Rhodococcus Opacus PD630 Research Articles

Sustainable Diesel Feedstock: a Comparison of Oleaginous Bacterial and Microalgal Model Systems

The key to sustainable and commercially viable biodiesel production relies primarily on species selection. Oleaginous species with high biomass productivity, lipid content, and lipid productivity are desirable. High growth rate of the species results in high biomass productivity, which leads to high lipid productivity. It is known that algal oil technology lacks commercial feasibility predominantly due to low biomass productivity and other factors. The use of a faster-growing organism, such as oleaginous bacteria, could offset this major disadvantage. Thus, the current study analyzes two model oleaginous systems: Rhodococcus opacus PD630 (a bacterium) and Chlorella vulgaris NIOT5 (a microalga) for their growth rate and lipid productivity. It was found that the bacterial growth rate was 25-fold the microalgal growth rate. The bacterium also showed 57-fold higher biomass productivity and 75-fold higher biodiesel productivity. Further, the analysis of a large number of literature data from relevant studies under different cultivation conditions showed that R. opacus PD630 has productivities far higher than various autotrophic microalgae. Similarly, a frequency distribution of data collected from the literature showed that Rhodococcus sp. has productivities in the higher range as compared to heterotrophic microalgae. Thus, bacteria could serve as a better alternative to microalgae toward developing a commercially viable biofuel technology. Further, the biodiesel characterization study showed that the quality of diesel from the bacterium was better than that from the microalga.

Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii.

The occurrence of NADP+-dependent malic enzymes (NADP+-MEs) in several Rhodococcus strains was analysed. The NADP+-ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP+-ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP+-ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP+-dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP+-ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP+-MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms.

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strains as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. This study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.

Enhanced production of carotenoids and lipids by Rhodococcus opacus PD630

AbstractBACKGROUNDLow‐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.RESULTSSubmerged 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.CONCLUSIONGlycerol 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

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.

Characterization of a Novel Tectivirus Phage Toil and Its Potential as an Agent for Biolipid Extraction

The oleaginous bacterium Rhodococcus opacus PD630 is metabolically diverse and can be cultivated on various renewable resources to serve as a sustainable triacylglycerol (TAG) feedstock for biodiesel production. Current methods for TAG extraction are costly, but infection of cultures by lytic bacteriophages (phages) may be a viable approach for achieving release of intracellular lipid from oleaginous bacteria such as R. opacus. This study reports the novel tectiviral phage Toil capable of releasing intracellular contents including a fluorescent protein marker and TAGs into the supernatant after phage infection of R. opacus PD631, a domesticated derivative of strain PD630. Phage Toil is placed in the Tectiviridae by its morphology, the presence of a lipid membrane, its genome architecture and the presence of terminal covalently-linked proteins. Toil is the first tectivirus capable of infecting a member of the Actinobacteria. Microscopy shows that infected cells do not undergo sudden lysis but instead maintain their original shape for several hours, with the cellular morphology gradually deteriorating. Approximately 30% of intracellular TAGs could be recovered from the culture supernatants of Toil-infected PD631 cells. Phage Toil has potential to be used as an agent in extraction of TAGs from oleaginous bacterium R. opacus. Importance: This study reported the first tectivirus (Phage Toil) capable of infecting a member of the Actinobacteria. In this study, we showed that Phage Toil can infect oleaginous bacterium Rhodococcus opacus to release intracellular contents such as a fluorescent protein marker and TAG lipid granules, which can serve as a starting material for biodiesel production. This study demonstrates a new method to extract TAGs by using this phage. Additionally, Phage Toil can be a new model phage to advance knowledge regarding phage infection mechanisms in Rhodococcus and other mycolic acid-containing bacteria such as Mycobacterium.

