CurtisLab (curtislab.org) is a biochemical engineering laboratory. Its hallmark is in applying bioprocess design and optimization towards energy conversion, environmental remediation, and medically relevant bioproducts with emphasis on realizing low-cost solutions. Specific fields of research include plant propagation, algal and cellulosic biofuels, metabolic engineering, protein expression, and environmental remediation.
We present our progress in developing technology for plant improvement based on transient genetic... more We present our progress in developing technology for plant improvement based on transient genetic manipulation of the developmental process of embryo formation as well as the environmental manipulation of the bioreactor in which the tissue culture takes place. Somatic embryogenesis (SE) is a way to make superior plants with a desired trait. SE allows the reprogramming of a somatic cell to form a new plant. This extremely complex system is tightly controlled by a network of regulating factors in the plant’s signaling mechanism. This network can be tweaked by environmental changes or more recently by the introduction of transcription factors to activate or repress specific pathways. This manipulation can be done through an agrobacterium mediated gene transfer however, very precise timing and control is necessary to prevent abnormal development or the production of transgenic plants. As a result, transient expression, the introduction of a gene into the plant for a short period of time...
We present our progress in developing technology for plant improvement based on transient genetic... more We present our progress in developing technology for plant improvement based on transient genetic manipulation of the developmental process of embryo formation as well as the environmental manipulation of the bioreactor in which the tissue culture takes place. Somatic embryogenesis (SE) is a way to make superior plants with a desired trait. SE allows the reprogramming of a somatic cell to form a new plant. This extremely complex system is tightly controlled by a network of regulating factors in the plant’s signaling mechanism. This network can be tweaked by environmental changes or more recently by the introduction of transcription factors to activate or repress specific pathways. This manipulation can be done through an agrobacterium mediated gene transfer however, very precise timing and control is necessary to prevent abnormal development or the production of transgenic plants. As a result, transient expression, the introduction of a gene into the plant for a short period of time...
... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is pre... more ... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is presented in a different session (#202d), this poster will focus on the genetic engineering strategy to move this hydrocarbon synthetic pathway to the model algae host Chlamydomonas ...
UV-vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating... more UV-vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating chromophore formation and cell concentration in liquid culture. OD wavelength is often chosen with little thought given to its effect on the quality of the measurement. Analysis of the contributions of absorption and scattering to the measured optical density provides a basis for understanding variability among spectrophotometers and enables a quantitative evaluation of the applicability of the Beer-Lambert law. This provides a rational approach for improving the accuracy of OD measurements used as a proxy for direct dry weight (DW), cell count, and pigment levels. For pigmented organisms, the choice of OD wavelength presents a tradeoff between the robustness and the sensitivity of the measurement. The OD at a robust wavelength is primarily the result of light scattering and does not vary with culture conditions; whereas, the OD at a sensitive wavelength is additionally dependent on lig...
... RESPIRATION IN PLANT ROOT CULTURES: IMPLICATIONS TO REACTOR DESIGN* Divakar RAMAKRISHNAN and ... more ... RESPIRATION IN PLANT ROOT CULTURES: IMPLICATIONS TO REACTOR DESIGN* Divakar RAMAKRISHNAN and Wayne R. CURTIS ... Demand, Meristem, Mass Transfer, Reactor Design Introduction Root cultures (especially Agrobacterium trans-formed roots) have been ...
... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is pre... more ... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is presented in a different session (#202d), this poster will focus on the genetic engineering strategy to move this hydrocarbon synthetic pathway to the model algae host Chlamydomonas ...
