Search

Effective utilization of waste paper as a substrate for biodiesel production has environmental and economic advantages. To study the feasibility of using waste office automation paper to produce microbial lipids, waste paper hydrolysate (WPH) was prepared by enzymatic hydrolysis. WPH was fed to Rhodococcus opacus PD630 for microbial lipid production. Among the nitrogen sources tested, the medium with NH4Cl yielded significantly high biomass and lipid of 4.92 and 1.29 g/L, respectively. When the ratio of C/N increases from 10 to 80, the lipid accumulation increases and the lipid content also increases from 18.92 to 43.41%. Under optimized conditions, the maximum biomass, lipid yield, and lipid content obtained were 6.51 g/L, 2.8 g/L, and 43% respectively. The extracted lipids exhibited similarities to those of vegetable oils. The carbon chain length of accumulated fatty acids ranged from C10 to C21, mainly oleic acid (31%). Moreover, biodiesel properties based on lipid profile yielded cetane number (59.88), iodine value (42.29 mg I2/g), high heating value (44.26 MJ/kg), and density (0.9 g/cm3) were in the permissible limits of international standards. The present study gives a sustainable biorefinery approach for the production of biodiesel from waste office paper hydrolysate. [ABSTRACT FROM AUTHOR]

The study was to investigate the structure and function of LPD06283 protein of Rhodococcus opacus PD630. Bioinformatics methods were used to analyze the physicochemical properties, hydrophilicity and hydrophobicity, amphiphilic α helix, homology, signal peptide, transmembrane region, secondary structure, domain, tertiary structure, phosphorylation modification sites and interacting proteins of LPD06283 protein. The results showed that LPD06283 protein was a stable acidic hydrophilic protein, the N-terminal hydrophobic region of which formed an amphiphilic α-helix, closely related to MLDS protein of Rhodococcus jostii RHA1, and contained no signal peptide or transmembrane region. The secondary structure of LPD06283 protein was composed of α-helix (81.88%), β-turn (3.62%) and random coil (14.49%), there was an apolipoprotein domain at N-terminus, the tertiary structure was predicted by comprehensive method and was of high quality; LPD06283 protein contained 19 phosphorylation sites, the strongest interacting protein was XRE family DNA-binding protein. The study indicates that LPD06283 protein may be involved in lipid metabolism of Rhodococcus opacus, which can be used as a potential target for microbial lipid research and development. [ABSTRACT FROM AUTHOR]

Abstract The current extraction and use of fossil fuels has been linked to extensive negative health and environmental outcomes. Lignocellulosic biomass-derived biofuels and bioproducts are being actively considered as renewable alternatives to the fuels, chemicals, and materials produced from fossil fuels. A major challenge limiting large-scale, economic deployment of second-generation biorefineries is the insufficient product yield, diversity, and value that current conversion technologies can extract from lignocellulose, in particular from the underutilized lignin fraction. Rhodococcus opacus PD630 is an oleaginous gram-positive bacterium with innate catabolic pathways and tolerance mechanisms for the inhibitory aromatic compounds found in depolymerized lignin, as well as native or engineered pathways for hexose and pentose sugars found in the carbohydrate fractions of biomass. As a result, R. opacus holds potential as a biological chassis for the conversion of lignocellulosic biomass into biodiesel precursors and other value-added products. This review begins by examining the important role that lignin utilization will play in the future of biorefineries and by providing a concise survey of the current lignin conversion technologies. The genetic machinery and capabilities of R. opacus that allow the bacterium to tolerate and metabolize aromatic compounds and depolymerized lignin are also discussed, along with a synopsis of the genetic toolbox and synthetic biology methods now available for engineering this organism. Finally, we summarize the different feedstocks that R. opacus has been demonstrated to consume, and the high-value products that it has been shown to produce. Engineered R. opacus will enable lignin valorization over the coming years, leading to cost-effective conversion of lignocellulose into fuels, chemicals, and materials.

Abstract Background Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered Rhodococcus strains (e.g., R. opacus PD630, R. jostii RHA1, and R. jostii RHA1 VanA−) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin. Results In this study, a co-fermentation module of natural and engineered Rhodococcus strains with significant improved lignin degradation and/or lipid biosynthesis capacities was established, which enabled simultaneous conversion of glucose, lignin, and its derivatives into lipids. Although Rhodococci sp. showed preference to glucose over lignin, nearly half of the lignin was quickly depolymerized to monomers by these strains for cell growth and lipid synthesis after glucose was nearly consumed up. Profiles of metabolites produced by Rhodococcus strains growing on different carbon sources (e.g., glucose, alkali lignin, and dilute acid flowthrough-pretreated poplar wood slurry) confirmed lignin conversion during co-fermentation, and indicated novel metabolic capacities and unexplored metabolic pathways in these organisms. Proteome profiles suggested that lignin depolymerization by Rhodococci sp. involved multiple peroxidases with accessory oxidases. Besides the β-ketoadipate pathway, the phenylacetic acid (PAA) pathway was another potential route for the in vivo ring cleavage activity. In addition, deficiency of reducing power and cellular oxidative stress probably led to lower lipid production using lignin as the sole carbon source than that using glucose. Conclusions This work demonstrated a potential strategy for efficient bioconversion of both lignin and glucose into lipids by co-culture of multiple natural and engineered Rhodococcus strains. In addition, the involvement of PAA pathway in lignin degradation can help to further improve lignin utilization, and the combinatory proteomics and bioinformatics strategies used in this study can also be applied into other systems to reveal the metabolic and regulatory pathways for balanced cellular metabolism and to select genetic targets for efficient conversion of both lignin and carbohydrates into biofuels.

Abstract Background Lignin valorization has recently been considered to be an essential process for sustainable and cost-effective biorefineries. Lignin represents a potential new feedstock for value-added products. Oleaginous bacteria such as Rhodococcus opacus can produce intracellular lipids from biodegradation of aromatic substrates. These lipids can be used for biofuel production, which can potentially replace petroleum-derived chemicals. However, the low reactivity of lignin produced from pretreatment and the underdeveloped fermentation technology hindered lignin bioconversion to lipids. In this study, combinatorial pretreatment with an optimized fermentation strategy was evaluated to improve lignin valorization into lipids using R. opacus PD630. Results As opposed to single pretreatment, combinatorial pretreatment produced a 12.8–75.6% higher lipid concentration in fermentation using lignin as the carbon source. Gas chromatography–mass spectrometry analysis showed that combinatorial pretreatment released more aromatic monomers, which could be more readily utilized by lignin-degrading strains. Three detoxification strategies were used to remove potential inhibitors produced from pretreatment. After heating detoxification of the lignin stream, the lipid concentration further increased by 2.9–9.7%. Different fermentation strategies were evaluated in scale-up lipid fermentation using a 2.0-l fermenter. With laccase treatment of the lignin stream produced from combinatorial pretreatment, the highest cell dry weight and lipid concentration were 10.1 and 1.83 g/l, respectively, in fed-batch fermentation, with a total soluble substrate concentration of 40 g/l. The improvement of the lipid fermentation performance may have resulted from lignin depolymerization by the combinatorial pretreatment and laccase treatment, reduced inhibition effects by fed-batch fermentation, adequate oxygen supply, and an accurate pH control in the fermenter. Conclusions Overall, these results demonstrate that combinatorial pretreatment, together with fermentation optimization, favorably improves lipid production using lignin as the carbon source. Combinatorial pretreatment integrated with fed-batch fermentation was an effective strategy to improve the bioconversion of lignin into lipids, thus facilitating lignin valorization in biorefineries.

