Rhodococcus Opacus Strain PD630 Research Articles

Bio-oil production by hydrothermal liquefaction of Rhodococcus opacus biomass utilizing refinery wastewater: Biomass valorization and process optimization

In this research, a pure culture of Rhodococcus opacus strain PD630 was used to treat wastewater generated from a refinery and the lipid rich bacterial biomass obtained was converted to bio-oil. A very high removal (70 ± 5%) of organics present in the wastewater was achieved at pH 7 within 96 h along with lipid accumulation of 50 ± 4% w ∕ w by the bacteria. The lipid containing biomass procured by using the wastewater as the sole substrate was converted into bio-oil by hydrothermal liquefaction (HTL) technique. Different operational parameters viz . temperature, time and biomass/water ratio were optimized employing Response Surface Methodology (RSM) to achieve maximum bio-oil yield. The optimum conditions for maximum bio-oil yield (25.53%) were found to be 215 °C, 125 min and 0.25 biomass/water ratio; the bio-oil had a high heating value (HHV) of 20.73MJ/Kg, and it also contained a very low amount of water soluble products in it. GC–MS analysis of the obtained bio-oil product revealed the presence of a variety of compounds, mainly aldehydes, ketones and fatty acids, whereas the water soluble product contained mainly alcohols and phenolic compounds. Further analysis of the aqueous phase obtained as a byproduct concluded its potential for reuse owing to the presence of nutritional compounds. FTIR spectra of bio-oil revealed the presence of C–H bonds due to alkanes as the predominant functional group present in the product. FAME analysis results and other properties of the transesterified bio-oil matched well with the ASTM standard thus indicating its potential for bio-fuel application. This experimental study thus demonstrated that the lipid rich R. opacus biomass grown on cheaply available refinery wastewater is highly suited for potential bio-oil production. • High organic removal from refinery wastewater was achieved by R. opacus . • The lipid-rich bacterial biomass was converted to bio-oil. • Optimization of process parameters increased the bio-oil yield up to 25%. • The bio-oil showed excellent potential as a bio-fuel. • Water-soluble by-product of bio-oil production showed reuse potential.

Automation Highlights from the Literature

Automation Highlights from the Literature

Integrated omics study delineates the dynamics of lipid droplets in Rhodococcus opacus PD630

Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.

Optimization of macroelement concentrations, pH and osmolarity for triacylglycerol accumulation in Rhodococcus opacus strain PD630.

The refinement of biodiesel or renewable diesel from bacterial lipids has a great potential to make a contribution for energy production in the future. This study provides new data concerning suitable nutrient concentrations for cultivation of the Gram-positive Rhodococcus opacus PD630, which is able to accumulate large amounts of lipids during nitrogen limitation. Enhanced concentrations of magnesium have been shown to increase the final optical density and the lipid content of the cells. Elevated phosphate concentrations slowed down the onset of the accumulation phase, without a clear effect on the final optical density and the cell’s lipid content. A robust growth of R. opacus was possible in the presence of ammonium concentrations of up to 1.4 g l-1 and sucrose concentrations of up to 240 g l-1, with an optimum regarding growth and lipid storage observed in the range of 0.2 to 0.4 g l-1 ammonium and 20 to 40 g l-1 sucrose, respectively. Moreover, R. opacus showed tolerance to high salt concentrations.

Neutral lipid production in Alcanivorax borkumensis SK2 and other marine hydrocarbonoclastic bacteria

AbstractTriacylglycerols (TAG) and wax esters (WE) constitute together with polyhydroxyalkanoates (PHA) the major storage lipophilic compounds in prokaryotes. Recently, the production of neutral lipids such as TAG and WE has been reported in species of the genus Alcanivorax, which belongs to the group of obligate hydrocarbonoclastic bacteria (OHCB). We analyzed the production of such neutral lipids by different marine hydrocarbonoclastic bacteria growing on pyruvate or hexadecane as sole carbon source, and compared it to other bacteria such as Rhodococcus opacus strain PD630 and Acinetobacter baylyi strain ADP1, which are two well‐studied strains for production of neutral lipids. Alcanivorax borkumensis SK2 synthesized mainly TAG when cells are cultivated on pyruvate, while biosynthesis and accumulation of WE was mainly observed in cells growing on hexadecane. Alcanivorax jadensis T9 synthesized and accumulated mainly WE if cells were cultivated with hexadecane, while both TAG and WE were observed if cells were cultivated with pyruvate as sole carbon source, respectively. Predominantly production as well as export of WE was observed in Marinobacter hydrocarbonoclasticus SP17 growing on pyruvate or hexadecane as sole carbon source. The chemical structures of TAG and WE produced by A. borkumensis SK2 were analyzed by gas chromatography and/or mass spectrometry, and first studies to investigate the influence of different C/N ratio (7, 50 or 150) on the production of neutral lipids were performed.

The Rhodococcus opacus PD630 heparin-binding hemagglutinin homolog TadA mediates lipid body formation.

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.

Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress

Rhodococcus opacus PD630 was investigated for physiological and morphological changes under water stress challenge. Gluconate- and hexadecane-grown cells were extremely resistant to these conditions, and survival accounted for up to 300 and 400 days; respectively, when they were subjected to slow air-drying. Results of this study suggest that strain PD630 has specific mechanisms to withstand water stress. Water-stressed cells were sensitive to the application of ethanol, high temperatures and oxidative stress, whereas they exhibited cross-protection solely against osmotic stress during the first hours of application. Results indicate that the resistance programme for water stress in R. opacus PD630 includes the following physiological and morphological changes, among others: (1) energetic adjustments with drastic reduction of the metabolic activity (≈39% decrease during the first 24 h and about 90% after 190 days under dehydration), (2) endogenous metabolism using intracellular triacylglycerols for generating energy and precursors, (3) biosynthesis of different osmolytes such as trehalose, ectoine and hydroxyectoine, which may achieve a water balance through osmotic adjustment and may explain the overlap between water and osmotic stress, (4) adjustments of the cell-wall through the turnover of mycolic acid species, as preliminary experiments revealed no evident changes in the thickness of the cell envelope, (5) formation of short fragmenting-cells as probable resistance forms, (6) production of an extracellular slime covering the surface of colonies, which probably regulates internal and external changes in water potential, and (7) formation of compact masses of cells. This contributes to understanding the water stress resistance processes in the soil bacterium R. opacus PD630.

High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale.

The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-1 scale in a stirred tank bioreactor at 30 degrees C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-1 pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.

In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630.

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the beta-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation.