Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

废弃食用油脂生物合成鼠李糖脂研究进展

碳源的成本过高限制了鼠李糖脂的工业化生产及应用,废弃食用油脂作为一种廉价易得的碳源,越来越多的研究者开始关注用它发酵生产鼠李糖脂.废弃食用油脂的种类、投加量对鼠李糖脂的产量、结构、性质均会产生影响,目前研究中用废弃食用油脂作碳源,鼠李糖脂产量最高可达24.61g/L、表面张力最低达到24mN/m、产物CMC最低可达40.19mg/L.此外,本文还总结了菌株、氮源、微量元素、pH、溶氧及培养方式等因素对废弃食用油脂生产鼠李糖脂的影响,并展望了利用废弃食用油脂生产鼠李糖脂实现产业化的重点研究方向.     

Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37°C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.     

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54?±?0.023?g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48?hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74?±?0.06?g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.     

Evaluation of critical nutritional parameters and their significance in the production of rhamnolipid biosurfactants from Pseudomonas aeruginosa BS‐161R

Eleven biosurfactant producing bacteria were isolated from different petroleum‐contaminated soil and sludge samples. Among these 11 isolates, two were identified as promising, as they reduced the surface tension of culture medium to values below 27 mN m^?1. Besides biosurfactant production property, they exhibited good flocculating activity. Microbacterium sp. was identified as a new addition to the list of biosurfactant and bioflocculant‐producers. Optimization of various conditions for rhamnolipid production was carried out for one of the promising isolate, Pseudomonas aeruginosa BS‐161R. Bioglycerol (2.5%), as a cheap renewable carbon source, attained better rhamnolipid yield, while sodium nitrate appeared to be the preferable nitrogen source. The optimum carbon to nitrogen (C/N) and carbon to iron (C/Fe) ratios achieved were 15 and 28,350, respectively, which favored rhamnolipid production. Physical parameters like pH, temperature, and agitation speed also affected the production of rhamnolipids. Results from shake flask optimization indicated that the concentration of bioglycerol, sodium nitrate, and iron were the most significant factors affecting rhamnolipid production, which was supported by the results of central composite rotatable design. After optimization of the culture conditions, the production of rhamnolipids increased by ninefold from 0.369 to 3.312 g L^?1. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l⁻¹) compared to the wild-type strain (1.28 g l⁻¹). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Characterization of rhamnolipid production by Burkholderia glumae

Aims: To investigate if Burkholderia glumae can produce rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Methods and Results: Burkholderia glumae AU6208 produces a large spectrum of mono‐ and di‐rhamnolipid congeners with side chains varying between C₁₂‐C₁₂ and C₁₆‐C₁₆, the most abundant being Rha‐Rha‐C₁₄‐C₁₄.The effects on rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000·7 mg l^?1 was reached after 6 days. These rhamnolipids display a critical micelle concentration of 25–27 mg l^?1 and decrease the interfacial tension against hexadecane from 40 to 1·8 mN m^?1. They also have excellent emulsifying properties against long chain alkanes. Conclusions: Burkholderia glumae AU6208 can produce considerable amounts of rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. Significance and Impact of the Study: In contrast with the classical rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of rhamnolipids with this species could be easier than with Ps. aeruginosa.     

Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts

The high-level production of rhamnolipid biosurfactants is a unique feature of Pseudomonas aeruginosa and is strictly regulated in response to environmental conditions. The final step in rhamnolipid biosynthesis is catalyzed by the rhlAB genes encoding a rhamnosyltransferase. The expression of the cloned rhlAB genes was studied in heterologous hosts, either under the control of the rhlR and rhlI rhamnolipid regulatory elements or under the control of the tac promoter. A recombinant P. fluorescens strain harboring multiple plasmid-encoded copies of the rhamnolipid gene cluster produced rhamnolipids (0.25 g liter(sup-1)) when grown under nitrogen-limiting conditions. The highest yields (0.6 g liter(sup-1)) and productivities (24 mg liter(sup-1) h(sup-1)) were obtained in a recombinant Pseudomonas putida strain, KT2442, harboring promoterless rhlAB genes fused to the tac promoter on a plasmid. Active rhamnosyltransferase was synthesized, but no rhamnolipids were produced, by recombinant Escherichia coli upon induction of rhlAB gene expression.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Effect of the carbon source on biosurfactant production byPseudomonas aeruginosa 44T1

Summary Pseudomonas aeruginosa 44T1 produces rhamnolipids when grown on C₁₂ n-alkane but not with other hydrocarbons tested. Best results were obtained with olive oil as carbon source; a final production of 7.65 g rhamnolipid/l with a production yield of 38.2% was detected.     

