Rhamnolipid Biosynthesis Research Articles

Utilization of palm sludge oil for rhamnolipid biosynthesis by Pseudomonas aeruginosa USM-AR2 in a stirred tank reactor.

The study focused on rhamnolipid production by batch fermentation of Pseudomonas aeruginosa USM-AR2 in a 3-L stirred-tank reactor (STR) using palm sludge oil (PSO) as the sole carbon source. The impact of various agitation rates towards the dispersion of PSO in the medium was evaluated to improve biomass growth and rhamnolipid production. A mechanical foam collection and recycling system was designed and retrofitted to the STR to overcome severe foam formation during fermentation. The maximum biomass produced was 11.29 ± 0.20g/L obtained at 400rpm, while themaximum rhamnolipid production was 5.06 ± 1.17g/L at 600rpm, giving a rhamnolipid productivity of 0.023g/L/h. High agitation enhances substrate availability by breaking the hydrophobic semi-solid PSO into smaller substrate particles, increasing surface contact area, thus facilitating the PSO utilisation by P. aeruginosa USM-AR2, thereby inducing rhamnolipid production. This study further demonstrates the ability of rhamnolipid to solubilize and disperse sludge oil, which typically remains a solid at room temperature, in the liquid medium. GCMS analysis showed that five fatty acids, namely palmitic acid, myristic acid, stearic acid, methyl ester and linoleic acid, have been utilised. The rhamnolipid showed an oil spreading test result of 160mm of waste engine oil displacement compared to control using distilled water that remained non-displaced, and a critical micelle concentration (CMC) of 17mg/L. In emulsification index (E24) assay, the rhamnolipid was shown to emulsify toluene (66.7% ± 7.2), waste engine oil (58.3% ± 7.2), kerosene (41.8% ± 4.8) and n-hexane (33.1% ± 5.7). UPLC analysis on rhamnolipid revealed a congener mixture of rhamnolipid, namely di-rhamnolipid and mono-rhamnolipid mixture. This is the first report on the employment of an integrated foam control reactor system with PSO as the carbon source for rhamnolipid production by P. aeruginosa USM-AR2 culture.

ZnO nanoparticle engineered Pseudomonas aeruginosa SOA1 towards enhanced Rhamnolipid production

Rhamnolipids, the potential biosurfactants, have widespread applications in pharmaceutics, food, and agriculture fields. However, its low biosynthesis efficiency limits surfactant industrialization. In this study, different nanoparticles (NPs) were immobilized over Pseudomonas aeruginosa (SOA1) and targeted towards increased rhamnolipids production by increasing the secretion (key step of rhamnolipids biosynthesis). It was observed that, upon adding ZnO NPs (4.0 g/L), rhamnolipids titer increased 4.87 times (from 4.1 to 19.98 g/L) in the 10 L bioreactor. This is due to an increment in the expression of the gene (present in the rhamnolipids biosynthesis process) with the introduction of ZnO NPs to SOA1. Further, with ZnO NPs, few rhamnolipid biosynthesis genes (rhlA and rhlB) were significantly upregulated. In contrast, the rhamnose biosynthetic genes (rmlA, rmlC, and algC) were down regulated during the early and late exponential growth phases as studied in Quantitative real-time PCR. The biosurfactant generation is regulated by quorum-sensing genes, which exhibit a hierarchical expression profile. Specifically, in the early log phase (ZnO NPs cultivation), the last gene was elevated, but both pqsE and rhlI were stimulated in the late log phase. This experiment paves a new path toward improving the biosynthesis of microbial metabolites via. NPs engineering.

Increase proportion of di‐rhamnolipids biosynthesized from Pseudomonas aeruginosa and evaluation of relationship between activity and di‐rhamnolipids proportion

AbstractRhamnolipids, simply divided into mono‐rhamnolipids and di‐rhamnolipids, their physicochemical properties are structure‐dependent. This study explored to increase the proportion of di‐rhamnolipids biosynthesized from Pseudomonas aeruginosa and evaluate the relationship between activity and di‐rhamnolipids proportions. P. aeruginosa SGrhlC was constructed by increasing the rhlC gene in P. aeruginosa SG. HPLC‐MS results indicated that SGrhlC produced more di‐rhamnolipids (62.32%) than that of the wild‐type strain SG (45.24%). Both the species and proportion of di‐rhamnolipids were increased, mainly Rha‐Rha‐C8‐C10 and Rha‐Rha‐C10‐C10. The rhamnolipids produced by SGrhlC was thermostable and salt‐tolerant. The SGrhlC rhamnolipids decreased surface tension of water to 27.0 mN/m with a critical micelle concentration (CMC) of 50 mg/L and emulsified crude oil with an emulsification index of 66.9 ± 1.5%. The SGrhlC rhamnolipids exhibited antimicrobial activity against Staphylococcus aureus and Cladosporium sp. with IC50 (half maximal inhibitory concentration) values less than 15 mg/L, and washed 73.02% ± 1.77% of oil from petroleum‐contaminated soil. Physicochemical activities of three bacterial rhamnolipids varied with their di‐rhamnolipids proportions. Results indicated that the higher proportions of di‐rhamnolipids were, the lower CMC, better surface activity and higher washing oil rate were, while the weaker emulsifying activity and lower antimicrobial activity were. The SGrhlC rhamnolipids showed better surface activity and a lower critical micelle concentration, which was superior for wetting, foaming, desorbing and dispersing. This study revealed that P. aeruginosa can be genetically regulated to biosynthesize rhamnolipids with specific structure. Perspectives of the customized biosynthesis and applications of rhamnolipids were also discussed.

Получение гетерологичных продуцентов рамнолипидов для промышленного производства высокоэффективного биосурфактанта

Rhamnolipids are promising microbial glycolipid biosurfactants that can be used as preparations for soil bioremediation in oil pollution. The expression of the rhamnolipid biosynthesis gene cluster in the main producer, Pseudomonas aeruginosa, is regulated by quorum sensing signals (QS). Complex biosynthesis regulation does not allow achieving high product yields. The production of heterologous rhamnolipid producers is a promising way to overcome the complex QS-dependent regulation of rhamnolipid biosynthesis, and also avoids the use of opportunistic Pseudomonas aeruginosa for the industrial production of rhamnolipids. Heterologous Escherichia coli BLWT-based producers with a transformant E14 productivity of 3.40±0.96 g/L were obtained on the basis of the plasmid vector pAl2–T with a construct from the native promoter and rhlAB genes, and Pseudomonas viridiflava AlC1223-based ones with the productivity of transformants P5, P6, P8 332.00±0.10, 200.00±0.10 and 180.00±0.10 g/L, respectively. The presence of rhlAB genes in the expression vector allows the obtaining monorhamnolipids for further use as an agent for the solubilization of soil hydrophobic pollutant (e. g. petroleum products), which will intensify bioremediation processes.

Microbe cultivation guidelines to optimize rhamnolipid applications

In the growing landscape of interest in natural surfactants, selecting the appropriate one for specific applications remains challenging. The extensive, yet often unsystematized, knowledge of microbial surfactants, predominantly represented by rhamnolipids (RLs), typically does not translate beyond the conditions presented in scientific publications. This limitation stems from the numerous variables and their interdependencies that characterize microbial surfactant production. We hypothesized that a computational recipe for biosynthesizing RLs with targeted applicational properties could be developed from existing literature and experimental data. We amassed literature data on RL biosynthesis and micellar solubilization and augmented it with our experimental results on the solubilization of triglycerides (TGs), a topic underrepresented in current literature. Utilizing this data, we constructed mathematical models that can predict RL characteristics and solubilization efficiency, represented as logPRL = f(carbon and nitrogen source, parameters of biosynthesis) and logMSR = f(solubilizate, rhamnolipid (e.g. logPRL), parameters of solubilization), respectively. The models, characterized by robust R2 values of respectively 0.581–0.997 and 0.804, enabled the ranking of descriptors based on their significance and impact—positive or negative—on the predicted values. These models have been translated into ready-to-use calculators, tools designed to streamline the selection process for identifying a biosurfactant optimally suited for intended applications.

In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding

Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.