Highlights • Lipid-rich biomass of Rhodococcus opacus could be grown on molasses. • Inhibition by molasses was overcome to displace expensive pure C-substrates. • Lipid content of microbial biomass exceeded 35% w/w after 170 h of culture. • Osmotic stress and other factors enhanced the lipid content in the biomass. Abstract Rhodococcus opacus PD630 was used to produce biomass and lipids in molasses-based media with and without osmotic stress. In a 7-day aerobic batch culture at 30 °C, the biomass and lipid concentrations were maximized using an initial molasses concentration of 80 g/L and ammonium acetate (nitrogen source) concentration of 2.14 g/L. At a fixed initial molasses concentration of 80 g/L, the concentration of the nitrogen source was further fine-tuned to 2.25 g/L, to maximize the lipid content of the biomass to around 30% by dry mass. This medium was used to test the effects of stressing salts (sodium acetate, magnesium sulfate, sodium chloride) on production of lipids and biomass. A two-step bolus feeding with magnesium sulfate and sodium acetate, enhanced the final biomass concentration to around 19 g/L (a 50% increase relative to control), but the lipid content in the biomass was reduced to around 16% w/w. A 33% enhancement in lipid concentration relative to control, was achieved by feeding magnesium sulfate and sodium acetate. Sugarcane molasses could be effectively used to produce biomass and lipids instead of using the much more expensive pure carbon sources such as glucose and sucrose. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Low‐value waste organics can be upgraded to higher‐value products using microorganisms. The bacterium Rhodococcus opacus PD630 was used to produce colorants (carotenoids) and lipids (potential fuel oils) from glycerol. RESULTS: Submerged culture of R. opacus PD630 produced carotenoids and lipids on glycerol as the primary carbon source, although supplementation with glucose was necessary. A 1:1 mixture (50 g L‐1 each) of glucose and glycerol in a batch culture afforded a final biomass concentration of nearly 9.0 g L‐1, a carotenoids concentration of 0.75 mg L‐1 and a lipids concentration of nearly 2.2 g L‐1. Compared with using only glycerol (100 g L‐1) in the medium, the final biomass concentration was nearly 4‐fold greater, the carotenoids concentration was 7.5‐fold greater and the lipids concentration was 8.6‐fold greater when a 1:1 mixture of glucose and glycerol was used. A combination of glucose (70 g L‐1), glycerol (30 g L‐1) and ammonium acetate (4.32 g L‐1) further increased the final biomass concentration to nearly 10.2 g L‐1. With this formulation, the carotenoids concentration rose to 0.99 mg L‐1 and lipids concentration rose to nearly 2.4 g L‐1 in batch culture. A repeated fed‐batch operation with an optimal feeding regimen raised the final biomass concentration to 12.1 g L‐1. The final concentration of carotenoids was 0.7 mg L‐1 and the final lipids concentration was nearly 4.9 g L‐1. CONCLUSION: Glycerol can partly displace the more expensive glucose in production of carotenoids and lipids from R. opacus PD630. Biomass with ∼40% lipids (dry basis) can be produced. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background: Lignin valorization has recently been considered to be an essential process for sustainable and costeffective biorefineries. Lignin represents a potential new feedstock for value-added products. Oleaginous bacteria such as Rhodococcus opacus can produce intracellular lipids from biodegradation of aromatic substrates. These lipids can be used for biofuel production, which can potentially replace petroleum-derived chemicals. However, the low reactivity of lignin produced from pretreatment and the underdeveloped fermentation technology hindered lignin bioconversion to lipids. In this study, combinatorial pretreatment with an optimized fermentation strategy was evaluated to improve lignin valorization into lipids using R. opacus PD630. Results: As opposed to single pretreatment, combinatorial pretreatment produced a 12.8-75.6% higher lipid concentration in fermentation using lignin as the carbon source. Gas chromatography-mass spectrometry analysis showed that combinatorial pretreatment released more aromatic monomers, which could be more readily utilized by lignin-degrading strains. Three detoxification strategies were used to remove potential inhibitors produced from pretreatment. After heating detoxification of the lignin stream, the lipid concentration further increased by 2.9-9.7%. Different fermentation strategies were evaluated in scale-up lipid fermentation using a 2.0-l fermenter. With laccase treatment of the lignin stream produced from combinatorial pretreatment, the highest cell dry weight and lipid concentration were 10.1 and 1.83 g/l, respectively, in fed-batch fermentation, with a total soluble substrate concentration of 40 g/l. The improvement of the lipid fermentation performance may have resulted from lignin depolymerization by the combinatorial pretreatment and laccase treatment, reduced inhibition effects by fed-batch fermentation, adequate oxygen supply, and an accurate pH control in the fermenter. Conclusions: Overall, these results demonstrate that combinatorial pretreatment, together with fermentation optimization, favorably improves lipid production using lignin as the carbon source. Combinatorial pretreatment integrated with fed-batch fermentation was an effective strategy to improve the bioconversion of lignin into lipids, thus facilitating lignin valorization in biorefineries. [ABSTRACT FROM AUTHOR]

