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Showing papers in "Biotechnology and Bioengineering in 2008"


Journal ArticleDOI
TL;DR: This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes and the strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process.
Abstract: Butanol is an aliphatic saturated alcohol having the molecular formula of C4H9OH Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications Moreover, butanol has been considered as a potential fuel or fuel additive Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed Biotechnol Bioeng 2008;101: 209-228 © 2008 Wiley Periodicals, Inc

1,017 citations


Journal ArticleDOI
TL;DR: Algae or cyanobacteria may be the best option to produce bioenergy at rates high enough to replace a substantial fraction of the authors' society's use of fossil fuels.
Abstract: Global warming can be slowed, and perhaps reversed, only when society replaces fossil fuels with renewable, carbon-neutral alternatives. The best option is bioenergy: the sun's energy is captured in biomass and converted to energy forms useful to modern society. To make a dent in global warming, bioenergy must be generated at a very high rate, since the world today uses approximately 10 TW of fossil-fuel energy. And, it must do so without inflicting serious damage on the environment or disrupting our food supply. While most bioenergy options fail on both counts, several microorganism-based options have the potential to produce large amounts of renewable energy without disruptions. In one approach, microbial communities convert the energy value of various biomass residuals to socially useful energy. Biomass residuals come from agricultural, animal, and a variety of industrial operations, as well as from human wastes. Microorganisms can convert almost all of the energy in these wastes to methane, hydrogen, and electricity. In a second approach, photosynthetic microorganisms convert sunlight into biodiesel. Certain algae (eukaryotes) or cyanobacteria (prokaryotes) have high lipid contents. Under proper conditions, these photosynthetic microorganisms can produce lipids for biodiesel with yields per unit area 100 times or more than possible with any plant system. In addition, the non-lipid biomass can be converted to methane, hydrogen, or electricity. Photosynthetic microorganisms do not require arable land, an advantage because our arable land must be used to produce food. Algae or cyanobacteria may be the best option to produce bioenergy at rates high enough to replace a substantial fraction of our society's use of fossil fuels.

659 citations


Journal ArticleDOI
TL;DR: Evidence is provided supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls.
Abstract: Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.

598 citations


Journal ArticleDOI
TL;DR: In a membrane bioreactor (MBR), fast growth of anammox bacteria was achieved with a sludge residence time of 12 days, resulting in a—for anamm ox bacteria—unprecedented purity of the enrichment of 97.6%.
Abstract: In a membrane bioreactor (MBR), fast growth of anammox bacteria was achieved with a sludge residence time (SRT) of 12 days. This relatively short SRT resulted in a—for anammox bacteria—unprecedented purity of the enrichment of 97.6%. The absence of a selective pressure for settling, and dedicated cultivation conditions led to growth in suspension as free cells and the complete absence of flocs or granules. Fast growth, low levels of calcium and magne- sium, and possibly the presence of yeast extract and a low shear stress are critical for the obtainment of a completely suspended culture consisting of free anammox cells. During cultivation, a population shift was observed from Candida- tus ''Brocadia'' to Candidatus ''Kuenenia stuttgartiensis.'' It is hypothesized that the reason for this shift is the higher affinity for nitrite of ''Kuenenia.'' The production of ana- mmox bacteria in suspension with high purity and produc- tivity makes the MBR a promising tool for the cultivation and study of anammox bacteria. Biotechnol. Bioeng. 2008;101: 286-294. 2008 Wiley Periodicals, Inc.

