Rhamnolipid production by Pseudomonas aeruginosa immobilised in polyvinyl alcohol beads

A marine bacterium, Pseudomonas aeruginosa BYK-2 (KCTC 18012P), was immobilised by entrapment in 10% (w/v) polyvinyl alcohol beads and optimized for the continuous production of rhamnolipid. The relative activity of rhamnolipid production was maintained at 80 approximately 90% of the initial production during 15 cycles in a repeated batch culture. Continuous culture was performed in a 1.8 1 airlift bioreactor, yielding 0.1 g rhamnolipid h(-1) at a dilution rate of 0.0 18 h(-1), 25 degrees C, initial pH 7, and 0.5 vvm aeration rate with a 1.21 working volume.     

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.     

Fermentative production of rhamnolipid and purification by adsorption chromatography

Glycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24?hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96?hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8?g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.     

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.     

Different feeding strategy for the production of biosurfactant from Pseudomonas aeruginosa USM AR2 in modified bioreactor

A locally-isolated Pseudomonas aeruginosa USM AR2 possessing the ability to produce glycolipid-type biosurfactant (rhamnolipid) was used in this research to explore fermentation technology for rhamnolipid production. Rhamnolipid concentration in 2.5 L fed-batch fermentation was improved from 0.173 to 8.06 g/L by manipulating the feeding strategy and cultivation protocol. The culture was fed with petroleum diesel and complex medium. The highest rhamnolipid concentration was achieved when the culture was initially fed with both petroleum diesel and complex medium, followed by feeding of petroleum diesel only at the end of the stationary phase. Severe foaming problem was resolved by modifying and integrating a foam recycler to the bioreactor. This successfully extended the cultivation period and increased the yield of final rhamnolipid. No antifoam agent was added as this modified bioreactor allowed cultivation to proceed even under foam generation. The viscosity measurement, surface tension activity test, and drop-collapse test were performed as an indirect measure of rhamnolipid presence.     

Medium factors on anaerobic production of rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SG and a simplifying medium for in situ microbial enhanced oil recovery applications

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO₄ ^2? and Mg²⁺ are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO₃, 5.49 g/l KH₂PO₄, 6.9 g/l K₂HPO₄·3H₂O and 0.40 g/l MgSO₄ was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI₂₄ = 82.5 %. The simplifying medium was promising for in situ MEOR applications.     

Evaluation of rhamnolipid production capacity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874

A lack of understanding of the quantitative rhamnolipid production regulation in bioreactor cultivations of Pseudomonas aeruginosa and the absence of respective comparative studies are important reasons for achieving insufficient productivities for an economic production of these biosurfactants. The Pseudomonas strains DSM 7108 and DSM 2874 are described to be good rhamnolipid over-producers. The strain PAO1 on the other hand is the best analyzed type strain for genetic regulation mechanisms in the species P. aeruginosa. These three strains were cultivated in a 30-L bioreactor with a medium containing nitrate and sunflower oil as sole C-source at 30 and 37 °C. The achieved maximum rhamnolipid concentrations varied from 7 to 38 g/L, the volumetric productivities from 0.16 to 0.43 g/(L·h), and the cellular yield from 0.67 to 3.15 g/g, with PAO1 showing the highest results for all of these variables. The molar di- to mono-rhamnolipid ratio changed during the cultivations; it was strain dependent but not significantly influenced by the temperature. This study explicitly shows that the specific rhamnolipid synthesis rate per cell follows secondary metabolite-like courses coinciding with the transition to the stationary phase of typical logistic growth behavior. However, the rhamnolipid synthesis was already induced before N-limitation occurred.     

Enhanced crude oil biodegradability of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ZJU after preservation in crude oil-containing medium

This study investigated the enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU, a strain isolated from the Shengli oil field (Shandong Province, China), after preservation in a crude oil-containing medium. This strain previously could not emulsify crude oil during preservation, but after switching to a subculture in a glycerol medium for passages, it expressed increased biodegradation of crude oil within the first six passages and this biodegradation sharply decreased after the seventh passage. It is noticed that about 70% of crude oil was degraded by Pseudomonas aeruginosa ZJU in the third passage while this biodegradability was less than 19% in the seventh passage. Similar to the trend on biodegradation of crude oil, rhamnolipid production increased during the first six passages and later sharply decreased. Thus, it seems that biodegradability was proportionally related to the rhamnolipid productivity in each passage in glycerol medium. Interestingly, both rhamnolipid production and crude oil biodegradation were maintained if this strain was continuously preserved in crude oil and could be retrieved if this strain was then re-preserved in crude oil-containing medium for seven days after the significant decline in these two characteristics previously observed in the seventh passage.     

蝉拟青霉液体发酵条件优化

采用液体发酵蝉拟青霉,对蝉拟青霉的发酵条件进行优化,以提高蝉拟青霉胞外多糖产量及生物量。摇瓶发酵条件下,在单因素基础上设计正交实验确定各因素的最佳组合。优化后得最佳发酵培养基:蔗糖8%,牛肉膏0.75%,酵母膏0.125%,MgSO_4·7H_2O 0.3%,KH_2PO_4 0.2%,麸皮0.5%。该条件下胞外多糖产量为5.96 g/L,生物量为42 g/L,较优化前提高了1倍。采用发酵罐进行扩大培养,对分批发酵时的初糖浓度进行了优化,并分析了补料分批发酵对发酵过程的影响。发酵罐培养时最适初糖浓度为5%,此时生物量最高为38 g/L,多糖含量最高为5.5 g/L;采用补料分批发酵时,多糖产量最高为5.89 g/L,生物量最高为40 g/L,效果优于分批发酵。     

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography–mass spectrophotometry (MS) and by high-performance liquid chromatography–MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l⁻¹. Under these conditions, the surface tension of the culture decreased to 30 mN m⁻¹, and the critical micelle concentration was 26.5 mg l⁻¹. It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.     