Surface properties and sub-surface aggregate assimilation of rhamnolipid surfactants in different aqueous systems

Tensioactive properties of rhamnolipids produced by a Pseudomonas aeruginosa strain were investigated in the presence or absence of Sr²⁺ or Pb²⁺. Surface and interfacial properties, and aggregate forming properties and morphologies were studied by various techniques including scanning electron microscopy. When the pH of a rhamnolipid aqueous solution (40 mg/l) was increased from 5 to 8, irregular vesicles gradually took the shape of oligo-vesicles, then regular vesicles and finally smaller spherical vesicles. Addition of metal ions controlled the aggregates’ morphology and stability, and influenced the surface and interfacial behavior of rhamnolipid solutions.     

Dependence of Pseudomonas aeruginosa continous culture biosurfactant production on nutritional and environmental factors

Summary Continuous culture studies with Pseudomonas aeruginosa were performed in order to establish nutritional and environmental conditions necessary for high production of biosurfactants. Empirical adjustments of the mineral medium formulation showed that better yields of the active compounds, rhamnolipids, are obtained by minimizing the concentration of the respective salts of magnesium, calcium, potassium, sodium and the trace elements. Improvements in performance were more evident when the intial substrate concentration, glucose, was increased up to 73 gl^-1. Further, the ranges for pH (6.2 to 6.4) and temperature (32° to 34°C) that yield high biosurfactant biosynthesis were established. Concerning the physiological state of the microorganism, rhamnolipid formation was restricted to specific growth rates lower than D=0.14 h^-1. By applying the conditions elaborated up to 300 mg rhamnose l^-1 h^-1 (equivalent to 685 mg rhamnolipid l^-1 h^-1) were obtained in a continuous production process.     

Synthesis of itaconic acid from agricultural waste using novel Aspergillus niveus

Abstract

Filamentous fungi from the genus Aspergillus are of high importance for the production of organic acids. Itaconic acid (IA) is considered as an important component for the production of synthetic fibers, resin, plastics, rubber, paints, coatings, adhesives, thickeners and binders. Aspergillus niveus MG183809 was isolated from the soil sample (wastewater unit) which was collected from Avadi, Chennai, India. In the present study, itaconic acid was successfully produced by isolated A. niveus by submerged batch fermentation. In the fermentation process, various low-cost substrates like corn starch, wheat flour and sweet potato were used for itaconic acid production. Further, the factor influencing parameters such as substrate concentration and incubation period were optimized. Maximum yield of itaconic acid (15.65?±?1.75?g/L) was achieved by using A. niveus from corn starch at a concentration of 120?g/L after 168?hr (pH 3.0). And also extraction of itaconic acid from the fermentation was performed with 91.96?±?1.57 degree of extraction.     

Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems

Rhamnolipids are biosurfactants with interesting physico-chemical properties. However, the main obstacles towards an economic production are low productivity, high raw-material costs, relatively expensive downstream processing, and a lack of understanding the rhamnolipid production regulation in bioreactor systems. This study shows that the sequenced Pseudomonas aeruginosa strain PAO1 is able to produce high quantities of rhamnolipid during 30 L batch bioreactor cultivations with sunflower oil as sole carbon source and nitrogen limiting conditions. Thus PAO1 could be an appropriate model for rhamnolipid production in pilot plant bioreactor systems. In contrast to well-established production strains, PAO1 allows knowledge-based systems biotechnological process development combined with the frequently used heuristic bioengineering approach. The maximum rhamnolipid concentration obtained was 39 g/L after 90 h of cultivation. The volumetric productivity of 0.43 g/Lh was comparable with previous described production strains. The specific rhamnolipid productivity showed a maximum between 40 and 70 h of process time of 0.088 g_RL/g_BDMh. At the same time interval, a shift of the molar di- to mono-rhamnolipid ratio from 1:1 to about 2:1 was observed. PAO1 not only seems to be an appropriate model, but surprisingly has the potential as a strain of choice for actual biotechnological rhamnolipid production.     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Designer rhamnolipids by reduction of congener diversity: production and characterization

Background

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.

Results

Here, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.

Conclusions

The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.

     

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na⁺, 2Na⁺ and K⁺), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.     

Enhanced rhamnolipid production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> under phosphate limitation

Rhamnolipid biosurfactants are effective antimicrobial agents and provide a promising alternative to synthetic medicine. Rhamnolipid accumulation by Pseudomonas aeruginosa ATCC 9027, and associated antimicrobial activity, was quantified during phosphate limited culture. The onset of rhamnolipid production occurred below 0.35 mg phosphate/l. Thereafter rhamnolipid accumulated during phosphate exhaustion where nitrogen remained above 0.9 g/l. A maximum 4.261 g rhamnolipid/l (measured as 1.333 g rhamnose/l) was attained at a productivity of 0.013 g rhamnose/l/h. Rhamnolipid accumulation under conditions of phosphate exhaustion and nitrogen excess suggests a non-specificity of the limiting nutrient, and that rhamnolipids will be synthesised provided carbon is in excess of the metabolic capacity. Antimicrobial activity was demonstrated against Mycobacterium aurum, a surrogate for M. tuberculosis, the causal agent of most forms of tuberculosis, by a 45 mm zone of M. aurum inhibition around a well of supernatant containing 3.954 g rhamnolipid/l.