484 citations


Journal ArticleDOI
TL;DR: This study shows how current density generated by an acclimated ARB biofilm was limited by proton transport out of the biofilm, and discusses the implications of the proton‐transport limitation in the field of microbial fuel cells and microbial electrolytic cells.
Abstract: Anode-respiring bacteria (ARB) in a biofilm anode carry out an oxidation half-reaction of organic matter, producing an electrical current from renewable biomass, including wastes. At the same time, ARB produce protons, usually one proton for every electron. Our study shows how current density generated by an acclimated ARB biofilm was limited by proton transport out of the biofilm. We determined that, at high current densities, protons were mainly transported out of the biofilm by protonating the conjugate base of the buffer system; the maximum current generation was directly related to the transport of the buffer, mainly by diffusion, into and out of the biofilm. With non-limiting acetate concentrations, the current density increased with higher buffer concentrations, going from 2.21 +/- 0.02 A m(-2) with 12.5-mM phosphate buffer medium to 9.3 +/- 0.4 A m(-2) using a 100-mM phosphate buffer at a constant anode potential of E(anode) = -0.35 V versus Ag/AgCl. Increasing the concentration of sodium chloride in the medium (0-100 mM) increased current density by only 15%, indicating that ion migration was not as important as diffusion of phosphate inside the biofilm. The current density also varied strongly with medium pH as a result of the buffer speciation: The current density was 10.0 +/- 0.8 A m(-2) at pH 8, and the pH giving one-half the maximum rate was 6.5. A j-V curve analysis using 100 mM phosphate buffer showed a maximum current density of 11.5 +/- 0.9 A m(-2) and half-saturation potential of -0.414 V versus Ag/AgCl, a value that deviated only slightly from the standard acetate potential, resulting in small anode-potential losses. We discuss the implications of the proton-transport limitation in the field of microbial fuel cells and microbial electrolytic cells.

484 citations


Journal ArticleDOI
TL;DR: Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes by combination of gene deletions and metabolic evolution.
Abstract: Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622–733 mM of succinate with molar yields of 1.2–1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized. Biotechnol. Bioeng. 2008;99: 1140–1153. © 2007 Wiley Periodicals, Inc.

382 citations


Journal ArticleDOI
TL;DR: Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied and it was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment.
Abstract: Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied. It was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment. At low temperature, the pretreatment chemicals, either NaOH alone or NaOH-urea mixture solution, can slightly remove lignin, hemicelluloses, and cellulose in the lignocellulosic materials, disrupt the connections between hemicelluloses, cellulose, and lignin, and alter the structure of treated biomass to make cellulose more accessible to hydrolysis enzymes. Moreover, the wood fiber bundles could be broken down to small and loose lignocellulosic particles by the chemical treatment. Therefore, the enzymatic hydrolysis efficiency of untreated mechanical fibers can also be remarkably enhanced by NaOH or NaOH/urea solution treatment. The results indicated that, for spruce, up to 70% glucose yield could be obtained for the cold temperature pretreatment (-15 degrees C) using 7% NaOH/12% urea solution, but only 20% and 24% glucose yields were obtained at temperatures of 23 degrees C and 60 degrees C, respectively, when other conditions remained the same. The best condition for the chemical pretreatment regarding this study was 3% NaOH/12% urea, and -15 degrees C. Over 60% glucose conversion was achieved upon this condition.

353 citations


Journal ArticleDOI
TL;DR: Digester performance was affected negatively by mixing intensity during startup of the digesters, with lower biogas production rates and higher volatile fatty acids concentrations observed for the 1,500‐RPM digester.
Abstract: We determined the effect of different mixing intensities on the performance, methanogenic population dynamics, and juxtaposition of syntrophic microbes in anaerobic digesters treating cow manure from a dairy farm. Computer automated radioactive particle tracking in conjunction with computational fluid dynamics was performed to quantify the shear levels locally. Four continuously stirred anaerobic digesters were operated at different mixing intensities of 1,500, 500, 250, and 50 revolutions per min (RPM) over a 260-day period at a temperature of 34 +/- 1 degrees C. Animal manure at a volatile solids (VS) concentration of 50 g/L was fed into the digesters daily at five different organic loading rates between 0.6 and 3.5 g VS/L day. The different mixing intensities had no effect on the biogas production rates and yields at steady-state conditions. A methane yield of 0.241 +/- 0.007 L CH(4)/g VS fed was obtained by pooling the data of all four digesters during steady-state periods. However, digester performance was affected negatively by mixing intensity during startup of the digesters, with lower biogas production rates and higher volatile fatty acids concentrations observed for the 1,500-RPM digester. Despite similar methane production yields and rates, the acetoclastic methanogenic populations were different for the high- and low-intensity mixed digesters with Methanosarcina spp. and Methanosaeta concilii as the predominant methanogens, respectively. For all four digesters, epifluorescence microscopy revealed decreasing microbial floc sizes beginning at week 4 and continuing through week 26 after which no microbial flocs remained. This decrease in size, and subsequent loss of microbial flocs did not, however, produce any long-term upsets in digester performance.

299 citations


Journal ArticleDOI
TL;DR: Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products with growth‐based selection for increased ATP production to create a strain devoid of foreign DNA.
Abstract: Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production The resulting strain, KJ073, produced 12 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB) KJ091 was further engineered for improvements in succinate production Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (13 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073 Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (15 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (09 g L(-1) h(-1)) Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA) Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium Both may be useful as biocatalysts for the commercial production of succinate

285 citations


Journal ArticleDOI
TL;DR: It is concluded that ammonia losses from the anode chamber were driven primarily by physical–chemical factors that are increased with electricity generation, although some losses may occur through biological nitrification and denitrification.
Abstract: Ammonia losses during swine wastewater treatment were examined using single- and two-chambered microbial fuel cells (MFCs). Ammonia removal was 60% over 5 days for a single-chamber MFC with the cathode exposed to air (air-cathode), versus 69% over 13 days from the anode chamber in a two-chamber MFC with a ferricyanide catholyte. In both types of systems, ammonia losses were accelerated with electricity generation. For the air-cathode system, our results suggest that nitrogen losses during electricity generation were increased due to ammonia volatilization with conversion of ammonium ion to the more volatile ammonia species as a result of an elevated pH near the cathode (where protons are consumed). This loss mechanism was supported by abiotic tests (applied voltage of 1.1 V). In a two-chamber MFC, nitrogen losses were primarily due to ammonium ion diffusion through the membrane connecting the anode and cathode chambers. This loss was higher with electricity generation as the rate of ammonium transport was increased by charge transfer across the membrane. Ammonia was not found to be used as a substrate for electricity generation, as intermittent ammonia injections did not produce power. The ammonia-oxidizing bacterium Nitrosomonas europaea was found on the cathode electrode of the single-chamber system, supporting evidence of biological nitrification, but anaerobic ammonia-oxidizing bacteria were not detected by molecular analyses. It is concluded that ammonia losses from the anode chamber were driven primarily by physical-chemical factors that are increased with electricity generation, although some losses may occur through biological nitrification and denitrification.

260 citations


Journal ArticleDOI
TL;DR: This work applied elementary metabolite unit (EMU) theory to NMFA, dramatically reducing computational difficulty, and introduced block decoupling, a new method that systematically and comprehensively divides EMU systems of equations into smaller subproblems to further reduce computational difficulty.
Abstract: Nonstationary metabolic flux analysis (NMFA) is at present a very computationally intensive exercise, especially for large reaction networks. We applied elementary metabolite unit (EMU) theory to NMFA, dramatically reducing computational difficulty. We also introduced block decoupling, a new method that systematically and comprehensively divides EMU systems of equations into smaller subproblems to further reduce computational difficulty. These improvements led to a 5000-fold reduction in simulation times, enabling an entirely new and more complicated set of problems to be analyzed with NMFA. We simulated a series of nonstationary and stationary GC/MS measurements for a large E. coli network that was then used to estimate parameters and their associated confidence intervals. We found that fluxes could be successfully estimated using only nonstationary labeling data and external flux measurements. Addition of near-stationary and stationary time points increased the precision of most parameters. Contrary to prior reports, the precision of nonstationary estimates proved to be comparable to the precision of estimates based solely on stationary data. Finally, we applied EMU-based NMFA to experimental nonstationary measurements taken from brown adipocytes and successfully estimated fluxes and some metabolite concentrations. By using NFMA instead of traditional MFA, the experiment required only 6 h instead of 50 (the time necessary for most metabolite labeling to reach 99% of isotopic steady state).

Journal ArticleDOI
TL;DR: The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes and adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesqiterpene production.
Abstract: The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.

Journal ArticleDOI
TL;DR: Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions and final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth.
Abstract: In this study, bioethanol production from steam-exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentose. Red Star is a robust hexose-fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. the highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) an an SSF process, it showed an ethanol yield on cunsumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/l h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furtermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced.