Production of carotenoids by Rhodococcus opacus PD630 is reported. A modified mineral salt medium formulated with glycerol as an inexpensive carbon source was used for the fermentation. Ammonium acetate was the nitrogen source. A dry cell mass concentration of nearly 5.4 g/L could be produced in shake flasks with a carotenoid concentration of 0.54 mg/L. In batch culture in a 5 L bioreactor, without pH control, the maximum dry biomass concentration was ~30 % lower than in shake flasks and the carotenoids concentration was 0.09 mg/L. Both the biomass concentration and the carotenoids concentration could be raised using a fed-batch operation with a feed mixture of ammonium acetate and acetic acid. With this strategy, the final biomass concentration was 8.2 g/L and the carotenoids concentration was 0.20 mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation. [ABSTRACT FROM AUTHOR]

Objectives: To explore the role of thioesterases in Rhodococcus opacus PD630 by endogenously overexpression in this bacteria for increased lipid production. Results: Overexpression of four thioesterases from R. opacus PD630 in E. coli led to a 2- to 8-fold increase in C16:1 and C18:1 fatty acids while, when overexpressed in R. opacus PD630, only two recombinants had significant effect on the quantities and compositions of total fatty acid. The contents of total fatty acids (FAs) in two recombinants, pJTE2 (OPAG_00508 thioesterase) and pJTE4 (WP_012687673.1 thioesterase), were 400-460 mg/g (CDW) which is 1.5 times of wild-type strain PD630 (300-350 mg/g CDW), and 20-30 % (w/w) more than that of the control strain PDpJAM2 (330-370 mg/g CDW). The contents of 17:1 and 18:1 fatty acids increased by about 27 and 35 %, respectively, in pJTE2 and by 35 and 20 %, respectively, in pJTE4 compared with the control strain. Conclusions: The engineered strains showed improved production of lipid (as total fatty acids), and could also tailor the composition of the fatty acid profile when cultured in mineral salts medium using glucose as sole carbon source. [ABSTRACT FROM AUTHOR]

Triacylglycerols (TAGs) can be produced via bacterial fermentation by the oleaginous Gram-positive microorganism Rhodococcus opacus strain PD630 in regulated, nutrient-deprived conditions with sufficient carbon supply. Microbially produced TAGs may be further refined via transesterification into biodiesel and glycerol, with 3 mole of biodiesel and 1 mole of glycerol produced from every 1 mole of TAG by chemical conversion. Large-scale industrial production of biodiesel has been conducted for over a decade, yet microbially derived biodiesel has been, up to this point, absent from the biodiesel market. The use of a novel feedstock, chitin, from New England fishery waste may present a viable, cost-effective, unexplored carbon feedstock source for local biodiesel development. Availability and implementation of chitin as a feedstock, along with analysis of potential fuel characteristics, yield promising results for future industrial development of biodiesel production from R. opacus PD630 TAGs in regional locations with large lobster, shrimp, and crab harvesting operations around the world. With declining resources of fossil fuels and increased societal awareness of carbon emissions and climate change, an analytical review of this nature is critically relevant. [ABSTRACT FROM AUTHOR]

Highlights: [•] Strain PD630 can tolerate low concentration of five model inhibitory compounds. [•] Strain PD630 can use vanillic acid and TPCA for cell growth and TAG accumulation. [•] Vanillin can be used as a carbon source, but TAGs are not accumulated. [•] Strain PD630 can grow on three lignocellulosic hydrolysates and accumulate TAGs. [Copyright &y& Elsevier]

