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.

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N-Nitroglycine by Variovorax sp. Strain JS1663.

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (nnlA) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed ß-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives.IMPORTANCE The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N-Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the isolation of a Gram-negative soil bacterium, Variovorax sp. strain JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a ß-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help identify other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives.

Selection for growth on 3-nitrotoluene by 2-nitrotoluene-utilizing Acidovorax sp. strain JS42 identifies nitroarene dioxygenases with altered specificities.

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

Circadian regulation of chloroplast transcription in Chlamydomonas is accompanied by little or no fluctuation in RPOD levels or core RNAP activity.

In Chlamydomonas growing under 24 h light-dark cycles, chloroplast transcription is under circadian clock control, and peaks early in the morning. The peak (but not trough) requires ongoing cytoplasmic translation, as it is sensitive to cycloheximide (CH). The chloroplast transcriptional apparatus in Chlamydomonas is simpler than in land plants, with only one type of RNA polymerase (RNAP, bacterial) and apparently only one sigma factor (RPOD). Core RNAP can be assayed in vitro with a non-sigma factor dependent template, and is sensitive to rifampicin. We developed a membrane-based assay for RNAP activity, and used it to determine that core activity is only weakly affected by pre-treating cells with CH. Moreover, core chloroplast RNAP activity was steady during a 24 h light-dark cycle. Levels of the sigma factor (RPOD) were examined using western blots, and found to fluctuate less than 25 % during light-dark cycles. These data indicate that circadian regulation of chloroplast transcription is distinct from regulation by sulfur availability, which involves significant changes in RPOD levels. The implications of this data for hypotheses that purport to explain the circadian control mechanism are discussed.

Annotated Genome Sequence of the High-Biomass-Producing Yellow-Green Alga Tribonema minus.

Here, we report the annotated genome sequence for a heterokont alga from the class Xanthophyceae. This high-biomass-producing strain, Tribonema minus UTEX B 3156, was isolated from a wastewater treatment plant in California. It is stable in outdoor raceway ponds and is a promising industrial feedstock for biofuels and bioproducts.

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.

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.

Draft Genome Sequence of Streptomyces vitaminophilus ATCC 31673, a Producer of Pyrrolomycin Antibiotics, Some of Which Contain a Nitro Group.

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. The 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content.