Journal ArticleDOI
TL;DR: In this article, a lab-scale sequencing batch reactor, seeded with granular sludge developed using synthetic wastewater, was operated for 13 months under alternating anaerobic and aerobic conditions.
Abstract: The biological removal of nitrogen and phosphorus from nutrient-rich abattoir wastewater using granular sludge has been investigated. A lab-scale sequencing batch reactor, seeded with granular sludge developed using synthetic wastewater, was operated for 13 months under alternating anaerobic and aerobic conditions. It is demonstrated that the granules could be sustained and indeed further developed with the use of abattoir wastewater. The organic, nitrogen, and phosphorus loading rates applied were 2.7 gCOD L-1 day-1, 0.43 gN L-1 day-1, and 0.06 gP L-1 day-1, respectively. The removal efficiency of soluble COD, soluble nitrogen and soluble phosphorus were 85%, 93%, and 89%, respectively. However, the high suspended solids in the effluent limited the overall removal efficiency to 68%, 86%, and 74% for total COD, TN, and TP, respectively. This good nutrient removal was achieved through the process known as simultaneous nitrification, denitrification, and phosphorus removal, likely facilitated by the presence of large anoxic zones in the center of the granules. The removal of nitrogen was likely via nitrite optimizing the use of the limited COD available in the wastewater. Accumulibacter spp. were found to be responsible for most of the denitrification, further reducing the COD requirement for nitrogen and phosphorus removal. Mineral precipitation was evaluated and was not found to significantly contribute to the overall nutrient removal. It is also shown that the minimum HRT in a granular sludge system is not governed by the sludge settleability, as is the case with floccular sludge systems, but likely by the limitations associated with the transfer of substrates in granules. Biotechnol. Bioeng. 2008;100: 529-541. © 2007 Wiley Periodicals, Inc.

Journal ArticleDOI
TL;DR: Evaluating factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation lead to the optimization of a batch-binding technique.
Abstract: The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.

Journal ArticleDOI
TL;DR: The ability of washed G. sulfurreducens cells to immediately produce electrical current was consistent with the external surface of this bacterium possessing a pathway linking oxidative metabolism to extracellular electron transfer, and illustrates a strategy for preparation of catalytic electrodes and study of Geobacter under defined conditions.
Abstract: Bacteria able to transfer electrons to conductive surfaces are of interest as catalysts in microbial fuel cells, as well as in bioprocessing, bioremediation, and corrosion. New procedures for immobilization of Geobacter sulfurreducens on graphite electrodes are described that allow routine, repeatable electrochemical analysis of cell-electrode interactions. Immediately after immobilizing G. sulfurreducens on electrodes, electrical current was obtained without addition of exogenous electron shuttles or electroactive polymers. Voltammetry and impedance analysis of pectin-immobilized bacteria transferring electrons to electrode surfaces could also be performed. Cyclic voltammetry of immobilized cells revealed voltage-dependent catalytic current similar to what is commonly observed with adsorbed enzymes, with catalytic waves centered at -0.15 V (vs. SHE). Electrodes maintained at +0.25 V (vs. SHE) initially produced 0.52 A/m(2) in the presence of acetate as the electron donor. Electrical Impedance Spectroscopy of coatings was also consistent with a catalytic mechanism, controlled by charge transfer rate. When electrodes were maintained at an oxidizing potential for 24 h, electron transfer to electrodes increased to 1.75 A/m(2). These observations of electron transfer by pectin-entrapped G. sulfurreducens appear to reflect native mechanisms used for respiration. The ability of washed G. sulfurreducens cells to immediately produce electrical current was consistent with the external surface of this bacterium possessing a pathway linking oxidative metabolism to extracellular electron transfer. This electrochemical activity of pectin-immobilized bacteria illustrates a strategy for preparation of catalytic electrodes and study of Geobacter under defined conditions.

Journal ArticleDOI
TL;DR: The IL was found to exhibit a stability improving effect on both enzymes and a solubility enhancing effect on hydrophobic substrates, which meant the conversion and volumetric productivity of ADH catalyzed reduction of acetophenone could be increased significantly.
Abstract: The ionic liquid (IL) Ammoeng110 contains cations with oligoethyleneglycol units and was found to be highly effective for the formation of aqueous two-phase systems (ATPS) that can be used for the biocompatible purification of active enzymes. Above critical concentrations of the IL and an inorganic salt in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a salt-enriched lower phase. For the optimization of the composition of IL-based ATPS with regard to the extraction of catalytically active enzymes, the Box-Wilson method of experimental design was successfully applied; IL-based ATPS proved to be suitable for the purification and stabilization of two different alcohol dehydrogenases (from Lactobacillus brevis and a thermophilic bacterium). Both enzymes were enriched in the IL-containing upper phase resulting in an increase of specific activity by a factor of 2 and 4 respectively. Furthermore, the presence of IL within the system provided the opportunity to combine the extraction process with the performance of enzyme-catalyzed reactions. The IL was found to exhibit a stability improving effect on both enzymes and a solubility enhancing effect on hydrophobic substrates. Thus the conversion and volumetric productivity of ADH catalyzed reduction of acetophenone could be increased significantly.

