the biomass yield on light energy is high but still lower than the theoretical maximal yield which must be related to photosaturation and thermal dissipation of absorbed light energy.
Abstract: Maximal productivity of a 14 mm light-path panel photobioreactor under high irradiance was determined. Under continuous illumination of 2,100 micromol photons m(-2) s(-1) with red light emitting diodes (LEDs) the effect of dilution rate on photobioreactor productivity was studied. The light intensity used in this work is similar to the maximal irradiance on a horizontal surface at latitudes lower than 37 degrees . Chlorella sorokiniana, a fast-growing green microalga, was used as a reference strain in this study. The dilution rate was varied from 0.06 to 0.26 h(-1). The maximal productivity was reached at a dilution rate of 0.24 h(-1), with a value of 7.7 g dw m(-2) h(-1) (m(2) of illuminated photobioreactor surface) and a volumetric productivity of 0.5 g dw L(-1) h(-1). At this dilution rate the biomass concentration inside the reactor was 2.1 g L(-1) and the photosynthetic efficiency was 1.0 g dw mol photons. This biomass yield on light energy is high but still lower than the theoretical maximal yield of 1.8 g mol photons(-1) which must be related to photosaturation and thermal dissipation of absorbed light energy.
TL;DR: It is suggested that in many cases, air‐bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production.
Abstract: There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates of between 20,000 and 250,000 s-1 for between 5 minutes and 30 ms using a parallel-plate and capillary rheometer respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s-1 and 0.06 pN respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20 to 150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production.
TL;DR: The results show that chloroplasts have the ability to fold and assemble full‐length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins.
Abstract: Monoclonal antibodies can be effective therapeutics against a variety of human diseases, but currently marketed antibody-based drugs are very expensive compared to other therapeutic options. Here, we show that the eukaryotic green algae Chlamydomonas reinhardtii is capable of synthesizing and assembling a full-length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts. This antibody, 83K7C, is derived from a human IgG1 directed against anthrax protective antigen 83 (PA83), and has been shown to block the effects of anthrax toxin in animal models. Here we show that 83K7C heavy and light chain proteins expressed in the chloroplast accumulate as soluble proteins that assemble into complexes containing two heavy and two light chain proteins. The algal-expressed 83K7C binds PA83 in vitro with similar affinity to the mammalian-expressed 83K7C antibody. In addition, a second human IgG1 and a mouse IgG1 were also expressed and shown to properly assemble in algal chloroplast. These results show that chloroplasts have the ability to fold and assemble full-length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins.