Rhodococcus and Yarrowia-Based Lipid Production Using Lignin-Containing Industrial Residues.

Improvement in biorefining technologies coupled with development of novel fermentation strategies and analysis will be paramount in establishing supplementary and sustainable biofuel pathways. Oleaginous microorganisms that are capable of accumulating triacylglycerides (TAGs) and fatty acid methyl esters (FAMEs), such as Rhodococcus and Yarrowia species, can be used to produce second-generation biofuels from non-food competing carbon sources. These "microbiorefineries" provide a pathway to upgrade agricultural and industrial waste streams to fungible fuels or precursors to chemicals and materials. Here we provide a general overview on cultivating Rhodococcus and Yarrowia on agro-waste/industrial biomass pretreatment waste streams to produce single-cell oils/lipids and preparing samples for FAME detection.

Production of single cell protein from agro-waste using Rhodococcus opacus.

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.

Diagnosing the Neural Circuitry of Reading.

We summarize the current state of knowledge of the brain's reading circuits, and then we describe opportunities to use quantitative and reproducible methods for diagnosing these circuits. Neural circuit diagnostics-by which we mean identifying the locations and responses in an individual that differ significantly from measurements in good readers-can help parents and educators select the best remediation strategy. A sustained effort to develop and share diagnostic methods can support the societal goal of improving literacy.

Utilization of simultaneous saccharification and fermentation residues as feedstock for lipid accumulation in Rhodococcus opacus.

Use of oleaginous microorganisms as "micro-factories" for accumulation of single cell oils for biofuel production has increased significantly to mitigate growing energy demands, resulting in efforts to upgrade industrial waste, such as second-generation lignocellulosic residues, into potential feedstocks. Dilute-acid pretreatment (DAP) is commonly used to alter the physicochemical properties of lignocellulosic materials and is typically coupled with simultaneous saccharification and fermentation (SSF) for conversion of sugars into ethanol. The resulting DAP residues are usually processed as a waste stream, e.g. burned for power, but this provides minimal value. Alternatively, these wastes can be utilized as feedstock to generate lipids, which can be converted to biofuel. DAP-SSF residues were generated from pine, poplar, and switchgrass. High performance liquid chromatography revealed less than 0.13% monomeric sugars in the dry residue. Fourier transform infrared spectroscopy was indicative of the presence of lignin and polysaccharides. Gel permeation chromatography suggested the bacterial strains preferred molecules with molecular weight ~ 400-500 g/mol. DAP-SSF residues were used as the sole carbon source for lipid production by Rhodococcus opacus DSM 1069 and PD630 in batch fermentations. Depending on the strain of Rhodococcus employed, 9-11 lipids for PD630 and DSM 1069 were observed, at a final concentration of ~ 15 mg/L fatty acid methyl esters (FAME) detected. Though the DAP-SSF substrate resulted in low FAME titers, novel analysis of solid-state fermentations was investigated, which determined that DAP-SSF residues could be a viable feedstock for lipid generation.

Sortase-mediated ligation of PsaE-modified photosystem I from Synechocystis sp. PCC 6803 to a conductive surface for enhanced photocurrent production on a gold electrode.

Sortase-mediated ligation was used to attach the photosystem I (PSI) complex from Synechocystis sp. PCC 6803 in a preferential orientation to enhance photoinduced electron flow to a conductive gold surface. Ideally, this method can result in a uniform monolayer of protein, covalently bound unidirectionally to the electrode surface. The exposed C-termini of the psaE subunits of the PSI trimer were targeted to contain an LPETG-sortase recognition sequence to increase noncompeting electron transfer by uniformly orienting the PSI stromal side proximal to the surface. Surface characterization with atomic force microscopy suggested that monolayer formation and optimal surface coverage occurred when the gold surfaces were incubated with peptide at 100 to 500 µM concentrations. When photochronoamperometry with potassium ferrocyanide and ferricyanide as redox mediators was used, photocurrents in the range of 100 to 200 nA/cm(2) were produced, which is an improvement over other attachment techniques for photosystem monolayers that produce approximately 100 nA/cm(2) or less. This work demonstrated that sortase-mediated ligation aided in the control of PSI orientation on modified gold surfaces with a distribution of 94% stromal side proximal and 6% lumenal side proximal to the surface for current-producing PSI.

Analysis of the solution structure of Thermosynechococcus elongatus photosystem I in n-dodecyl-ß-D-maltoside using small-angle neutron scattering and molecular dynamics simulation.

Small-angle neutron scattering (SANS) and molecular dynamics (MD) simulation were used to investigate the structure of trimeric photosystem I (PSI) from Thermosynechococcus elongatus (T. elongatus) stabilized in n-dodecyl-ß-d-maltoside (DDM) detergent solution. Scattering curves of detergent and protein-detergent complexes were measured at 18% D2O, the contrast match point for the detergent, and 100% D2O, allowing observation of the structures of protein/detergent complexes. It was determined that the maximum dimension of the PSI-DDM complex was consistent with the presence of a monolayer belt of detergent around the periphery of PSI. A dummy-atom reconstruction of the shape of the complex from the SANS data indicates that the detergent envelope has an irregular shape around the hydrophobic periphery of the PSI trimer rather than a uniform, toroidal belt around the complex. A 50 ns MD simulation model (a DDM ring surrounding the PSI complex with extra interstitial DDM) of the PSI-DDM complex was developed for comparison with the SANS data. The results suggest that DDM undergoes additional structuring around the membrane-spanning surface of the complex instead of a simple, relatively uniform belt, as is generally assumed for studies that use detergents to solubilize membrane proteins.

Adsorption and molecular rearrangement of amphoteric species at oil-water interfaces.

The formation of stable water-in-petroleum emulsions is a costly challenge when transporting, processing, and refining heavy crude oil and bitumen. The stability of these emulsions is attributed to interfacial films with well-documented viscoelastic properties that are known to vary with concentration, solvent quality, and asphaltene chemistry. In this study, we explore the impact of aqueous phase pH and salinity on the transient interfacial rheological properties of asphaltenic films. Using two chemically unique asphaltenes, interfacial shear rheology revealed an apparent salt-induced retardation of the interfacial consolidation processes that ultimately engender elasticity to the film. For Hondo asphaltenes at pH 7, a linear dependence of this retardation on the Debye parameter (kappa) suggested that shielding of electrostatic attraction was responsible. Further investigation with dynamic oscillating drop tensiometry at pH 3, 7, and 10 illustrated that intralayer repulsive and attractive electrostatic interactions can significantly influence the evolution of the interfacial structure. More specifically, the transient tension and dilatational modulus profiles indicated several interfacial processes were affected by the addition of salt, including (i) interfacial activity and the extent of adsorption, (ii) interfacial rearrangement and consolidation, and (iii) interfacial transport or displacement or both. Furthermore, the observed asphaltene interfacial behavior was consistent with those published for interfacial structure-forming amphoteric proteins, such as lysozyme and beta-casein.