Rewiring neutral lipids production for the de novo synthesis of wax esters in Rhodococcus opacus PD630

Rhodococcus opacus PD630 accumulates significant amounts of triacylglycerols (TAG), but is not able to de novo synthesize wax esters (WE) from structural unrelated carbon sources, such as gluconate. In this study, strain PD630 was engineered to produce WE by heterologous expression of maqu_2220 gene, which encodes a fatty acyl-CoA reductase for the production of fatty alcohols in Marinobacter hydrocarbonoclasticus. Recombinant cells produced ca. 46% of WE and 54% of TAG (of total WE+TAG) from gluconate compared with the wild type, which produced 100% of TAG. Cell growth was not affected by the heterologous expression of MAQU_2220. Several saturated and monounsaturated WE species were produced by cells, with C18:C16, C16:C16 and C16:C18 as main species. The fatty acid composition of WE fraction in PD630maqu_2220 was enriched with C16:0, C18:0, whereas C16:0, C18:0 and C18:1 predominated in the TAG fraction. Significant amounts of WE and TAG were accumulated by PD630maqu_2220 from whey, an inexpensive waste material from dairy industries, without affecting cell biomass production. This is the first report on WE synthesis by R. opacus from gluconate, which demonstrates that lipid metabolism of this bacterium is flexible enough to assimilate heterologous components for the production of new lipid derivatives with industrial interest.

Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a nonmodel, Gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R.opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R.opacus, including: (1) six fluorescent reporters for quantifying promoter output, (2) three chemically inducible promoters for variable gene expression, and (3) two classes of metabolite sensors derived from native R.opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R.opacus. Overall, this work expands the ability to control and characterize gene expression in R.opacus for future lignocellulose-based fuel and chemical production.

Real-time lipid production and dairy wastewater treatment using Rhodococcus opacus in a bioreactor under fed-batch, continuous and continuous cell recycling modes for potential biodiesel application

ABSTRACTThis study focused on lipid production from dairy wastewater using Rhodococcus opacus PD630 in a bioreactor. Simultaneous wastewater COD removal, biomass production and lipid accumulation by R. opacus were evaluated using the bioreactor operated under three different culture modes: fed-batch, continuous and continuous cell recycling. To support the R. opacus growth, the dairy wastewater was supplemented with 25% (v/v) mineral salt media. Results from the fed batch operated bioreactor revealed that a maximum COD removal efficiency of 93% could be achieved along with 1.1 g L-1 of lipid accumulation by the bacteria. Continuous mode experiments provided the best results with almost 100% COD removal efficiency and a maximum lipid accumulation of 1.8 g L-1 at a hydraulic retention time (HRT) of 6.6 h. However, lipid production was even more superior under continuous cell recycle mode with a maximum lipid accumulation of 3.4 g L-1. Thus, the present study proved that R. opacus can be successfully scaled up for efficient dairy wastewater treatment along with lipid accumulation for potential biofuel application.

Construction of a BioBrick™ compatible vector system for Rhodococcus

Throughout the past decade, the field of synthetic biology has grown rapidly. By using assembly platforms such as BioBricks™, scientists can quickly and easily build gene circuits or multi-step pathways. One limitation, however, is that most of these parts were designed and characterized with Escherichia coli as the target chassis. As a consequence, there exists a lack of standardized and well characterized or BioBrick™ compatible plasmid backbones that replicate in other potential non-model chassis organisms. The Gram-positive bacteria of the genus Rhodococcus represent an interesting chassis for biotechnological applications due to their tremendous metabolic capabilities. In this report we describe our progress toward developing a BioBrick™ compatible plasmid system for Rhodococcus. We demonstrate its utility for heterologous protein expression through flow cytometric analysis of the lac promoter in the oleaginous strain Rhodococcus opacus PD630.

Production of potential fuel oils by Rhodococcus opacus grown on petroleum processing wastewaters

The wastewater of a petroleum processing facility was used to grow the oleaginous bacterium Rhodococcus opacus PD630 to produce microbial oils. Undiluted wastewater supplemented with molasses (20 g/l) and ammonium chloride (1 g/l) provided a maximum dry biomass concentration of nearly 6 g/l in a 96 h batch culture. The lipid concentration in the bacterial broth exceeded 3 g/l and the lipid content in the dry biomass was nearly 52% by weight. Biomass and oil production were further improved using a 96-h fed-batch fermentation instead of a batch culture. The final biomass concentration exceeded 7.2 g/l and the lipid concentration was nearly 4 g/l. The lipid content of the dry biomass exceeded 54% and the lipid yield on sugars was 0.33 g/g. The lipids were similar to vegetable oils. They contained mainly long chain C16 and C18 fatty acids. Potentially such lipids may be used to replace vegetable oils in production of biodiesel.

