Wayne Roger Curtis

Pennsylvania State University | Penn State · Department of Chemical Engineering

Pseudomonas aeruginosa strain SW1 is an aerobic, motile, Gram-negative, and rod-shaped bacterium isolated from degraded seaweeds. Based on the 16S rRNA gene sequence and MALDI TOF analysis, strain SW1 exhibits 100% similarity to P. aeruginosa DSM 50,071, its closest phylogenetic neighbor. The complete genome of strain SW1 consists of a single circu...

Objectives: To carry out mass propagation of superior plants to improve agricultural and silvicultural production though advancements in plant cell totipotency, or the ability of differentiated somatic plant cells to regenerate an entire plant. Results: The first demonstration of a titratable control over somatic embryo formation in a commercial...

479621 Constructing and Testing a Photobioreactor Designed for Precise Characterizations of Growth of Microalgae for Biofuel Applications Monday, November 14, 2016 Grand Ballroom B (Hilton San Francisco Union Square) Lucas A Nugent, Chemical Engineering, The Pennsylvania State Univeristy, University Pk, PA, Benjamin Geveke, Chemical Engineering, Th...

A crucial challenge associated with high-density, commercial-scale, outdoor microalgal cultivation is maintaining pH stability without excessive use of CO2 buffering. This includes a media-dependent, intracellular metabolic proton imbalance leading to the alkalization or acidification of growth media that results when algae consume, respectively, n...

Metagenomics has the potential to facilitate the discovery of novel enzymes; however, to date, only a few alkaline proteases have been characterized from environmentally-sourced DNA. We report the identification and characterization of an alkaline serine protease designated as Prt1A from the metagenomic library of tannery activated sludge. Sequence...

The colony algae Botryococcus braunii produces large amounts of C30+ triterpene hydrocarbons. Recent discovery of the associated biosynthetic genes has facilitated the metabolic engineering of these triterpene hydrocarbons in alternative hosts ? where squalene has served as an analytical standard and a closely associated model hydrocarbon biosynthe...

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic-driven TIB (Hy-TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move t...

A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for environmental microbiology. The present work describes an improved method of DNA extraction namely " powdered glass method " from diverse soils. The method involves the use of sterile glass powder for cell lysis followed by addition of 1% powdered activated...

Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bact...

Theobroma 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. Manipula...

The ability of autotrophic organisms to fix CO2 presents an opportunity to utilize this 'greenhouse gas' as an inexpensive substrate for biochemical production. Unlike conventional heterotrophic microorganisms that consume carbohydrates and amino acids, prokaryotic chemolithoautotrophs have evolved the capacity to utilize reduced chemical compounds...

Membrane protein overexpression is often hindered by toxic effects on the expression host, limiting achievable volumetric productivity. Moreover, protein structure and function may be impaired due to inclusion body formation and proteolytic degradation. To address these challenges, we employed the photosynthetic bacterium, Rhodobacter sphaeroides f...

Intimin is the primary adhesin protein of enterohemorrhagic Escherichia coli O157:H7, a pathogen carried by cattle and transmitted to humans via contaminated food and water. This work describes the rapid production of 1.3 kg dry weight of transgenic tobacco cells expressing the C-terminal 261 amino acids of intimin (Int261) for subsequent oral vacc...

The use of algae as a potential platform for fuels or biochemical production requires process design and control that can be implemented at agronomic scales. Toward achieving pH control in large unmixed systems, we present a rigorous set of direct measurements of non-buffered proton uptake and efflux during growth on ammonium and nitrate, observing...

Triterpene hydrocarbon biosynthesis of the ancient algae Botryococcus braunii was installed into Rhodobacter capsulatus to explore the production of C30 hydrocarbon in a host capable of diverse growth habits - utilizing carbohydrate, sunlight or hydrogen (with CO2 fixation) as alternative energy feedstocks. Engineering an enhanced MEP pathway was a...

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 an...

Background Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embr...

Biofilm formation is a critical component to the lifestyle of many naturally occurring cellulose-degrading microbes. In this work, cellular aggregation and biofilm formation of Clostridium phytofermentans, a cellulolytic anaerobic bacterium, was investigated using a combination of microscopy and analytical techniques. Aggregates included thread-lik...

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 grams dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5 to 15% of the respective cu...

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, ex...

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 al...

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 consi...

We demonstrate that a novel aquaporin from Rhodobacter sphaeroides 2.4.1., RsAqpZ, displays superior water transport characteristics in phospholipid vesicles as compared to well-studied E.coli AqpZ and most of the other orthodox aquaporins. BACKGROUND:Most biological cells are surrounded by phospholipid bilayer protecting them from their surroundi...