Background The Gram-positive actinomycete Rhodococcus opacus is widely studied for its innate ability to store large amounts of carbon in the form of triacylglycerol (TAG). Several groups have demonstrated that R. opacus PD630 is capable of storing anywhere from 50 to 76% of its cell dry weight as TAG. While numerous studies have focused on phenomenological aspects of this process, few have sought to identify the underlying molecular and biochemical mechanisms responsible for the biosynthesis and storage of this molecule.Results Herein we further our previous efforts to illuminate the black box that is lipid metabolism in actinomycetes using a genetic approach. Utilizing a simple, colorimetric genetic screen, we have identified a gene, referred to herein as tadD (triacylglycerol accumulation deficient), which is critical for TAG biosynthesis in R. opacus PD630. Furthermore, we demonstrate that the purified protein product of this gene is capable of oxidizing glyceraldehyde-3-phosphate, while simultaneously reducing NAD(P)+ to NAD(P)H. Supporting this biochemical data, we observed that the ratio of NAD(P)H to NAD(P)+ is elevated in wildtype cultures grown under lipid production conditions as compared to cultures grown under vegetative growth conditions, while the mutant strain demonstrated no change irrespective of growth conditions. Finally, we demonstrate that over-expressing a putative phosphorylative glyceraldehyde-3- phosphate dehydrogenase leads to decreased TAG production during growth on TAG accumulation conditions. Conclusion Taken together, the data support the identification of a key metabolic branch point separating vegetative growth and lipid accumulation lifestyles in Rhodococcus. [ABSTRACT FROM AUTHOR]

• CELF lignin facilitates Rhodococcus opacus PD630 growth and lipid accumulation. • CELF cleaves β -O-4 and β -5 bonds completely. • Increases in carboxylic acid OH group of CELF lignin benefit microbial conversion. • CELF lignin is a promising candidate for producing lipid building blocks. Cosolvent Enhanced Lignocellulosic Fractionation (CELF) is an emerging solvolysis pretreatment to fractionate lignocellulosic biomass. Herein, the bioconversion performance of CELF lignin was fully evaluated for the first time. Results showed that CELF lignin possessed higher content of carboxylic acid OH, lower molecular weight, and disappeared β -O-4 and β -5 linkages compared to other two technical lignins including a conventional ethanol organosolv lignin (EOL) and a kraft lignin (KL). Rhodococcus opacus PD630 cell count from CELF lignin fermentation reached the highest value of 3.9 × 107 CFU/mL, representing a 62.5% and 77.3% improvement over EOL and KL, respectively. Correspondingly, lipid yield reached 143 mg/L from CELF lignin, which was 36.2% and 26.5% higher than from EOL and KL, respectively. Principal component analysis (PCA) revealed that more carboxylic acid groups and lower molecular weight contributed to the enhanced bioconversion performance of CELF lignin. This study demonstrates that CELF lignin is a promising candidate for bioconversion. [ABSTRACT FROM AUTHOR]

Abstract: Biodiesel, monoalkyl esters of long-chain fatty acids with short-chain alcohols derived from triacylglycerols (TAGs), can be produced from renewable biomass sources. Recently, there has been interest in producing microbial oils from oleaginous microorganisms. Rhodococcus opacus PD630 is known to accumulate large amounts of TAGs. Following on these earlier works we demonstrate that R. opacus PD630 has the uncommon capacity to grow in defined media supplemented with glucose at a concentration of 300gl−1 during batch-culture fermentations. We found that we could significantly increase concentrations of both glucose and (NH4)2SO4 in the production medium resulting in a dramatic increase in fatty acid production when pH was controlled. We describe the experimental design protocol used to achieve the culture conditions necessary to obtain both high-cell-density and TAG accumulation; specifically, we describe the importance of the C/N ratio of the medium composition. Our bioprocess results demonstrate that R. opacus PD630 grown in batch-culture with an optimal production medium containing 240gl−1 glucose and 13.45gl−1 (NH4)2SO4 (C/N of 17.8) yields 77.6gl−1 of cell dry weight composed of approximately 38% TAGs indicating that this strain holds great potential as a future source of industrial biodiesel on starchy cellulosic feedstocks that are glucose polymers. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.