ABSTRACT We present our progress in producing triterpene fuel precursors from CO2, H2 and O2as pa... more ABSTRACT We present our progress in producing triterpene fuel precursors from CO2, H2 and O2as part of the ARPA-E electrofuels program. While increasing product titer provides an obvious improvement to process economic feasibility, we also present that the maintenance energy of bacteria, or the energy needed to keep it alive, is a significant economic consideration for fuel production. The Advanced Research Projects Agency (ARPA) program seeks to produce liquid transportation fuels from renewable electricity and CO2. Botryococcenes (C30 hydrocarbons) from Botryococcus braunii is a very good potential fuel molecule for this purpose due to its high energy density, hydrophobicity and similarity to petroleum crude. A primary research milestone is the genetic engineering of the isoprene metabolic pathway through isopentenyl diphosphate (IPP) to triterpenes. This is being accomplished using the genes from Mevalonate and MEP pathways as well as triterpene synthase genes from an algae, Botryococcus braunii, known to produce hydrocarbons. We were able to use the vector systems that have been used earlier to transform other Rhodobacterspecies with constitutive promoters (pLac) and native promoters to successfully produce isoprenes. Current efforts are undertaking a "breadth screen" for different vectors, promoters, knock-in and hosts to examine the "landscape" for triterpene production. A "depth" screen is underway to better understand the rate limiting enzymes and metabolic fluxes. The current status of these screens will be presented. We are using economic considerations of the envisioned process to help drive research priorities in addition to the goal of genetically engineering the botryococcenes pathway from B. braunii into a chemolithoautotrophic bacteria – Rhodobacter capsulatus. Since the process involves the production of a moderate value fuel molecule from useful gaseous substrates (H2 and CO2), the growth of the bacteria has to be minimized to maximize the yield to to final product. Therefore the maintenance coefficient of the bacterial host becomes important as this is the absolute minimum energy wasted in the process without generating any product. To this end, we have undertaken the measurement of the growth yield and maintenance coefficient of Rhodobacter capsulatus and Ralstonia eutropha (another well-studied autotrophic production host) and determine their relative impact on the overall economics of the process. While it is relatively straight-forward to close a mass balance in the liquid phase, it is quite difficult to do it accurately in the gas phase because of effects of temperature, pressure and composition on the measurements. As such, literature references for growth yield and maintenance coefficient of autrophic bacteria are scarce. We have carried out chemostat studies under various conditions using an elaborate setup to continuously measure the inlet and outlet gas flow rate and composition from the reactor to ensure accurate determination of the parameter values. Our results indicate that under oxygen limited conditions the maintenance coefficient of R. capsulatus is significantly lower than that of R. eutropha. On the other hand, the growth yield of R. eutropha is much better than R. capsulatus. This represents critical trade-offs in terms of process design. The other important parameter affecting the process is the gas-liquid mass transfer of the bioreactor employed as this would affect the volumetric productivity of the process and thus the capital cost. We have carried out a detailed process design and economic analysis (incorporating operating, fixed and capital costs at large industrial scale) to determine the overall feasibility of the process. There exist multiple routes of going from gaseous substrates to liquid fuels. We will present the results of the different economic scenarios incorporating both literature and experimentally obtained values for the organism and reactor performance. This analysis will be used to draw conclusions about the most probable path forward for this technology. The results of this project thus far challenge the typical paradigm of working with organisms that are inherently fast growing and easy to culture. We suggest that as genetic engineering capabilities extend to more difficult organisms, some of the more fringe ‘tougher, more efficient’ organisms may be better suited for fuels production than their fast-growing ‘easy to manipulate’ counterparts.
ABSTRACT We present several lines of research focused on improving algae oil production. Secondar... more ABSTRACT We present several lines of research focused on improving algae oil production. Secondary Ion Mass Spectrometry (SIMS) is being used to better understand the extracellular matrix of the hydrocarbon-producing alga Botryococcus braunii race B which consists of triterpene oils as well as a surrounding polysaccharide network. Nile red provides a crude means of correlating oil content for screening of different algae lines as well as different growth conditions for oil formation. These two methods therefore represent the opposite ends of the spectrum in terms of in depth, data-intensive chemical profiling and rapid, crude production screening. SIMS CHEMICAL IMAGING: Botryococcus braunii is a hydrocarbon-rich microalga that has a poorly understood and complex extracellular matrix within which the algae grow as a colony of cells. Additionally, a polysaccharide network surrounding B. braunii colonies increases the difficulty of studying and cultivating these valuable algae. SIMS provides an unprecedented