Journal ArticleDOI
TL;DR: The results suggest that new strategies to address this kinetic constraint of the anode bio‐electrochemical reaction and its influence on the overall power output of the MFC system, even in the high internal resistance system used in this study.
Abstract: Electrochemical impedance spectroscopy (EIS) was used to study the behavior of a microbial fuel cell (MFC) during initial biofilm growth in an acetate-fed, two-chamber MFC system with ferricyanide in the cathode. EIS experi- ments were performed both on the full cell (between cathode and anode) as well as on individual electrodes. The Nyquist plots of the EIS data were fitted with an equivalent electrical circuit to estimate the contributions of various intrinsic resistances to the overall internal MFC impedance. During initial development of the anode biofilm, the anode polar- ization resistance was found to decrease by over 70% at open circuit and by over 45% at 27 mA/cm 2 , and a simultaneous increase in power density by about 120% was observed. The exchange current density for the bio-electrochemical reaction on the anode was estimated to be in the range of 40-60 nA/cm 2 for an immature biofilm after 5 days of closed circuit operation, which increased to around 182 nA/cm 2 after more than 3 weeks of operation and stable performance in an identical parallel system. The polarization resistance of the anode was 30-40 times higher than that of the ferricyanide cathode for the conditions tested, even with an established biofilm. For a two-chamber MFC system with a Nafion 1 117 membrane and an inter-electrode spacing of 15 cm, the membrane and electrolyte solution dominate the ohmic resistance and contribute to over 95% of the MFC internal impedance. Detailed EIS analyses provide new insights into the dominant kinetic resistance of the anode bio-electrochemical reaction and its influence on the overall power output of the MFC system, even in the high internal resistance system used in this study. These results suggest that new strategies to address this kinetic constraint of the anode bio-electrochemical reactions are needed to com- plement the reduction of ohmic resistance in modern designs.

Journal ArticleDOI
TL;DR: The results demonstrate good cytocompatibility together with unequivocal BNNT cellular uptake by an energy-dependent endocytic process.
Abstract: Boron nitride nanotubes (BNNTs) have unique physical properties, which can be exploited in the biomedical field. Hence, the surprising lack of reported studies on their biocompatibility and interactions with living cells, addressed by the present paper which deals the results of such an investigation based on 72 h culture of human neuroblastoma cell line (SH-SY5Y) in the presence of an aqueous suspension of polyethyleneimine (PEI)-coated BNNTs. BNNTs conjugated with fluorescent markers (quantum dots) are employed to enable tracking of their uptake by living cells. The results demonstrate good cytocompatibility together with unequivocal BNNT cellular uptake by an energy-dependent endocytic process.

Journal ArticleDOI
TL;DR: A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed and could be economically viable for the development of a biological route for succinic Acid production.
Abstract: A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.

Journal ArticleDOI
TL;DR: The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time‐consuming need to re‐engineer either the antibody molecule or the host cell line.
Abstract: Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.

Journal ArticleDOI
TL;DR: Cell culture experiments showed that the encapsulation of HAp could enhance cell attachment to scaffolds and lower cytotoxicity, and it was shown that BMP‐2 protein successfully maintained its integrity and natural conformations after undergoing the process of electrospinning.
Abstract: A protein loaded three-dimensional scaffold can be used for protein delivery and bone tissue regeneration. The main objective of this project was to develop recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded poly(D,L-lactide-co-glycolide)/hydroxylapatite (PLGA/HAp) composite fibrous scaffolds through a promising fabrication technique, electrospinning. In vitro release of BMP-2 from these scaffolds, and the attachment ability and viability of marrow derived messenchymal stem cells (MSCs) in the presence of the scaffolds were investigated. The PLGA/HAp composite scaffolds developed in this study exhibit good morphology and it was observed that HAp nanoparticles were homogeneously dispersed inside PLGA matrix within the scaffold. The composite scaffolds allowed sustained (2-8 weeks) release of BMP-2 whose release rate was accelerated with increasing HAp content. It was also shown that BMP-2 protein successfully maintained its integrity and natural conformations after undergoing the process of electrospinning. Cell culture experiments showed that the encapsulation of HAp could enhance cell attachment to scaffolds and lower cytotoxicity.

