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     

Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.     

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 degrees 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.     

一株产脂肽类表面活性剂的碱性Dietzia菌及特性研究

【目的】筛选降解性能良好的产生物表面活性剂的菌株,对其进行分类学鉴定,确定所产表面活性剂物质并对各影响因素进行评价。【方法】利用液体石蜡为底物筛选降解性能良好的产生物表面活性剂菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等实验确定菌株的分类地位。通过排油圈活性、表面张力值、薄层层析等方法确定生物表面活性剂的性质,分析碳、氮源和温度、pH、盐浓度各因素对菌株产生物表面活性剂的影响。【结果】从大连新港采集的样品中分离得到一株产表面活性剂的嗜碱菌株3372,经分类鉴定表明其是Dietzia cercidiphylli的新菌株。嗜碱菌3372发酵液粗提物的排油直径为6.1 cm,表面张力可从67.62 mN/m降到32.95 mN/m,经薄层层析分析,初步鉴定为脂肽类表面活性剂。综合各因素对发酵液表面活性的影响,菌株3372在pH为9.0、适盐浓度为3%的培养基中,经30°C培养可将发酵液表面张力值降到最低。【结论】嗜碱菌3372是脂肽类生物表面活性剂产生菌的新成员,其在高盐碱条件下产生表面活性剂的特性在工业应用上有一定的潜力。     

Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by <Emphasis Type="Italic">Pseudomonas nitroreducens</Emphasis> isolated from soil

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from petroleum-contaminated soil was investigated. The effects of carbon, nitrogen and carbon to nitrogen ratio on biosurfactant production were examined using mineral salts medium as the growth medium. The tenso-active properties (surface activity and critical micelle concentrations of the produced biosurfactant were also evaluated. The best carbon source, nitrogen source were glucose and sodium nitrate giving rhamnolipid yields of 5.28 and 4.38 g l⁻¹, respectively. The maximum rhamnolipid production of 5.46 g l⁻¹ was at C/N (glucose/sodium nitrate) of 22. The rhamnolipid biosurfactant reduced the surface tension of water from 72 to ~37 mN/m. It also has critical micelle concentration of ~28 mg l⁻¹. Thus, the results presented in our reports show that the produced rhamnolipid can find wide applications in various bioremediation activities such as enhanced oil recovery and petroleum degradation.     

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1: batch versus chemostat production

Functionalized medium-chain-length polyhydroxyalkanoates (mclPHAs) have gained much interest in research on biopolymers because of their ease of chemical modification. Tailored olefinic mclPHA production from mixtures of octanoic acid and 10-undecenoic acid was investigated in batch and dual (C,N) nutrient limited chemostat cultures of Pseudomonas putida GPo1 (ATCC 29347). In a batch culture, where P. putida GPo1 was grown on a mixture of octanoic acid (58 mol%) and 10-undecenoic acid (42 mol%), it was found that the fraction of aliphatic monomers was slightly lower in mclPHA produced during exponential growth than during late stationary phase. Thus, the total monomeric composition changed over time indicating different kinetics for the two carbon substrates. Chemostat experiments showed that the dual (C,N) nutrient limited growth regime (DNLGR) for 10-undecenoic acid coincided with the one for octanoic acid. Five different chemostats on equimolar mixtures of octanoic acid and 10-undecenoic acid within the DNLGR revealed that the monomeric composition of mclPHA was not a function of the carbon to nitrogen (C(0)/N(0)) ratio in the feed medium but rather of the dilution rate. The fraction of aliphatic monomers in the accumulated mclPHA was slightly lower at high dilution rates and increased towards low dilution rates, again indicating different kinetics for the two carbon substrates in P. putida GPo1.     