Genome analysis of a coral-associated bacterial consortium highlights complementary hydrocarbon degradation ability and other beneficial mechanisms for the host

Here we report the oil degradation genetic potential of six oil-degrading bacteria (ODB), previously used as a bioremediation consortium, isolated from the hydrocoral Millepora alcicornis and seawater. The strains were identified as Halomonas sp. (LC_1), Cobetia sp. (LC_6), Pseudoalteromonas shioyasakiensis (LC_2), Halopseudomonas aestusnigri (LC_3), Shewanella algae (LC_4), and Brucella intermedia (LC_5). The taxonomic identification differed from that of the original paper when we used whole genome gene markers instead of just 16S rRNA gene. Genes responsible for the degradation of aromatic hydrocarbons and n-alkanes were found in all genomes, although different (and complementary) steps of the metabolic pathways were unique to each strain. Genes for naphthalene and toluene degradation were found in various strains. We annotated quinate degradation genes in LC_6, while LC_3 and LC_5 presented genes for biosurfactant and rhamnolipid biosynthesis. We also annotated genes related to beneficial mechanisms for corals, such as genes involved in nitrogen and DMSP metabolism, cobalamin biosynthesis and antimicrobial compounds production. Our findings reinforce the importance of using bacterial consortia for bioremediation approaches instead of single strains, due to their complementary genomic arsenals. We also propose a genome-based framework to select complementary ODB that can provide additional benefits to coral health.

Extended Biosynthesis of Rhamnolipid by Immobilized Pseudomonas aeruginosa USM-AR2 Cells in a Fluidized Bed Bioreactor.

This study aims to evaluate rhamnolipid production by immobilized Pseudomonas aeruginosa USM-AR2 cells using waste cooking oil (WCO) as the carbon source. P. aeruginosa USM-AR2 cells were entrapped in polyvinyl alcohol (PVA)-alginate hydrogel beads. The performance of entrapped cells was compared with free cells in shake flasks before cultivation in a custom-designed fluidized bed reactor (FBR). A mass of 1 g wet cells entrapped in PVA-alginate hydrogel beads were successfully recycled 3 times in shake flasks at 200 rpm, producing between 0.66-1.34 g L-1 rhamnolipid after 120 h. Meanwhile, cultivation of entrapped cells in FBR with broth recirculation showed that the suitable hydrogel beads to medium ratio was 1:20 ratio at an aeration rate of 0.5 vvm, producing between 0.77 to 1.58 g L-1 rhamnolipid, degrading 8.67 to 20.93 g L-1 of waste cooking oil in 15 cycles of repeated batch cultivation. Entrapped P. aeruginosa USM-AR2 cells achieved a higher rhamnolipid production, by 1.03-fold during cycle 3 in shake flasks and 1.19-fold during cycle 11 in an FBR, compared to free cells. These results show that entrapment enables reusability of viable cells and maintains stability of rhamnolipid production throughout the extended cultivation, increasing cell tolerance to perturbations in fermentation conditions.

Improving Rhamnolipids Biosynthesis in Pseudomonas sp. L01 through Atmospheric and Room-Temperature Plasma (ARTP) Mutagenesis.

Biosurfactants have significant applications in various industries, including microbial-enhanced oil recovery (MEOR). While the state-of-the-art genetic approaches can generate high-yield strains for biosurfactant production in fermenters, there remains a critical challenge in enhancing biosurfactant-producing strains for use in natural environments with minimal ecological risks. The objectives of this work are enhancing the strain's capacity for rhamnolipids production and exploring the genetic mechanisms for its improvement. In this study, we employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the biosynthesis of rhamnolipids in Pseudomonas sp. L01, a biosurfactant-producing strain isolated from petroleum-contaminated soil. Following ARTP treatment, we identified 13 high-yield mutants, with the highest yield of 3.45 ± 0.09 g/L, representing a 2.7-fold increase compared to the parent strain. To determine the genetic mechanisms behind the enhanced rhamnolipids biosynthesis, we sequenced the genomes of the strain L01 and five high-yield mutants. A comparative genomic analysis suggested that mutations in genes related to the synthesis of lipopolysaccharides (LPS) and the transport of rhamnolipids may contribute to the improved biosynthesis. To the best of our knowledge, this is the first instance of utilizing the ARTP approach to improve rhamnolipid production in Pseudomonas strains. Our study provides valuable insights into the enhancement of biosurfactant-producing strains and the regulatory mechanisms of rhamnolipids biosynthesis.

RhlA Exhibits Dual Thioesterase and Acyltransferase Activities during Rhamnolipid Biosynthesis

RhlA Exhibits Dual Thioesterase and Acyltransferase Activities during Rhamnolipid Biosynthesis

Bioremediation of Petroleum-Contaminated Soils with Biosurfactant-Producing Degraders Isolated from the Native Desert Soils.