Advanced analytical methods of Sum Frequency Generation (SFG) spectroscopy, Secondary Ion Mass Spectrometry (SIMS), InfraRed microspectroscopy are used to quantify difference in the dynamics of degradation of lignocellulosic substrates by two different wood-degrading fungi. Wood is a composite that undergoes natural degradation in different ways de...

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...

We describe the use of fluorescent protein fusions as a method for optimizing and accurately quantifying membrane protein expression in Rhodobacter. Membrane proteins (MP) are a difficult class of proteins to study; however, their medical and biotechnical importance makes them compelling to address the associated challenges. Difficulties of express...

Background Lack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production sy...

Background Lignocellulosic 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 sing...

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...

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic-driven TIB (Hy-TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move t...

Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral...

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. C...

Our overall ARPA-E project goal seeks to produce a renewable hydrocarbon feedstock by the genetic engineering of a C34 branched hyrdrocarbon of Botrococcus braunii into Rhodobacter capsulatus. We have shown the ability of this genetically engineered autotrophic organism to produce C30+ isoprenes from H2, O2 and CO2. My specific component of this pr...

Colony blots of an Agaricus bisporus fruiting body cDNA library. A cDNA library consisting of a total of 4,608 cDNA clones was screened by colony blot hybridization using gene-specific DNA oligos (60 nt) as probes. Based on probing∼4,800 bacterial colonies, the frequencies of cDNA sequences were: >110 for hydrophobin A (HYPA),>50 for fruiting body-...

GUS enzyme activity in mycelium colonizing the lower compost layer. For each treatment, three filter paper discs were exposed to the compost substrate during the Agaricus bisporus colonization period in the mushroom cultivation cycle and then subjected to a histological GUS assay. Compost inoculant treatments were: non-inoculated (None); WT-inocula...

HPT activity assay of basidiospores derived from fruiting bodies. Indicated is the upper layer inoculant/lower layer inoculant for the bi-layered cultivation substrate. WT: wild-type line; LGS: transgenic GUS line carrying the laccase 2 promoter; HPT: hygromycin phosphotransferase. Basidiospores were collected from fruiting bodies grown using the i...

Structural organization of the GUS expression cassette. The HPT gene, which conferred hygromycin B resistance as a selectable marker, and GUS reporter gene (GUSPlus; CambiaLabs) [33] were situated between the left (T-DNA LB) and right (T-DNA RB) border sequences of the Agrobacterium T-DNA. The GUS gene was joined to either the native hydrophobin A...

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologo...

Agaricus bisporus (button mushroom) is a promising platform for large-scale therapeutic protein production due to the advent of a facile gene transfer method, coupled with exisiting mature technologies for the commercial-scale and economical cultivation of biomass in the form of its edible fruiting body. In an effort to maximize heterologous protei...

Plant tissue culture provides a means to proliferate plants without the requirement of going through seeds, thus enabling the rapid propagation of superior plants such as sterile hybrids or disease resistant varieties. We are developing this technology for increasing the productivity of cultivated plants including fungal resistant Theobroma cacao (...

The Department of Energy (DOE) has become very interested in using microbial systems to remediate radioactive metal contaminants, such as Technetium (Tc) and Uranium (U). These materials are found naturally in their soluble form. Water sources, such as lakes, rivers, and aquifers, contaminated with these and other metal traces represent an emerging...

Geobacter Sulfurreducens, an iron-reducing microbe, has great potential for remediation of metal-contaminated water. The ability of Geobacter to reduce and precipitate metals provides a means to sequester toxic ions that would otherwise move freely in water systems. This is done by feeding the organism sufficient carbon to maintain respiratory acti...

In this paper, we present progress on the development of Rhodobacter sphaeroides as a platform for high-level expression of functional membrane proteins. The enhanced capacity of Rhodobacter for membrane protein expression when compared to traditional hosts results from the dense intracellular membrane infoldings synthesized by this organism under...

Algae can be used to capture and reduce CO2 to hydrocarbons using photosynthetic energy. 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 Chl...

This paper presents progress on the development of Rhodobacter sphaeroides as a platform for high-level expression of functional membrane proteins. The high capacity of Rhodobacter for membrane protein expression stems from the dense intracellular membranes synthesized by this organism under anaerobic photoheterotrophic conditions. Using plasmids w...

This presentation focuses on characterization of growth and hydrocarbon production from Botryococcus braunii as part of an overall project to develop the tools to migrate the unique metabolic capacity of this organism for the production of C30+ isoprene hydrocarbons to other algae and higher plants. This work compliments our ongoing photobioreactor...

Batch-to-batch variations in protein expression levels pose an intractable and often unreported technical hurdle in the development of rapid plant-based heterologous protein expression platforms. Our group is developing protein expression in non-transgenic plant tissue culture which is grown and then co-cultured with an Agrobacterium tumefaciens au...

The elicitation of Hyoscyamus muticus root and cell suspension cultures by fungal elicitor from Rhizoctonia solani causes dramatic changes in respiration, nutrient yields, and growth. Cells and mature root tissues have similar specific oxygen uptake rates (SOUR) before and after the onset of the elicitation process. Cell suspension SOUR were 11 and...

The reporter gene beta-glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through co-culture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to tran...

Mechanical forces generated by prosthetic heart devices (artificial valves, artificial hearts, ventricular assist devices) have been known to cause damage and destruction of erythrocytes. Turbulent flow within such devices generates shear stresses and can induce cell damage. Current models of cell damage rate utilize only the power input per unit m...

A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector....

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 wil...

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...

In this Letter, we report a straightforward method to produce multiwalled carbon nanotubes using magnetic nanoparticles of Magnetospirillium magnetotacticum as catalysts. Carbon nanotube growth on these nanoparticles resulted in multiwalled carbon nanotubes of an average diameter of 13 nm showing a narrow distribution in diameter. The magnetic char...

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate...

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nut...

Transgenic "hairy" roots of Hyoscyamus muticus, immobilized within an ebb-and-flow bioreactor (EFBR), were cultured for a period of 18 d. The growth results showed that a 2.5-L EFBR, with a productivity of 0.481 g DW/L-d and an average specific growth rate (μ) of 0.211/d, duplicated the growth performance of the standard 50-mL shake-flask control,...

The ebb-and-flow bioreactor (EFBR) is characterized by the repetitive ebbing and flowing or periodic rising and falling of its liquid medium. An experimental 2.5-L bench-top EFBR prototype was fabricated for growing immobilized Hyoscyamus muticus "hairy root" cultures for the production of secondary metabolites. Ideal mathematical models were formu...

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column...

Plant cells can be grown in an undifferentiated state as cell suspensions, or as differentiated tissues including transformed root culture. This paper summarizes the highlights of our efforts over the past 10 years in the design and operation of these reactor systems. The following assertions are presented: 1) Plant cell suspensions can be grown in...

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 o...

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture....

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 mu M pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. Th...

When treated with nitroblue tetrazolium the hairy root cultures of Beta vulgaris (golden and red varieties), Brassica juncea, Cosmos sulphureus, Hyoscyamus muticus, Ocimum basilicum and Nicotiana tabaccum in Gamborg's liquid medium showed rapid and intense staining at the apical meristem and lateral root initials; this is indicative of elevated res...

This paper presents a technique for accurate estimation of growth in root culture systems. Biomass correlations, were used to estimate fresh weight time course data in shake flasks and reactors based on a model of liquid nutrient uptake and osmolality, to account for changing specific water content of roots. This mass balance technique has been dev...

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. The roots were incubated on callus-inducing medium to generate transformed cells. Transformants were selected on medium contain...

Cell suspensions inoculated at low cell concentrations displayed a typical growth reduction, whereas root cultures displayed an improvement in growth. Specific growth rate ofHyoscyamus muticus cell suspensions decreased from 0.25 to 0.12 d−1 as inoculum concentration was reduced from 4.0 to 0.02 g fresh weight per liter. In contrast, roots show an...

Growth of plant cell cultures is demonstrated in an uncontrolled, simple, and inexpensive plastic-lined vessel. Sustained specific growth rates of 0.22 day−1 for Hyoscyamus muticus cell suspension cultures are achieved in a low-cost gas-sparged bioreactor configuration (6.5 L working volume, wv) which is comparable to an “optimized” 5 L wv mechanic...

Except for very high value food additives, even the ‘best case’ scenario of highly productive, fast growing cultures, is not likely to be economically feasible in existing technology. The problems of feasibility are not generally due to inherent engineering scale-up problems, or inadequate cellular productivities. There are many examples where reas...

Design equations for oxygen transfer rates were developed to examine the influence of poor mixing in large-scale reactors for root culture. The experimentally observed condition of plug flow of the gas and axially stratified liquid was compared to the typical bioreactor design case of a well mixed gas and liquid phase. In addition, the effect of hy...

Great potential exists in the use of Plant Cell and Tissue Culture (PCTC) technology for the production of food ingredients. Tremendous progress has been made in recent years in the basic understanding of plant metabolic pathways and regulatory mechanisms, in the developments of processes for increasing yields and in the design and operation of bio...

Commercial development of cultured-derived food ingredients has attracted interna­ tional interest. As consumers have become more health conscious in recent years, the de­ mand for natural food ingredients and disease-preventative phytochemicals has increased tremendously. Plant Cell and Tissue Culture provides an alternative method for controlled...

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...