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.     

产鼠李糖脂菌株种子培养条件和C、N源的优化

对自行筛选的3个可利用废弃油脂进行发酵生产鼠李糖脂的铜绿假单胞菌菌株进行评价,并进行了种子培养条件和摇瓶发酵部分条件的优化。种子培养优化实验表明,当培养基pH 6～8,培养温度为30 ℃时最利于菌体生长。菌株均具有一定的耐盐性,在5%的盐度下生长未受到明显抑制,因此在沿海地区采用盐水或海水发酵具有较广阔的应用前景。通过排油圈、表面张力、苯酚-H2SO4比色法比较了这3个菌株的表面活性剂表面活性的大小,以表现较好的Z41进行了摇瓶发酵条件的优化。单因素实验表明,发酵较优条件为发酵温度30 ℃,接种量5%。在此基础上,通过正交试验对Z41菌株发酵培养基中的C、N源进行了研究,实验结果表明,在考虑因素间交互作用和发酵成本的情况下,最佳C源为3%炸货油,最佳N源为3.5 g/L尿素。在此发酵条件下,糖脂产量较高13.024 g/L,且成本较低。     

Production of Rhamnolipid Biosurfactant by Fed-batch Culture of Pseudomonas aeruginosa Using Glucose as a Sole Carbon Source

The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant. With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.     

Effects of carbon and nitrogen sources on the proteome of Pseudomonas aeruginosa PA1 during rhamnolipid production

The PA1 strain of Pseudomonas aeruginosa isolated from oil waste produces rhamnolipid, a biodegradable surfactant with applications in several industrial and environmental fields. The metabolic pathway and genetic regulation of rhamnolipid production in P. aeruginosa are poorly understood. Herein, several proteins directly or indirectly related to rhamnolipid production and their genetic regulations were identified by comparative proteomic. We compared the proteome of P. aeruginosa PA1 after fermentation in two different conditions of carbon and nitrogen sources: condition A allowed rhamnolipid production and condition B prevented it. Protein extracts from cellular pellets were compared using 2D-PAGE stained with colloidal Coomassie followed by MALDI-TOF/TOF mass spectrometry. We identified 21 differentially expressed proteins, including those involved in secretion, quorum sensing, oxidative response and metabolism.     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

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.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l⁻¹) and at 72 h (751 mg l⁻¹) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30°C and 100 rpm. It reached 8373 mg l⁻¹ when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

预水解发酵碳源对铜绿假单胞菌NY3产鼠李糖脂特性及应用效能的影响研究

发酵碳源对铜绿假单胞菌NY3(Pseudomonas aeruginosa NY3)产鼠李糖脂(Rhamnolipids,Rha)的特性影响较大。研究了利用废弃动物油作为发酵碳源时,其碱预水解和酶预水解对NY3菌发酵产鼠李糖脂产量、产物结构和性能的影响,从碳源水解酸值与水解产物、鼠李糖脂组分结构和实际应用效果进行了研究。碱、酶预水解实验发现,碳源酸值由初始的19.81 mg/g分别提高到72.04 mg/g和73.75 mg/g,气质联用(GC-MS)分析检测结果表明,碱、酶预水解后,碳源均释放7种C14-C18碳链的脂肪酸,鼠李糖脂产量由未预水解的8.28 g/L分别提高到15.35 g/L和17.63 g/L。液质联用(LCMS-IT-TOF)分析结果表明,用未预水解及碱、酶预水解碳源发酵时,NY3菌所产鼠李糖脂中单糖脂含量分别为62.07%、65.67%、87.32%。利用NY3菌在中试条件下处理高浓度石化企业油污泥,发现鼠李糖脂能促进NY3菌去除油污泥中的石油烃,且促进作用强弱顺序为未预水解产Rha碱预水解产Rha酶预水解产Rha。     

Optimization of Nutrient Requirements and Culture Conditions for the Production of Rhamnolipid from Pseudomonas aeruginosa (MTCC 7815) using Mesua ferrea Seed Oil

Environmental awareness has led to a serious consideration for biological surfactants and hence non-edible vegetable oils may serve as a substitute carbon source for bio-surfactant production (rhamnolipid) which might be an alternative to complex synthetic surfactants. There are reports of rhamnolipid production from plant based oil giving higher production than that of glucose because of their hydrophobicity and high carbon content. Therefore the contribution of non-edible oil such as Mesua ferrea seed oil could serve as a good carbon source for rhamnolipid production. Moreover the use of rhamnolipid production from non-edible plant based seed oil has not been reported elsewhere. The present work focus on the optimal production of rhamnolipid by considering both micro and macro nutrients and culture conditions using response surface methodology. The study observes that micronutrients play a significant role in rhamnolipid production from Pseudomonas aeruginosa (MTCC 7815). The investigation results with the statistically optimize parameters able to produce a higher rhamnolipid production and this methodology could be used to optimize the nutrients requirements and culture conditions. The present findings would assist in bioremediation of crude oil contaminated ecosystems.