A crude oil spill in 2014 resulted in extensive soil contamination of the hyper arid Evrona Nature Reserve in Israel's Negev Desert. The contaminated soils became highly hydrophobic, threatening the existence of plants in the habitat. We hypothesized that bioaugmenting the soil with indigenous biosurfactant-producing, hydrocarbon-degrading bacteria (HDB) would accelerate the reduction in the soil's hydrophobicity. We aimed to isolate and characterize biosurfactant-producing HDBs from the desert-contaminated soil and test if they can be used for augmenting the soil. Twelve hydrocarbon-degrading strains were isolated, identified as Pseudomonas, and classified as biosurfactants "producing" and "nonproducing". Inoculating 109 CFU/g of "producing" strains into the polluted soil resulted in a 99.2% reduction in soil hydrophobicity within seven days. At the same time, nonproducing strains reduced hydrophobicity by only 17%, while no change was observed in the untreated control. The microbial community in the inoculated soil was dominated by the introduced strains over 28 days, pointing to their persistence. Rhamnolipid biosynthesis gene rhlAB remained persistent in soil inoculated with biosurfactants, indicating in situ production. We propose that the success of the treatment is due to the use of inoculum enriched from the polluted soil.

Metabolic engineering of Pseudomonas taiwanensis VLB120 for rhamnolipid biosynthesis from biomass-derived aromatics

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

Achieving “Non-Foaming” Rhamnolipid Production and Productivity Rebounds of Pseudomonas aeruginosa under Weakly Acidic Fermentation

The rhamnolipid production of Pseudomonas aeruginosa has been impeded by its severe foaming; overcoming the bottleneck of foaming has become the most urgent requirement for rhamnolipid production in recent decades. In this study, we performed rhamnolipid fermentation under weakly acidic conditions to address this bottleneck. The results showed that the foaming behavior of rhamnolipid fermentation broths was pH-dependent with the foaming ability decreasing from 162.8% to 28.6% from pH 8 to 4. The “non-foaming” rhamnolipid fermentation can be realized at pH 5.5, but the biosynthesis of rhamnolipids was significantly inhibited. Further, rhamnolipid yield rebounded from 8.1 g/L to 15.4 g/L after ultraviolet and ethyl methanesulfonate compound mutagenesis. The mechanism study showed that the species changes of rhamnolipid homologs did not affect the foaming behavior of the fermentation but had a slight effect on the bioactivity of rhamnolipids. At pH 8.0 to 5.0, increased surface tension, decreased viscosity and zeta potential, and aggregation of rhamnolipid molecules contributed to the “non-foaming” rhamnolipid fermentation. This study provides a promising avenue for the “non-foaming” rhamnolipid fermentation and elucidates the mechanisms involved, facilitating the understanding of pH-associated foaming behavior and developing a more efficient strategy for achieving rhamnolipid production.

BY THE 85TH ANNIVERSARY OF THE BIRTH OF ACADEMICIAN NIKOLAI ALEXANDROVICH KARTEL GENETICALLY MODIFIED PLANTS ― HISTORY AND PROSPECTS

The generalizing results of the activity of the laboratory of molecular genetics in the field of creation of genetically modified plants are presented. Under the leadership of academician N.A. Kartel created transgenic plants of tobacco, arabidopsis, potato, foxglove, rapeseed with genes rhlA and rhlB rhamnolipid biosynthesis, endochitinase chiA, cry3aM Bacillus thuringiensis encoding delta endotoxin, cyp11a1 cytochrome p450scc of animal origin, glucose oxidase, resistance to the herbicide glufosinate, etc. The prospects are considered in the field of plant transgenesis.