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TL;DR: It appears that the beetle killed LP is more receptive to organosolv pretreatment other than a slightly lower recovery of carbohydrates.
Abstract: Lodgepole pine (Pinus contorta) killed by mountain pine beetle (Dendroctonus ponderosae) (BLP) was compared with healthy lodgepole pine (HLP) for bioconversion to ethanol and high-value co-products. The BLP and HLP chips were pretreated using an ethanol organosolv process at a variety of severities. It was shown that the BLP was easier to pretreat and delignify than were the HLP chips. The resulting pretreated BLP substrate had a lower residual lignin, lower degree of polymerization of cellulose, lower cellulose crystallinity, smaller fiber size and thereby a better enzymatic hydrolysability than did the HLP substrates. However, under the same conditions, the BLP showed lower substrate yield and cellulose recovery than did the HLP, which likely resulted from the excessive hydrolysis and subsequent decomposition of the cellulose and hemicellulose during the pretreatment. The BLP wood yielded more ethanol organosolv lignin than was obtained with the HLP material. The HLP lignin had a lower molecular weight and narrower distribution than did the BLP lignin. It appears that the beetle killed LP is more receptive to organosolv pretreatment other than a slightly lower recovery of carbohydrates.

Journal ArticleDOI
TL;DR: A genome‐scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi‐automated reverse engineering algorithm, allowing for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle.
Abstract: A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.

Journal ArticleDOI
TL;DR: Results from the animal experiments indicate that the bioactivity of BMP‐2 released from the fibrous PLGA/HAp composite scaffolds is well maintained, which further improves the formation of new bone and the healing of segmental defects in vivo.
Abstract: Contemporary treatment of critical bone defect remains a significant challenge in the field of orthopedic surgery. Engineered biomaterials combined with growth factors have emerged as a new treatment alternative in bone repair and regeneration. Our approach is to encapsulate bone morphogenetic protein-2 (BMP-2) into a polymeric matrix in different ways and characterize their individual performance in a nude mouse model. The main objective of this study is to examine whether the PLGA/HAp composite fibrous scaffolds loaded with BMP-2 through electrospinning can improve bone regeneration. The hypothesis is that different loading methods of BMP-2 and different HAp contents in scaffolds can alternate the release profiles of BMP-2 in vivo, therefore modify the performance of scaffolds in bone regeneration. Firstly, mechanical strength of scaffolds and HAp nanoparticles distribution in scaffolds were investigated. Secondly, nude mice experiments extended to 6 weeks were carried out to test the in vivo performance of these scaffolds, in which measurements, like serum BMP-2 concentration, ALP activity, X-ray qualification, and H&E/IHC tissue staining were utilized to monitor the growth of new bone and the changes of the corresponding biochemical parameters. The results showed that the PLGA/HAp composite scaffolds developed in this study exhibited good morphology/mechanical strength and HAp nanoparticles were homogeneously dispersed inside PLGA matrix. Results from the animal experiments indicate that the bioactivity of BMP-2 released from the fibrous PLGA/HAp composite scaffolds is well maintained, which further improves the formation of new bone and the healing of segmental defects in vivo. It is concluded that BMP-2 loaded PLGA/HAp composite scaffolds are promising for bone healing.

