Wayne R. Curtis

Developing symbiotic consortia for lignocellulosic biofuel production

The search for petroleum alternatives has motivated intense research into biological breakdown of lignocellulose to produce liquid fuels such as ethanol. Degradation of lignocellulose for biofuel production is a difficult process which is limited by, among other factors, the recalcitrance of lignocellulose and biological toxicity of the products. Consolidated bioprocessing has been suggested as an efficient and economical method of producing low value products from lignocellulose; however, it is not clear whether this would be accomplished more efficiently with a single organism or community of organisms. This review highlights examples of mixtures of microbes in the context of conceptual models for developing symbiotic consortia for biofuel production from lignocellulose. Engineering a symbiosis within consortia is a putative means of improving both process efficiency and stability relative to monoculture. Because microbes often interact and exist attached to surfaces, quorum sensing and biofilm formation are also discussed in terms of consortia development and stability. An engineered, symbiotic culture of multiple organisms may be a means of assembling a novel combination of metabolic capabilities that can efficiently produce biofuel from lignocellulose.

Improving accuracy of cell and chromophore concentration measurements using optical density

BackgroundUV–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.ResultsFor 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 light absorption by the organism’s pigments. Suitably robust and sensitive wavelengths are identified for a wide range of organisms by comparing their spectra to the true absorption spectra of dyes. The relative scattering contribution can be reduced either by measurement at higher OD, or by the addition of bovine serum albumin. Reduction of scattering or correlation with off-peak light attenuation provides for more accurate assessment of chromophore levels within cells. Conversion factors between DW, OD, and colony-forming unit density are tabulated for 17 diverse organisms to illustrate the scope of variability of these correlations. Finally, an inexpensive short pathlength LED-based flow cell is demonstrated for the online monitoring of growth in a bioreactor at culture concentrations greater than 5 grams dry weight per liter which would otherwise require off-line dilutions to obtain non-saturated OD measurements.ConclusionsOD is most accurate as a time-saving proxy measurement for biomass concentration when light attenuation is dominated by scattering. However, the applicability of OD-based correlations is highly dependent on the measurement specifications (spectrophotometer model and wavelength) and culture conditions (media type; growth stage; culture stress; cell/colony geometry; presence and concentration of secreted compounds). These variations highlight the importance of treating literature conversion factors as rough approximations as opposed to concrete constants. There is an opportunity to optimize measurements of cell pigment levels by considering scattering and absorption-dependent wavelengths of the OD spectrum.

Plant cell and tissue culture for the production of food ingredients

1. Plant Cell and Tissue Culture for Food Ingredient Production: An Introduction.- 2. Plant Secondary Metabolism: Control Points and Prospects for Genetic Manipulation of Phenylpropanoid Biosynthesis.- 3. Production of Aromatic Amino Acid Derivatives through Metabolic Engineering of Crop Plants.- 4. Vanillin Biosynthetic Pathways: An Overview.- 5. Quantification of Metabolic Fluxes for Metabolic Engineering of Plant Products.- 6. Biosynthesis and Accumulation of Rosmarinic Acid in Plant Cell Cultures.- 7. Overview of Yield Improvement Strategies for Secondary Metabolite Production in Plant Cell Culture.- 8. Novel Approaches to Improve Plant Secondary Metabolite Production.- 9. Elicitation-Manipulating and Enhancing Secondary Metabolite Production.- 10. The Selectivity by Plant Biotransformation.- 11. Production of Betalains by Hairy Root Cultures of Beta vulgaris L.- 12. Yield Improvement in Plant Cell Cultures by In Situ Extraction.- 13. Reactor Design for Root Culture: Oxygen Mass Transfer Limitations.- 14. Medium Recycling as an Operational Strategy to Increase Plant Secondary Metabolite Formation: Continuous Taxol Production.- 15. Production of Bioactive Metabolites by Cell and Tissue Cultures of Marine Macroalgae in Bioreactor Systems.- 16. Trichosanthes kirilowii Plant Cell Culture in a 5 Liter Bioreactor.- 17. Economic Considerations for Food Ingredients Produced by Plant Cell and Tissue Culture.- 18. Commercial Production of Ginseng by Plant Tissue Culture Technology.- 19. Achieving Economic Feasibility for Moderate-Value Food and Flavor Additives: A Perspective on Productivity and Proposal for Production Technology Cost Reduction.- 20. Plant Cell and Tissue Culture for Food Ingredient Production: Safety Considerations.- 21. The Safety Assessment of Flavor Ingredients Derived from Plant Cell and Tissue Culture.- 22. European Guidelines for Safety Evaluation of Flavourings Produced by Plant Tissue Culture.- 23. Food Ingredients from Plant Cell and Tissue Culture: Regulatory Considerations.- 24. Regulatory Issues: Canadian Perspective.- 25. Regulations for Plant Cell Culture Derived Products in Japan.

Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture

BackgroundLignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity).ResultsEthanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana “protect” C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively.ConclusionThis work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.

Characterization of fluid-flow resistance in root cultures with a convective flow tubular bioreactor

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 als (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.

Comparison of Transient Protein Expression in Tobacco Leaves and Plant Suspension Culture

Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor‐intensive and time‐consuming process of generating stably transformed transgenic plants. Transient expression of the gus‐intron reporter gene was facilitated in three different tobacco species. Two different approaches to T‐DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotiana benthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture‐based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.

Inoculation and tissue distribution in pilot-scale plant root culture bioreactors

Inoculation of large-scale plant root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto process packing elements (e.g. distillation packings), randomly distributed within the reactor, in a bubble column operation. These two techniques have been successfully used to inoculate a 14 L pilot-scale reactor which is subsequently operated as a trickle bed reactor.

Intrinsic Oxygen Use Kinetics of Transformed Plant Root Culture

Root meristem oxygen uptake, root tip extension rate, and specific growth rate are assessed as a function of dissolved oxygen level for three transformed root cultures. The influence of hydrodynamic boundary layer was considered for all measurements to permit correlation of oxygen‐dependent kinetics with the concentration of oxygen at the surface of the root meristem. Oxygen uptake rate is shown to be saturated at ambient conditions, and a saturation level of approximately 300 μmole O2/(cm3 tissue·hr) was observed for all three of these morphologically diverse root types. In nearly all cases, the observation of a minimum oxygen pressure, below which respiration, extension, or root growth would not occur, could be accounted for as a boundary layer mass transfer resistance. The critical oxygen pressure below which respiration declines is below saturated ambient oxygen conditions. In contrast, critical oxygen pressures for root tip extension were much higher; extension was nearly linear for the two thicker root types (Hyoscyamus muticus, henbain; Solanum tuberosum, potato) above ambient oxygen levels. The performance of the thinnest root, Brassica juncea (Indian mustard) was consistent with reduced internal limitations for oxygen transport. Extension rates did not correlate with biomass accumulation. The fastest growing henbain culture (μ = 0.44 day−1) displayed the slowest extension rate (0.16 mm/hr), and the slowest growing mustard culture (μ = 0.22 day−1) had the fastest tip extension rate (0.3 mm/hr). This apparent paradox is explained in terms of root branching patterns, where the root branching ratio is shown to be dependent upon the oxygen‐limited mersitem extension rate. The implications of these observations on the performance of root culture in bioreactors is discussed.

The role of liquid mixing and gas-phase dispersion in a submerged, sparged root reactor.

An Agrobacterium-transformed root culture of Solanum tuberosum was grown in a 15-1 bubble column. The specific respiration rate decreased by a factor of ten as the tissue grew over a 25-day culture period. On days 5, 8, 13, and 21, respiration was shown to be independent of aeration rate over a range of 0.05-0.4 vvm (volume of air per volume of liquid min-1). Gas dispersion measured from argon tracer residence time distributions increased fourfold due to increased stagnation and channeling of gas through the bed of growing roots; however, introduction of an antifoam surfactant on day 20 greatly reduced dispersion with no accompanying change in respiration. Taken together, the gas dispersion and respiration studies suggest that the gas-liquid interface is not the dominant resistance to oxygen mass transfer. Liquid mixing time measured with a dye tracer increased from 1.45 +/- 0.45 min with no root tissue to 40.2 +/- 1.6 min with 180 g FW l-1 of roots in the column. In addition, the oxygen uptake rate of growing tips (5.2 +/- 0.2 mm) of individual root segments of S. tuberosum measured in a stirred microcell (600 microliters) increased with the oxygen tension of the medium. Based on these results, the role of liquid mixing, gas-phase dispersion, and diffusion in the tissue in the scaleup of root culture is discussed.

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor

BackgroundTheobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state.ResultsAn ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development.ConclusionOur work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.