GC-MS analysis and Molecular docking of Quercetin compounds of Phytolacca octandra with the target protein of infection causing Staphylococcus aureus

Phytolacca octandra is a perennial usually about 1m high herb, dense and erect in full sun. As only few reports were available on the studies about the bioactive compounds and various activities in the Phytolacca octandra, the present study focuses on the bio active compounds attributed to antibacterial activity in the plant extracts by Gas Chromatography – Mass Spectrometry (GCMS) and molecular docking methods. Antibacterial activity of Phytolacca octandra showed maximum inhibitory zones of 21mm, 18mm, 19mm, 19mm and 20mm against respective organisms for 25mg/ml of acetone extracts. The outcome of Phytolacca octandra extracts that was exposed to GC-MS analysis, showed the presence of 20 more compounds. The most identified compounds to have anti-oxidant activity are Dodecane, Octadecane and Octacosane. The other major compounds present in extract are Cyclohexen-oxopropyl, 1,2-Benzenedicarboxylic acid.The overall docking energies of the target protein, rhamnolipids biosynthesis 3-oxoacyl-[acyl-carrier-protein] reductase with quercetin with the number of hydrogen bonds were presented in the study; The docking report revealed –8.01Kcal/Mol binding energies and 8 hydrogen bonding between the Phytolacca octandra compound, quercetin and the target binding protein, rhlG of infection causing pathogen Staphylococcus aureus.

Glucose metabolism in Pseudomonas aeruginosa is cyclic when producing Polyhydroxyalkanoates and Rhamnolipids

Pseudomonas aeruginosa is an important chassis for production of polyhydroxyalkanoates (PHA) and rhamnolipids (RHL). Advances in the understanding of the biosynthesis metabolism of these biocompounds are crucial for increasing yield. 13C-Metabolic Flux Ratio Analysis (13C-MFA) is a technique to estimate in vivo metabolic fluxes ratios. PHA and RHL are essentially non-growth associated products of biotechnological interest and both contain hydroxyalkanoates (HAs), whose labeling patterns could be accessed by GC-MS. In this study, to reveal the relative contributions of the Entner-Doudoroff (ED) pathway and the non-oxidative Pentose Phosphate (PP) pathway to PHA and RHL production, 13C-MFA was performed in Pseudomonas aeruginosa LFM634 when supplied with labeled glucose. This bacterial strain lacks both functional EMP and the oxidative PP branch. Labeling patterns in HAs were measured. Experiments with [U-13C] glucose indicated a low flux though PP pathway. An optimal design of labeling experiment showed that [6-13C] glucose would be the best substrate to enable an estimation of the ED flux with high accuracy. Results of experiments performed with this isotope indicated that about two-thirds of glyceraldehyde 3-phosphate is recycled through a cyclic ED architecture, suggesting that P. aeruginosa utilizes that cycle to regulate the NADPH/Acetyl-CoA ratio for PHA and RHL biosynthesis.

Glycerol or crude glycerol as substrates make Pseudomonas aeruginosa achieve anaerobic production of rhamnolipids

BackgroundThe anaerobic production of rhamnolipids is significant in research and application, such as foamless fermentation and in situ production of rhamnolipids in the anoxic environments. Although a few studies reported that some rare Pseudomonas aeruginosa strains can produce rhamnolipids anaerobically, the decisive factors for anaerobic production of rhamnolipids were unknown.ResultsTwo possible hypotheses on the decisive factors for anaerobic production of rhamnolipids by P. aeruginosa were proposed, the strains specificity of rare P. aeruginosa (hypothesis 1) and the effect of specific substrates (hypothesis 2). This study assessed the anaerobic growth and rhamnolipids synthesis of three P. aeruginosa strains using different substrates. P. aeruginosa strains anaerobically grew well using all the tested substrates, but glycerol was the only carbon source that supported anaerobic production of rhamnolipids. Other carbon sources with different concentrations still failed for anaerobic production of rhamnolipids by P. aeruginosa. Nitrate was the excellent nitrogen source for anaerobic production of rhamnolipids. FTIR spectra analysis confirmed the anaerobically produced rhamnolipids by P. aeruginosa using glycerol. The anaerobically produced rhamnolipids decreased air-water surface tension to below 29.0 mN/m and emulsified crude oil with EI24 above 65%. Crude glycerol and 1, 2-propylene glycol also supported the anaerobic production of rhamnolipids by all P. aeruginosa strains. Prospects and bottlenecks to anaerobic production of rhamnolipids were also discussed.ConclusionsGlycerol substrate was the decisive factor for anaerobic production of rhamnolipids by P. aeruginosa. Strain specificity resulted in the different anaerobic yield of rhamnolipids. Crude glycerol was one low cost substrate for anaerobic biosynthesis of rhamnolipids by P. aeruginosa. Results help advance the research on anaerobic production of rhamnolipids, deepen the biosynthesis theory of rhamnolipids and optimize the anaerobic production of rhamnolipids.

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C10–C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.