depth of spatially-resolved chemical information in 2 to 3 dimensions of B. braunii. In this manner, one can develop a chemical image of several chemical species within the algae. This is particularly useful for biofilms, or colony forming algae such as B. braunii race B. Accordingly, high-resolution chemical imaging has been used to investigate the hydrocarbon storage and distribution of this strong candidate for alternative fuels. Organelles within the hydrocarbon-rich extracellular matrix surrounding the algal cells have been chemically imaged utilizing a 250 nm diameter C60+ primary ion beam on the J105 3D Chemical Imager (Ionoptika, LTD). Organelles of great interest are approximately 1 -2 µm round vesicles within the extracellular matrix, which have been proposed to contain C23 – C37 liquid hydrocarbons, termed “botryococcenes,” unique to B. braunii race B. They are revealed after depth profiling through the algal cell colonies, thus, enabling the acquisition of chemical information in three dimensions. Unique chemical components of B. braunii over m/z 600 may be chemically mapped. Chemical signatures for the polysaccharide network, algal cell wall, and the liquid hydrocarbon extracellular matrix have been identified. The botryococcenes and hydrocarbon-derivative chemical species are located in specific locations dependent on their function within the cell and in the colony. Peak identification has been aided by the use of tandem MS capabilities. With the use of this unique chemical imaging technique, questions surrounding the purpose and function of the B. braunii cell’s behavior and association with hydrocarbons may be investigated. ESTABLISHING AXENIC CULTURES: An outcome of the SIMS work was a better understanding of the polysaccharide matrix which appears to function in part by holding the colonies together. Since it is desirable to have axenic cultures of B. braunii to carry out chemical feeding and genetic transformation studies, the presence of cellulose in the surrounding matrix suggested we might dissipate the algae colonies with cellulase treatment. This has been substantiated by chemically mapping the presence of cellulose within B. braunii colonies. Accordingly, the use of hydrolytic enzymes with endoxylanase and cellulase activity to degrade the polysaccharide wall surrounding these algal colonies resulted in the isolation of single cells. These results indicate that an axenic culture may be obtained through dilution plating or laser capture microdissection of these single cells. The outcome of this work will enable the mixotrophic growth of B. braunii resulting in an efficient and expedient means to scaling up for large scale biofuels production. NILE RED SCREENING: Complementary to the data-intensive chemical profiling provided by SIMS, Nile red fluorescence staining provides a means of rapidly screening for the hydrocarbon content in microalgae. This method has been applied for the determination of the neutral hydrocarbon content in microalgae, where fluorescence intensity is correlated to the quantity of oil present. Conventional solvent extraction methods are time consuming and require large culture samples as compared to the staining of algae in a 96-well plate. With very little sample preparation and low sample volume, a fluorometer using 530 nm excitation and 575 nm emission enables the quick and accurate analysis of oil content in a given algal culture. The ability to sample small amounts of culture volume under a variety of culturing conditions enables high-throughput screening for maximum oil productivity.
Agrobacterium transformed root cultures of Hyoscyamus muticus were grown in a recirculating 2 L t... more Agrobacterium transformed root cultures of Hyoscyamus muticus were grown in a recirculating 2 L tubular bioreactor system. Performance of this convective flow reactor (CFR) was compared to a bubble column (BC) reactor of the same geometry: replicated CFR experiments produced an average tissue concentration of 556 +/- 4 grams fresh weight per liter in 30 d whereas the bubble column produced only 328 +/- 5 grams per liter corresponding to 25.3 +/- 0.0 and 14.3 +/- 0.5 grams dry weight per liter, respectively. Because media nutrient levels were maintained sufficiently high to saturate growth rate, the improved performance of the CFR is attributed to enhanced convective mass transfer. The pressure drops observed for flow through roots grown within the reactors were more than an order of magnitude higher than previously obtained by placing roots grown in shake culture into defined geometries. The experimentally observed flow resistance was much higher than would be predicted from correlations using the root diameter as the characteristic diameter for flow resistance. Several lines of evidence suggest that root hairs are a substantial contributor to the observed high flow resistance in these transformed root cultures. Pressure drop increased nonlinearly with velocity which could not be adequately described by a modified form of the Ergun equation. Kyan et al's (1970) equation, although predicting such curvature, relies almost exclusively on an empirical packing deflection term to describe the hydrodynamic behavior. Implications of these results to the design of submerged reactor systems for root culture are discussed. Copyright 1998 John Wiley & Sons, Inc.
Page 1. HAIRY ROOTS, BIOREACTOR GROWTH WAYNE R. CURTIS The Pennsylvania State University, Univers... more Page 1. HAIRY ROOTS, BIOREACTOR GROWTH WAYNE R. CURTIS The Pennsylvania State University, University Park, Pennsylvania INTRODUCTION A wide variety of bioreactor configurations have been used to cultivate plant roots. ...
Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the c... more Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast
ABSTRACT Natural elastomers made from protein extracts have received significant interest as eco-... more ABSTRACT Natural elastomers made from protein extracts have received significant interest as eco-friendly functional materials due to their unique mechanical and optical properties emanating from secondary structures. The next generation sequencing approach is used to identify protein sequences in a squid ring teeth complex extracted from Loligo vulgaris and the use of recombinant expression is demonstrated in the fabrication of a new generation of thermoplastic materials. Native and recombinant thermoplastic squid proteins exhibit reversible solid to melt phase transition, enabling them to be thermally shaped into 3D geometries such as fibers, colloids, and thin films. Direct extraction or recombinant expression of protein based thermoplastics opens up new avenues for materials fabrication and synthesis, which will eventually be competitive with the high-end synthetic oil based plastics.
Page 1. 173 OXYGEN TRANSPORT IN PLANT TISSUE CULTURE SYSTEMS Oxygen transport limitations WAYNE R... more Page 1. 173 OXYGEN TRANSPORT IN PLANT TISSUE CULTURE SYSTEMS Oxygen transport limitations WAYNE R. CURTIS1 AND AMALIE L. TUERK2 1108 Fenske Laboratory, The Pennsylvania State University, University ...
Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nic... more Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y(g)) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y(g) (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.
Page 1. PHYTOCHEMICAL ANALYSIS, VOL. 1, 70-73 (1990) Interference of Intracellular Inorganic Phos... more Page 1. PHYTOCHEMICAL ANALYSIS, VOL. 1, 70-73 (1990) Interference of Intracellular Inorganic Phosphate Analysis by Phosphatase in Papaver somniferum Cell Suspensions Wayne R. Curtis Department of Chemical EnginecringlBiotechnology ...
Calcium alginate immobilization of Hyoscyamus muticus plant tissue culture is shown to enhance th... more Calcium alginate immobilization of Hyoscyamus muticus plant tissue culture is shown to enhance the rapid formation of sesquiterpenes in response to exposure to extracts of the fungal pathogen Rhizoctonia solani. The studies were carried out with suspension-cultured cells and roots at low tissue loadings to demonstrate that the enhancement is not due to either the degree of differentiation or the
We present our progress in developing technology for plant improvement based on transient genetic... more We present our progress in developing technology for plant improvement based on transient genetic manipulation of the developmental process of embryo formation as well as the environmental manipulation of the bioreactor in which the tissue culture takes place. Somatic embryogenesis (SE) is a way to make superior plants with a desired trait. SE allows the reprogramming of a somatic cell to form a new plant. This extremely complex system is tightly controlled by a network of regulating factors in the plant’s signaling mechanism. This network can be tweaked by environmental changes or more recently by the introduction of transcription factors to activate or repress specific pathways. This manipulation can be done through an agrobacterium mediated gene transfer however, very precise timing and control is necessary to prevent abnormal development or the production of transgenic plants. As a result, transient expression, the introduction of a gene into the plant for a short period of time...
We present our progress in developing technology for plant improvement based on transient genetic... more We present our progress in developing technology for plant improvement based on transient genetic manipulation of the developmental process of embryo formation as well as the environmental manipulation of the bioreactor in which the tissue culture takes place. Somatic embryogenesis (SE) is a way to make superior plants with a desired trait. SE allows the reprogramming of a somatic cell to form a new plant. This extremely complex system is tightly controlled by a network of regulating factors in the plant’s signaling mechanism. This network can be tweaked by environmental changes or more recently by the introduction of transcription factors to activate or repress specific pathways. This manipulation can be done through an agrobacterium mediated gene transfer however, very precise timing and control is necessary to prevent abnormal development or the production of transgenic plants. As a result, transient expression, the introduction of a gene into the plant for a short period of time...
... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is pre... more ... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is presented in a different session (#202d), this poster will focus on the genetic engineering strategy to move this hydrocarbon synthetic pathway to the model algae host Chlamydomonas ...