Journal ArticleDOI
TL;DR: To the first time a prokaryote‐based in vitro transcription/translation system has generated a virus‐like particle, Escherichia coli‐based cell‐free protein synthesis is presented as a production technology to overcome many of the limitations of current VLP production processes.
Abstract: Virus-like particles (VLP) have received considerable attention for vaccine, drug delivery, gene therapy and material science applications. Although the number of unique VLP and their applications are rapidly growing, the positive impact of VLP applications is limited by the current diverse, expensive, and typically low-yielding production technologies available. These technologies, when scaled, often result in structurally and compositionally inconsistent products. We present Escherichia coli-based cell-free protein synthesis as a production technology to overcome many of the limitations of current VLP production processes. Using this technique, the MS2 bacteriophage coat protein VLP was produced at a yield 14 times the best published production yield. Also, a C-terminally truncated Hepatitis B core protein VLP was produced at similarly high yields (6 × 1013 VLP/mL). These VLP were found to have comparable characteristics to those produced in vivo. The scalability of this technology was tested without loss in production yields. To our knowledge, this is the first time a prokaryote-based in vitro transcription/translation system has generated a virus-like particle. Biotechnol. Bioeng. 2008;100: 28–37. Biotechnol. Bioeng. 2008;100: 28–37. © 2007 Wiley Periodicals, Inc.

Journal ArticleDOI
Lawrence W. Dick1, Difei Qiu1, David Mahon1, Michael Adamo1, Kuang-Chuan Cheng1 
TL;DR: It was determined that LC‐MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods, and evidence is shown that carboxypeptidase B or another basic carboxypesidase causes this variation.
Abstract: The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.

Journal ArticleDOI
TL;DR: With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process and are recommended as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.
Abstract: Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.

Journal ArticleDOI
TL;DR: Mathematical model calculations of siRNA‐mediated target protein knockdown and tumor growth inhibition are used to elucidate possible mechanisms to explain the observed effects and to provide guidelines for designing more effective si RNA‐based treatment regimens regardless of delivery methodology and tumor type.
Abstract: This study addresses issues of relevance for siRNA nanoparticle delivery by investigating the functional impact of tumor-specific targeting and dosing schedule. The investigations are performed using an experimental system involving a syngeneic mouse cancer model and a theoretical system based on our previously described mathematical model of siRNA delivery and function. A/J mice bearing subcutaneous Neuro2A tumors ∼100 mm3 in size were treated by intravenous injection with siRNA-containing nanoparticles formed with cyclodextrin-containing polycations (CDP). Three consecutive daily doses of transferrin (Tf)-targeted nanoparticles carrying 2.5 mg/kg of two different siRNA sequences targeting ribonucleotide reductase subunit M2 (RRM2) slowed tumor growth, whereas non-targeted nanoparticles were significantly less effective when given at the same dose. Furthermore, administration of the three doses on consecutive days or every 3 days did not lead to statistically significant differences in tumor growth delay. Mathematical model calculations of siRNA-mediated target protein knockdown and tumor growth inhibition are used to elucidate possible mechanisms to explain the observed effects and to provide guidelines for designing more effective siRNA-based treatment regimens regardless of delivery methodology and tumor type. Biotechnol. Bioeng. 2008; 99; 975–985. © 2007 Wiley Periodicals, Inc.

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TL;DR: It is shown for the first time that cofactor NAD(H) covalently attached to micro particles, which can be easily recovered and reused, effectively mediated multistep reactions catalyzed by enzymes that were also immobilized with the micro particles.
Abstract: Efficient cofactor regeneration and reuse are highly desired for many important biotransformation applications. Here we show for the first time that cofactor NAD(H) covalently attached to micro particles, which can be easily recovered and reused, effectively mediated multistep reactions catalyzed by enzymes that were also immobilized with the micro particles. Such an immobilized enzyme-cofactor catalytic system was examined for the production of methanol from CO(2) with in situ cofactor regeneration. Four enzymes including formate, formaldehyde, alcohol, and glutamate dehydrogenases were coimmobilized using the same particles as that used for cofactor immobilization (enzymes and cofactor were immobilized separately). Reactions were performed by bubbling CO(2) in a suspension solution of the particle-attached enzymes and cofactor. It appeared that the collision among the particles afforded sufficient interactions between the cofactor and enzymes, and thus enabled the sequential transformation of CO(2) to methanol along with cofactor regeneration. For a 30-min batch reaction, a productivity of 0.02 micromol methanol/h/g-enzyme was achieved. That was lower than but comparable to the 0.04 micromol methanol/h/g-enzyme observed for free enzymes and cofactor at the same reaction conditions. The immobilized system showed fairly good stabilities in reusing. Over 80% of their original productivity was retained after 11 reusing cycles, with a cumulative methanol yield based on the amount of cofactor reached 127%. That was a promising enhancement in cofactor utilization as compared to the single-batch yield of 12% observed with free enzymes and free cofactor.