Production of carotenoids and lipids by Rhodococcus opacus PD630 in batch and fed-batch culture.

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 5.4g/L could be produced in shake flasks with a carotenoid concentration of 0.54mg/L. In batch culture in a 5L bioreactor, without pH control, the maximum dry biomass concentration was ~30% lower than in shake flasks and the carotenoids concentration was 0.09mg/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 8.2g/L and the carotenoids concentration was 0.20mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation.

Functional divergence of HBHA from Mycobacterium tuberculosis and its evolutionary relationship with TadA from Rhodococcus opacus.

Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 are oleaginous bacteria able to synthesize and accumulate triacylglycerols (TAG) in lipid bodies (LB). Highly relevant to the structure of LB is a protein homologous to heparin-binding hemagglutinin (HBHA) (called TadA in rhodococci), which is a virulence factor found in Mycobacterium tuberculosis. HBHA is an adhesin involved in binding to non-phagocytic cells and extrapulmonary dissemination. We observed a conserved synteny of three genes encoding a transcriptional regulator (TR), the HBHA protein and a membrane protein (MP) between TAG-accumulating actinobacteria belonging to Rhodococcus, Mycobacterium, Nocardia and Dietzia genera, among others. A 354bp-intergenic spacing containing a SigF-binding site was found between hbha and the TR genes in M. tuberculosis, which was absent in genomes of other investigated actinobacteria. Analyses of available "omic" information revealed that TadA and TR were co-induced in rhodococci under TAG-accumulating conditions; whereas in M. tuberculosis and Mycobacterium smegmatis, HBHA and TR were regulated independently under stress conditions occurring during infection. We also found differences in protein lengths, domain content and distribution between HBHA and TadA proteins from mycobacteria and rhodococci, which may explain their different roles in cells. Based on the combination of results obtained in model actinobacteria, we hypothesize that HBHA and TadA proteins originated from a common ancestor, but later suffered a process of functional divergence during evolution. Thus, rhodococcal TadA probably has maintained its original role; whereas HBHA may have evolved as a virulence factor in pathogenic mycobacteria.

Valorization of lubricant-based wastewater for bacterial neutral lipids production: Growth-linked biosynthesis

Lipids produced by microorganisms are currently of great interest as raw material for either biofuels or oleochemicals production. Significant biosynthesis of neutral lipids, such as triacylglycerol (TAG) and wax esters (WE) are thought to be limited to a few strains. Hydrocarbonoclastic bacteria (HCB), key players in bioremediation of hydrocarbon contaminated ecosystems, are among this group of strains. Hydrocarbon rich wastewaters have been overlooked concerning their potential as raw material for microbial lipids production. In this study, lubricant-based wastewater was fed, as sole carbon source, to two HCB representative wild strains: Alcanivorax borkumensis SK2, and Rhodococcus opacus PD630. Neutral lipid production was observed with both strains cultivated under uncontrolled conditions of pH and dissolved oxygen. A. borkumensis SK2 was further investigated in a pH- and OD-controlled fermenter. Different phases were assessed separately in terms of lipids production and alkanes removal. The maximum TAG production rate occurred during stationary phase (4 mg-TAG/L h). The maximum production rate of WE-like compounds was 15 mg/L h, and was observed during exponential growth phase. Hydrocarbons removal was 97% of the gas chromatography (GC) resolved straight-chain alkanes. The maximum removal rate was observed during exponential growth phase (6 mg-alkanes/L h). This investigation proposes a novel approach for the management of lubricant waste oil, aiming at its conversion into valuable lipids. The feasibility of the concept is demonstrated under low salt (0.3%) and saline (3.3%) conditions, and presents clues for its technological development, since growth associated oil production opens the possibility for establishing continuous fermentation processes.

Rhodococcus opacus B4: a promising bacterium for production of biofuels and biobased chemicals.

