Proteomic and metabolomic analysis of the cellular biomarkers related to inhibitors tolerance in Zymomonas mobilis ZM4.

BACKGROUND: Toxic compounds present in both the hydrolysate and pyrolysate of lignocellulosic biomass severely hinder the further conversion of lignocellulose-derived fermentable sugars into useful chemicals by common biocatalysts like Zymomonas mobilis, which has remarkable advantages over yeast. Although the extra detoxification treatment prior to fermentation process can help biocatalysts to eliminate the inhibitory environment, it is not environment friendly and cost effective for industrial application. As also reported by previous studies, an ideal and holistic approach to solve this issue is to develop microbial strains with inhibitor tolerance. However, previously engineered strains had the limitation that they could not cope well with the synergistic effect of multiple inhibitors as they are resistant only to a single inhibitor. Hence, understanding the universal cellular responses of Z. mobilis to various inhibitors may guide the designing of rational strategies to obtain more robust engineered strains for biofuel production from lignocellulosic biomass. RESULTS: Quantitative proteomics and metabolomics approaches were used to determine the cellular responses of Z. mobilis ZM4 to representative biomass-derived inhibitors like formic acid, acetic acid, furfural, 5-hydroxymethylfurfural, and phenol. The differentially expressed proteins identified under the challenge of single and combined inhibitors were involved in cell wall/membrane biogenesis, energy production, DNA replication, DNA recombination, DNA repair, DNA transcription, RNA translation, posttranslational modification, biosynthesis of amino acids, central carbon metabolism, etc. Metabolomics analysis showed that the up- or down-regulation pattern of metabolites was changed consistently with that of relevant proteins. CONCLUSION: Fifteen up-regulated proteins (e.g., Isopropylmalate isomerase LeuC, transcription-repair-coupling factor Mfd, and phosphoglucose isomerase PGI) and thirteen down-regulated proteins (e.g., TonB-dependent transporter ZMO1522, transcription termination factor Rho, and S1/P1 nuclease ZMO0127) were identified as candidate proteins related to all the stress conditions, implying that these proteins are potential biomarkers for the improvement of Z. mobilis ZM4 to resist complex biomass-derived inhibitors. These data can be used to generate a database of inhibitor-tolerance biomarkers, which could provide a basis for engineering Z. mobilis that would be able to grow in the presence of multiple inhibitors and directly ferment the biomass-derived sugars into biofuels.

A review on microbial lipids as a potential biofuel.

Energy security, environmental concerns, and unstable oil prices have been the driving trifecta of demand for alternative fuels in the United States. The United States' dependence on energy resources, often from unstable oil-producing countries has created political insecurities and concerns. As we try to gain energy security, unconventional oil becomes more common, flooding the market, and causing the major downshift of the usual unstable oil prices. Meanwhile, consumption of fossil fuels and the consequent CO2 emissions have driven disruptions in the Earth's atmosphere and are recognized to be responsible for global climate change. While the significance of each of these three factors may fluctuate with global politics or new technologies, transportation energy will remain the prominent focus of multi-disciplined research. Bioenergy future depends on the price of oil. Current energy policy of the United States heavily favors petroleum industry. In this review, the current trend in microbial lipids as a potential biofuel is discussed.

Lipid accumulation by Rhodococcus rhodochrous grown on glucose.

Biodiesel is an alternative fuel made from costly vegetable oil feedstocks. Some microorganisms can accumulate lipids when nutrients are limited and carbon is in excess. Rhodococcus rhodochrous is a gram-positive bacterium most often used in bioremediation or acrylamide production. The purpose of this study was to investigate and characterize the lipid accumulation capabilities of R. rhodochrous. Shake flasks and a large-scale fermentation were used to cultivate R. rhodochrous in varying concentrations of glucose. R. rhodochrous achieved almost 50 % of dry cell mass as lipid when grown in 20 g/L of glucose. Wax esters and triglycerides were identified in R. rhodochrous lipid extract. The transesterified extractables of R. rhodochrous consisted of mostly palmitic (35 %) and oleic (42 %) acid methyl esters. This study shows R. rhodochrous to be an oleaginous bacterium with potential for application in alternative fuels.

Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198.

Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

Lipid-enhancement of activated sludges obtained from conventional activated sludge and oxidation ditch processes.

Lipid-enhancement of activated sludges was conducted to increase the amount of saponifiable lipids in the sludges. The sludges were obtained from a conventional activated sludge (CAS) and an oxidation ditch process (ODP). Results showed 59-222% and 150-250% increase in saponifiable lipid content of the sludges from CAS and ODP, respectively. The fatty acid methyl ester (FAMEs) obtained from triacylglycerides was 57-67% (of total FAMEs) for enhanced CAS and 55-73% for enhanced ODP, a very significant improvement from 6% to 10% (CAS) and 4% to 8% (ODP). Regardless of the source, the enhancement resulted in sludges with similar fatty acid profile indicating homogenization of the lipids in the sludges. This study provides a potential strategy to utilize existing wastewater treatment facilities as source of significant amount of lipids for biofuel applications.

The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis.

Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols (TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAG are broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inexpensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of different growth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24h on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids were extracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show a difference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone had the highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can be cultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as secondary or primary carbon substrate.

Development of a heterogeneous catalytic cracking reactor utilizing online mass spectrometry analysis.

A laboratory system has been designed, constructed, and validated that reduces the complexity, time required, and data variability associated with catalytic microreactors that require post reaction steps prior to product analysis. In this work, a Varian (Walnut Creek, CA, USA) 3600 GC (gas chromatography) system coupled with a Saturn quadrupole ion trap mass spectrometer was used to perform mass spectral analysis in real-time catalytic cracking reactions. As this was an integrated reactor/analyzer, the GC column was exposed to temperatures beyond the degradation point of the column, and so selective ion storage RF waveform was used to remove the siloxane masses from the spectra. This produced lower detection limits and full scan data for identification. Mass/charge segmentation of the mass spectrometer allowed the complete product identification for electron impact spectra. Hexane was reacted over H-ZSM-5 catalyst for instrument validation. This produced a series of alkanes, alkenes, and aromatics with distributions consistent with that reported for the cracking of hexane.

Isopropanol and acetone induces vinyl chloride degradation in Rhodococcus rhodochrous.

In situ bioremediation of vinyl chloride (VC)-contaminated waste sites requires a microorganism capable of degrading VC. While propane will induce an oxygenase to accomplish this goal, its use as a primary substrate in bioremediation is complicated by its flammability and low water solubility. This study demonstrates that two degradation products of propane, isoproponal and acetone, can induce the enzymes in Rhodococcus rhodochrous that degrade VC. Additionally, a reasonable number of cells for bioremediation can be grown on conventional solid bacteriological media (nutrient agar, tryptic soy agar, plate count agar) in an average microbiological laboratory and then induced to produce the necessary enzymes by incubation of a resting cell suspension with isopropanol or acetone. Since acetone is more volatile than isopropanol and has other undesirable characteristics, isopropanol is the inducer of choice. It offers a non-toxic, water-soluble, relatively inexpensive alternative to propane for in situ bioremediation of waste sites contaminated with VC.

Effects of n-hexadecane and PM-100 clay on trichloroethylene degradation by Burkholderia cepacia.

Trichloroethylene (TCE) is a non-flammable, volatile organochlorine compound which was a widely used degreasing agent, anesthetic, and coolant prior to 1960, but has since been placed on the Environmental Protection Agency's (EPA) list of priority pollutants. The inadequate disposal practices for TCE have created numerous TCE-contaminated superfund sites. The most commonly employed practice for remediating TCE-contaminated sites is to purge the contaminant from the source and trap it onto an adsorbent which is disposed of in a landfill or by incineration. This investigation was undertaken to evaluate the effectiveness of Burkholderia cepacia strain G4 (G4) to regenerate used sorbents by degrading TCE from the sorbent directly or indirectly. The results of this investigation showed that G4 was capable of reducing TCE attached to PM-100 clay but at significantly reduced rate due to the slow desorption rate. Conversely, it was shown that G4 was capable of degrading TCE dissolved in n-hexadecane at the same rate as systems without n-hexadecane present. The reduction in TCE degradation when the TCE is attached to the PM-100 clay could be overcome by solvent rinsing the TCE from the clay with subsequent removal of the TCE from the n-hexadecane by G4.