UV-vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating... more UV-vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating chromophore formation and cell concentration in liquid culture. OD wavelength is often chosen with little thought given to its effect on the quality of the measurement. Analysis of the contributions of absorption and scattering to the measured optical density provides a basis for understanding variability among spectrophotometers and enables a quantitative evaluation of the applicability of the Beer-Lambert law. This provides a rational approach for improving the accuracy of OD measurements used as a proxy for direct dry weight (DW), cell count, and pigment levels. For pigmented organisms, the choice of OD wavelength presents a tradeoff between the robustness and the sensitivity of the measurement. The OD at a robust wavelength is primarily the result of light scattering and does not vary with culture conditions; whereas, the OD at a sensitive wavelength is additionally dependent on lig...
... RESPIRATION IN PLANT ROOT CULTURES: IMPLICATIONS TO REACTOR DESIGN* Divakar RAMAKRISHNAN and ... more ... RESPIRATION IN PLANT ROOT CULTURES: IMPLICATIONS TO REACTOR DESIGN* Divakar RAMAKRISHNAN and Wayne R. CURTIS ... Demand, Meristem, Mass Transfer, Reactor Design Introduction Root cultures (especially Agrobacterium trans-formed roots) have been ...
... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is pre... more ... While our work with the native hydrocarbon producing algae strain Botryococcus braunii is presented in a different session (#202d), this poster will focus on the genetic engineering strategy to move this hydrocarbon synthetic pathway to the model algae host Chlamydomonas ...
ABSTRACT We present our progress in producing triterpene fuel precursors from CO2, H2 and O2as pa... more ABSTRACT We present our progress in producing triterpene fuel precursors from CO2, H2 and O2as part of the ARPA-E electrofuels program. While increasing product titer provides an obvious improvement to process economic feasibility, we also present that the maintenance energy of bacteria, or the energy needed to keep it alive, is a significant economic consideration for fuel production. The Advanced Research Projects Agency (ARPA) program seeks to produce liquid transportation fuels from renewable electricity and CO2. Botryococcenes (C30 hydrocarbons) from Botryococcus braunii is a very good potential fuel molecule for this purpose due to its high energy density, hydrophobicity and similarity to petroleum crude. A primary research milestone is the genetic engineering of the isoprene metabolic pathway through isopentenyl diphosphate (IPP) to triterpenes. This is being accomplished using the genes from Mevalonate and MEP pathways as well as triterpene synthase genes from an algae, Botryococcus braunii, known to produce hydrocarbons. We were able to use the vector systems that have been used earlier to transform other Rhodobacterspecies with constitutive promoters (pLac) and native promoters to successfully produce isoprenes. Current efforts are undertaking a "breadth screen" for different vectors, promoters, knock-in and hosts to examine the "landscape" for triterpene production. A "depth" screen is underway to better understand the rate limiting enzymes and metabolic fluxes. The current status of these screens will be presented. We are using economic considerations of the envisioned process to help drive research priorities in addition to the goal of genetically engineering the botryococcenes pathway from B. braunii into a chemolithoautotrophic bacteria – Rhodobacter capsulatus. Since the process involves the production of a moderate value fuel molecule from useful gaseous substrates (H2 and CO2), the growth of the bacteria has to be minimized to maximize the yield to to final product. Therefore the maintenance coefficient of the bacterial host becomes important as this is the absolute minimum energy wasted in the process without generating any product. To this end, we have undertaken the measurement of the growth yield and maintenance coefficient of Rhodobacter capsulatus and Ralstonia eutropha (another well-studied autotrophic production host) and determine their relative impact on the overall economics of the process. While it is relatively straight-forward to close a mass balance in the liquid phase, it is quite difficult to do it accurately in the gas phase because of effects of temperature, pressure and composition on the measurements. As such, literature references for growth yield and maintenance coefficient of autrophic bacteria are scarce. We have carried out chemostat studies under various conditions using an elaborate setup to continuously measure the inlet and outlet gas flow rate and composition from the reactor to ensure accurate determination of the parameter values. Our results indicate that under oxygen limited conditions the maintenance coefficient of R. capsulatus is significantly lower than that of R. eutropha. On the other hand, the growth yield of R. eutropha is much better than R. capsulatus. This represents critical trade-offs in terms of process design. The other important parameter affecting the process is the gas-liquid mass transfer of the bioreactor employed as this would affect the volumetric productivity of the process and thus the capital cost. We have carried out a detailed process design and economic analysis (incorporating operating, fixed and capital costs at large industrial scale) to determine the overall feasibility of the process. There exist multiple routes of going from gaseous substrates to liquid fuels. We will present the results of the different economic scenarios incorporating both literature and experimentally obtained values for the organism and reactor performance. This analysis will be used to draw conclusions about the most probable path forward for this technology. The results of this project thus far challenge the typical paradigm of working with organisms that are inherently fast growing and easy to culture. We suggest that as genetic engineering capabilities extend to more difficult organisms, some of the more fringe ‘tougher, more efficient’ organisms may be better suited for fuels production than their fast-growing ‘easy to manipulate’ counterparts.
ABSTRACT We present several lines of research focused on improving algae oil production. Secondar... more ABSTRACT We present several lines of research focused on improving algae oil production. Secondary Ion Mass Spectrometry (SIMS) is being used to better understand the extracellular matrix of the hydrocarbon-producing alga Botryococcus braunii race B which consists of triterpene oils as well as a surrounding polysaccharide network. Nile red provides a crude means of correlating oil content for screening of different algae lines as well as different growth conditions for oil formation. These two methods therefore represent the opposite ends of the spectrum in terms of in depth, data-intensive chemical profiling and rapid, crude production screening. SIMS CHEMICAL IMAGING: Botryococcus braunii is a hydrocarbon-rich microalga that has a poorly understood and complex extracellular matrix within which the algae grow as a colony of cells. Additionally, a polysaccharide network surrounding B. braunii colonies increases the difficulty of studying and cultivating these valuable algae. SIMS provides an unprecedented depth of spatially-resolved chemical information in 2 to 3 dimensions of B. braunii. In this manner, one can develop a chemical image of several chemical species within the algae. This is particularly useful for biofilms, or colony forming algae such as B. braunii race B. Accordingly, high-resolution chemical imaging has been used to investigate the hydrocarbon storage and distribution of this strong candidate for alternative fuels. Organelles within the hydrocarbon-rich extracellular matrix surrounding the algal cells have been chemically imaged utilizing a 250 nm diameter C60+ primary ion beam on the J105 3D Chemical Imager (Ionoptika, LTD). Organelles of great interest are approximately 1 -2 µm round vesicles within the extracellular matrix, which have been proposed to contain C23 – C37 liquid hydrocarbons, termed “botryococcenes,” unique to B. braunii race B. They are revealed after depth profiling through the algal cell colonies, thus, enabling the acquisition of chemical information in three dimensions. Unique chemical components of B. braunii over m/z 600 may be chemically mapped. Chemical signatures for the polysaccharide network, algal cell wall, and the liquid hydrocarbon extracellular matrix have been identified. The botryococcenes and hydrocarbon-derivative chemical species are located in specific locations dependent on their function within the cell and in the colony. Peak identification has been aided by the use of tandem MS capabilities. With the use of this unique chemical imaging technique, questions surrounding the purpose and function of the B. braunii cell’s behavior and association with hydrocarbons may be investigated. ESTABLISHING AXENIC CULTURES: An outcome of the SIMS work was a better understanding of the polysaccharide matrix which appears to function in part by holding the colonies together. Since it is desirable to have axenic cultures of B. braunii to carry out chemical feeding and genetic transformation studies, the presence of cellulose in the surrounding matrix suggested we might dissipate the algae colonies with cellulase treatment. This has been substantiated by chemically mapping the presence of cellulose within B. braunii colonies. Accordingly, the use of hydrolytic enzymes with endoxylanase and cellulase activity to degrade the polysaccharide wall surrounding these algal colonies resulted in the isolation of single cells. These results indicate that an axenic culture may be obtained through dilution plating or laser capture microdissection of these single cells. The outcome of this work will enable the mixotrophic growth of B. braunii resulting in an efficient and expedient means to scaling up for large scale biofuels production. NILE RED SCREENING: Complementary to the data-intensive chemical profiling provided by SIMS, Nile red fluorescence staining provides a means of rapidly screening for the hydrocarbon content in microalgae. This method has been applied for the determination of the neutral hydrocarbon content in microalgae, where fluorescence intensity is correlated to the quantity of oil present. Conventional solvent extraction methods are time consuming and require large culture samples as compared to the staining of algae in a 96-well plate. With very little sample preparation and low sample volume, a fluorometer using 530 nm excitation and 575 nm emission enables the quick and accurate analysis of oil content in a given algal culture. The ability to sample small amounts of culture volume under a variety of culturing conditions enables high-throughput screening for maximum oil productivity.