Chemical characterization and physical and biological activities of rhamnolipids produced by Pseudomonas aeruginosa BN10

Pseudomonas aeruginosa BN10 isolated from hydrocarbon-polluted soil was found to produce rhamnolipids when cultivated on 2% glycerol, glucose, n-hexadecane, and n-alkanes. The rhamnolipids were partially purified on silica gel columns and their chemical structures elucidated by combination of one- and two-dimensional 1H and 13C NMR techniques and ESI-MS analysis. Eight structural rhamnolipid homologues were identified: Rha-C10-C8, Rha-C10-C10, Rha-C10-C12:1, Rha-C10-C12, Rha2-C10-C8, Rha2-C10-C10, Rha2-C10-C12:1, and Rha2-C10-C12. The chemical composition of the rhamnolipid mixtures produced on different carbon sources did not vary with the type of carbon source used. The rhamnolipid mixture produced by Pseudomonas aeruginosa BN10 on glycerol reduced the surface tension of pure water from 72 to 29 mN m(-1) at a critical micellar concentration of 40 mg 1(-1), and the interfacial tension was 0.9 mN m(-1). The new surfactant product formed stable emulsions with hydrocarbons and showed high antimicrobial activity against Gram-positive bacteria. The present study shows that the new strain Pseudomonas aeruginosa BN10 demonstrates enhanced production of the di-rhamnolipid Rha2-C10-C10 on all carbon sources used. Due to its excellent surface and good antimicrobial activities the rhamnolipid homologue mixture from Pseudomonas aeruginosa BN10 can be exploited for use in bioremediation, petroleum and pharmaceutical industries.     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

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.     

Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida

A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5 g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4.7H2O and 0.025% CaCl2.2H2O, with shaking at 200 rpm for an incubation period of 100 h at 30 degrees C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes.     

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens immobilized on Ca2+ alginate beads and under resting cell condition

Pseudomonas nitroreducens MILB-8054A isolated from petroleum-contaminated soil, immobilized on calcium alginate beads, and under resting cell condition, produced biosurfactants. Immobilized cells gave a best yield of 5.6 g rhamnolipid l^?1 using sucrose as carbon source. Time course study using resting cells showed that 2 % v/v of palm oil (preculture carbon source) and 10 % diesel (carbon source) gave the best rhamnolipid yield of 5.1 g l^?1 at pH 8 and temperature of 30 °C. Carbon utilization by resting cells was compared with that of growing cells. The best biosurfactant recovery procedure was acetone extraction.     

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.     

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.     

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.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain

The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new Bacillus subtilis 22BN strain was investigated. The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l(-1)). Biosurfactant production was detected by surface tension lowering and emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 +/- 1% and 75 +/- 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first report of Bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant.     

Structural characterization of rhamnolipid produced by Pseudonomas aeruginosa strain FIN2 isolated from oil reservoir water

Biosurfactant-producing microorganisms inhabiting oil reservoirs are of great potential in industrial applications. Yet, till now, the knowledge about the structure and physicochemical property of their metabolites are still limited. The aim of this study was to purify and structurally characterize the biosurfactant from Pseudomonas aeruginosa strain FIN2, a newly isolated strain from an oil reservoir. The purification was conducted by silica gel column chromatography followed by pre-RP HPLC and the structural characterization was carried out by GC–MS combined with MS/MS. The results show that the biosurfactant produced by FIN2 is rhamnolipid in nature and its four main fractions were identified to be Rha-C10-C10(46.1 %), Rha–Rha-C10-C10(20.1 %), Rha-C8-C10 (7.5 %) and Rha-C10-C12:1(5.5 %), respectively. Meanwhile, the rarely reported rhamnolipid congeners containing β-hydroxy fatty acids of C6, C9, C10:1 and C11 were also proved to be present in the rhamnolipid mixture produced. The rhamnolipid mixture exhibited a strong surface activity by lowering the surface tension of distilled water to 28.6 mN/m with a CMC value of 195 mg/l.     

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.

     

Optimization of Xylanase Production by Sporotrichum thermophile Using Corn Cobs and Response Surface Methodology

A 3² central composite experimental design was performed with the aim of optimizing the production of xylanase by Sporotrichum thermophile grown on corn cobs in submerged cultures. Various carbon and nitrogen sources were consecutively optimized, and corn cobs and ammonium phosphate concentrations were selected as substrates to test the effect of two variables, i.e., both substrate concentrations, on xylanase production. A second‐order quadratic model and a response surface method showed that the optimum conditions for xylanase production were 2.7 % [w/v] corn cobs and 0.7 % [w/v] ammonium phosphate. These optimum conditions were transferred to 7 L bioreactors, where activities as high as 56 U/mL were obtained.