Bacterial lipids have relevant applications in the production of renewable fuels and biobased oleochemicals. The genus Rhodococcus is one of the most relevant lipid producers due to its capability to accumulate those compounds, mainly triacylglycerols (TAG), when cultivated on different defined substrates, namely sugars, organic acids and hydrocarbons but also on complex carbon sources present in industrial wastes. In this work, the production of storage lipids by Rhodococcus opacus B4 using glucose, acetate and hexadecane is reported for the first time and its productivity compared with Rhodococcus opacus PD630, the best TAG producer bacterium reported. Both strains accumulated mainly TAG from all carbon sources, being influenced by the carbon source itself and by the duration of the accumulation period. R. opacus B4 produced 0.09 and 0.14 g L−1 at 24 and 72 h, with hexadecane as carbon source, which was 2 and 3.3 fold higher than the volumetric production obtained by R. opacus PD630. Both strains presented similar fatty acids (FA) profiles in intact cells while in TAG produced fraction, R. opacus B4 revealed a higher variability in fatty acid composition than R. opacus PD630, when both strains were cultivated on hexadecane. The obtained results open new perspectives for the use of R. opacus B4 to produce TAG, in particular using oily (alkane-contaminated) waste and wastewater as cheap raw-materials. Combining TAG production with hydrocarbons degradation is a promising strategy to achieve environmental remediation while producing added value compounds.

Boosting fatty acid synthesis in Rhodococcus opacus PD630 by overexpression of autologous thioesterases.

To explore the role of thioesterases in Rhodococcus opacus PD630 by endogenously overexpression in this bacteria for increased lipid production. 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 400-460mg/g (CDW) which is 1.5 times of wild-type strain PD630 (300-350mg/g CDW), and 20-30% (w/w) more than that of the control strain PDpJAM2 (330-370mg/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. 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.

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630.

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (∼20 mg/l/h) and ∼2-fold higher lipid production from phenol than the wild-type strain. Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13C-metabolite fingerprinting.

For rapid analysis of microbial metabolisms, (13)C-fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous (13)C-metabolic flux analysis ((13)C-MFA), this method aims to provide metabolic insights without expensive flux measurements. Using (13)C-fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value-added chemicals. Specifically, seven metabolic insights were gathered as follows: (1) glucose metabolism mainly via the Entner-Doudoroff (ED) pathway; (2) lack of glucose catabolite repression during phenol co-utilization; (3) simultaneous operation of gluconeogenesis and the ED pathway for the co-metabolism of glucose and phenol; (4) an active glyoxylate shunt in acetate-fed culture; (5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase); (6) presence of alternative glycine synthesis pathway via glycine dehydrogenase; and (7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a (13)C-fingerprinting kit was developed for studying the central metabolism of non-model microbial species. This low-cost kit can be used to characterize microbial metabolisms and facilitate the design-build-test-learn cycle during the development of microbial cell factories.

Microbial lipid production by oleaginous Rhodococci cultured in lignocellulosic autohydrolysates.

Metabolic synthesis of single cell oils (SCOs) for biodiesel application by heterotrophic oleaginous microorganisms is being hampered by the high cost of culture media. This study investigated the possibility of using loblolly pine and sweetgum autohydrolysates as economic feedstocks for microbial lipid production by oleaginous Rhodococcus opacus (R. opacus) PD630 and DSM 1069. Results revealed that when the substrates were detoxified by the removal of inhibitors (such as HMF-hydroxymethyl-furfural), the two strains exhibited viable growth patterns after a short adaptation/lag phase. R. opacus PD630 accumulated as much as 28.6 % of its cell dry weight (CDW) in lipids while growing on detoxified sweetgum autohydrolysate (DSAH) that translates to 0.25 g/l lipid yield. The accumulation of SCOs reached the level of oleagenicity in DSM 1069 cells (28.3 % of CDW) as well, while being cultured on detoxified pine autohydrolysate (DPAH), with the maximum lipid yield of 0.31 g/l. The composition of the obtained microbial oils varied depending on the substrates provided. These results indicate that lignocellulosic autohydrolysates can be used as low-cost fermentation substrates for microbial lipid production by wild-type R. opacus species. Consequently, the variety of applications for aqueous liquors from lignocellulosic pretreatment has been expanded, allowing for the further optimization of the integrated biorefinery.