Agrobacterium transformed root cultures of Hyoscyamus muticus were grown in a recirculating 2 L t... more Agrobacterium transformed root cultures of Hyoscyamus muticus were grown in a recirculating 2 L tubular bioreactor system. Performance of this convective flow reactor (CFR) was compared to a bubble column (BC) reactor of the same geometry: replicated CFR experiments produced an average tissue concentration of 556 +/- 4 grams fresh weight per liter in 30 d whereas the bubble column produced only 328 +/- 5 grams per liter corresponding to 25.3 +/- 0.0 and 14.3 +/- 0.5 grams dry weight per liter, respectively. Because media nutrient levels were maintained sufficiently high to saturate growth rate, the improved performance of the CFR is attributed to enhanced convective mass transfer. The pressure drops observed for flow through roots grown within the reactors were more than an order of magnitude higher than previously obtained by placing roots grown in shake culture into defined geometries. The experimentally observed flow resistance was much higher than would be predicted from correlations using the root diameter as the characteristic diameter for flow resistance. Several lines of evidence suggest that root hairs are a substantial contributor to the observed high flow resistance in these transformed root cultures. Pressure drop increased nonlinearly with velocity which could not be adequately described by a modified form of the Ergun equation. Kyan et al's (1970) equation, although predicting such curvature, relies almost exclusively on an empirical packing deflection term to describe the hydrodynamic behavior. Implications of these results to the design of submerged reactor systems for root culture are discussed. Copyright 1998 John Wiley & Sons, Inc.
Page 1. HAIRY ROOTS, BIOREACTOR GROWTH WAYNE R. CURTIS The Pennsylvania State University, Univers... more Page 1. HAIRY ROOTS, BIOREACTOR GROWTH WAYNE R. CURTIS The Pennsylvania State University, University Park, Pennsylvania INTRODUCTION A wide variety of bioreactor configurations have been used to cultivate plant roots. ...
Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the c... more Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast
ABSTRACT Natural elastomers made from protein extracts have received significant interest as eco-... more ABSTRACT Natural elastomers made from protein extracts have received significant interest as eco-friendly functional materials due to their unique mechanical and optical properties emanating from secondary structures. The next generation sequencing approach is used to identify protein sequences in a squid ring teeth complex extracted from Loligo vulgaris and the use of recombinant expression is demonstrated in the fabrication of a new generation of thermoplastic materials. Native and recombinant thermoplastic squid proteins exhibit reversible solid to melt phase transition, enabling them to be thermally shaped into 3D geometries such as fibers, colloids, and thin films. Direct extraction or recombinant expression of protein based thermoplastics opens up new avenues for materials fabrication and synthesis, which will eventually be competitive with the high-end synthetic oil based plastics.
Page 1. 173 OXYGEN TRANSPORT IN PLANT TISSUE CULTURE SYSTEMS Oxygen transport limitations WAYNE R... more Page 1. 173 OXYGEN TRANSPORT IN PLANT TISSUE CULTURE SYSTEMS Oxygen transport limitations WAYNE R. CURTIS1 AND AMALIE L. TUERK2 1108 Fenske Laboratory, The Pennsylvania State University, University ...
Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nic... more Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y(g)) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y(g) (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.
Page 1. PHYTOCHEMICAL ANALYSIS, VOL. 1, 70-73 (1990) Interference of Intracellular Inorganic Phos... more Page 1. PHYTOCHEMICAL ANALYSIS, VOL. 1, 70-73 (1990) Interference of Intracellular Inorganic Phosphate Analysis by Phosphatase in Papaver somniferum Cell Suspensions Wayne R. Curtis Department of Chemical EnginecringlBiotechnology ...
Calcium alginate immobilization of Hyoscyamus muticus plant tissue culture is shown to enhance th... more Calcium alginate immobilization of Hyoscyamus muticus plant tissue culture is shown to enhance the rapid formation of sesquiterpenes in response to exposure to extracts of the fungal pathogen Rhizoctonia solani. The studies were carried out with suspension-cultured cells and roots at low tissue loadings to demonstrate that the enhancement is not due to either the degree of differentiation or the
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