Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum

Biosurfactants are microbial secondary metabolites. The most studied are rhamnolipids, which decrease the surface tension and have emulsifying capacity. In this study, the production of biosurfactants, with emphasis on rhamnolipids, and diesel oil degradation by 18 strains of bacteria isolated from waste landfill soil contaminated by petroleum was analyzed. Among the studied bacteria, gram‐positive endospore forming rods (39%), gram positive rods without endospores (17%), and gram‐negative rods (44%) were found. The following methods were used to test for biosurfactant production: oil spreading, emulsification, and hemolytic activity. All strains showed the ability to disperse the diesel oil, while 77% and 44% of the strains showed hemolysis and emulsification of diesel oil, respectively. Rhamnolipids production was observed in four strains that were classified on the basis of the 16S rRNA sequences as Pseudomonas aeruginosa. Only those strains showed the rhlAB gene involved in rhamnolipids synthesis, and antibacterial activity against Escherichia coli, P. aeruginosa, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora, and Ralstonia solanacearum. The highest production of rhamnolipids was 565.7 mg/L observed in mineral medium containing olive oil (pH 8). With regard to the capacity to degrade diesel oil, it was observed that 7 strains were positive in reduction of the dye 2,6‐dichlorophenolindophenol (2,6‐DCPIP) while 16 had the gene alkane mono‐oxygenase (alkB), and the producers of rhamnolipids were positive in both tests. Several bacterial strains have shown high potential to be explored further for bioremediation purposes due to their simultaneous ability to emulsify, disperse, and degrade diesel oil. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:262–270, 2016     

The Influence of Rhamnolipids on Aliphatic Fractions of Diesel Oil Biodegradation by Microorganism Combinations

Twelve different bacteria–yeast combinations were tested for determination of their ability to biodegrade diesel oil. The cell surface properties of the bacterial and yeast strains were correlated with the type of carbon source used in the experiments. The highest biodegradation of diesel oil after 7 days was obtained for the following combinations: Aeromonas hydrophila MR4–Yarrowia lipolytica EH 56 (87 %) and Xantomonas maltophila MRP7–Candida maltosa EH15 (90 %). Degradation performances of 10 of 12 combinations were enhanced by the presence of rhamnolipids. The highest increases were observed for A. hydrophila MR4–C. maltosa EH15 (from 34 to 67 %), A. hydrophila MR4–C. maltosa EH60 (from 47 to 76 %) and for Pseudomonas stutzeri MR7–C. maltosa EH60 (from 29 to 79 %). The addition of rhamnolipids to the system reduces the removal time of diesel oil from the contaminated water and changes the microbial adhesion to hydrocarbons. Modification of the cell surface of the tested strain during biodegradation is a very important factor determining the removal of hydrophobic compounds.     

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.     

Interactions between rhamnolipid biosurfactants and toxic chlorinated phenols enhance biodegradation of a model hydrocarbon-rich effluent

Surfactant-mediated treatment increases hydrocarbon solubilization and potentially facilitates biodegradation, unless toxic co-contaminants inhibiting microbial activity are present in the hydrocarbon mixture. We assessed the effect of rhamnolipids on the performance of a bacterial consortium degrading diesel fuel employed as a model hydrocarbon-rich effluent, co-contaminated with toxic phenol, 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP). This approach led to the unexpected finding that rhamnolipids reduced toxicity of 4-CP and 2,4-DCP to the hydrocarbon-degrading cells. The facts that rhamnolipids decreased diesel fuel - water partition coefficient (K_FW) of 4-CP and 2,4-DCP and modified aggregate size distribution profiles of the dispersed diesel fuel - chlorinated phenols solutions, suggest the existence of specific interactions between rhamnolipids and the co-contaminants. Due to the polar nature of 4-CP and 2,4-DCP, possible explanations involve adsorption of 4-CP and 2,4-DCP on the surface of biosurfactant aggregates. This property of rhamnolipids is of interest to those using biosurfactants for microbial treatment of hydrocarbon-rich wastewaters co-contaminated with toxic compounds.     

The impact of long-term contact of Achromobacter sp. 4(2010) with diesel oil – Changes in biodegradation,surface properties and hexadecane monooxygenase activity

A bacterial strain was isolated from soil that was contaminated with diesel oil and was used in our experiments. The strain was then phenotypically, biochemically and genetically tested and named as Achromobacter 4(2011). In order to examine the impact of long-term contact with diesel oil of bacterial cells, the strain was stored under different conditions – on standard nutrient agar plates and on agar plates with 50 μl diesel oil as a sole carbon and energy source. The results clearly indicated that longer contact with diesel oil led to changes in both the bacterial surface and biochemical properties, as well as the hexadecane monooxygenase activity. Moreover, the fatty acid profiles also changed, leading to an increased content of saturated fatty acids. In addition, the rates of biodegradation of diesel oil were higher even when supplemented with the surfactants – rhamnolipids and saponins. This work demonstrates that prolonged contact of microorganisms with diesel oil can lead to many changes, not only in biodegradation potential, but also in their surface and genetic properties.     

不同结构配比的鼠李糖脂乳化活性与油泥清洗效果

以大庆油田原油和含油污泥为对象,研究不同结构配比鼠李糖脂表面活性剂乳化活性及其对含油污泥清洗效果的影响,并优化清洗工艺参数。结果表明:单鼠李糖脂比例越高,其表面活性越好;双鼠李糖脂比例越高,其对原油的乳化能力越强;临界胶束浓度随着双鼠李糖脂比例的增加而逐渐加大;单、双鼠李糖脂配比不同的表活剂对油泥的清洗效果也不同,质量比为50∶50时清洗效率最高;鼠李糖脂浓度为1.0 g·L^-1、热洗时间为1.5h、热洗温度为65℃、洗脱强度为220 r·min^-1、固液质量比为1∶5条件下,油泥的清洗效率最高,可达81.3%;含油率为29.6%的落地油泥,经一级洗涤后油泥残油率降至5.5%,原油回收率达到87.3%,清洗出的原油无明显乳化,易于分离。由此可知,鼠李糖脂的单、双糖脂比例不同对其理化性质和清洗含油污泥的效果均有不同程度的影响。     

Structure,properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater

The properties and applications of rhamnolipid surfactants produced by Pseudomonas aeruginosa L2-1 from cassava wastewater added with waste cooking oil (CWO) as low-cost substrate, were investigated and compared with the commercial rhamnolipid mixture JBR599 (Jeneil Biosurfactant Co., Saukville, USA). The rhamnolipids produced by strain L2-1 were characterized by high performance liquid chromatography–mass spectrometry. Sixteen different rhamnolipid congeners were detected, with Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀ being the most abundant. The L2-1 rhamnolipids from CWO showed similar or better tensioactive properties than those from JBR599, with a minimal surface tension of 30 mN/m and a critical micelle concentration (CMC) of 30 mg/l. The L2-1 biosurfactants formed stable emulsions with several hydrocarbons and showed excellent emulsification of soybean oil (100%). These rhamnolipids removed 69% of crude oil present in contaminated sand samples at the CMC and presented antimicrobial activity against Bacillus cereus (32 μg/ml), Micrococcus luteus (32 μg/ml) and Staphylococcus aureus (128 μg/ml). These results demonstrate that the rhamnolipids produced in CWO can be useful for industrial applications, such as the bioremediation of oil spills.     

Biodegradation of diesel oil by an Arabian Sea sediment culture isolated from the vicinity of an oil field

Laboratory scale batch studies were performed to test the diesel oil biodegradation ability of ES1 cultures isolated from Arabian Sea sediments obtained from the vicinity of an oil field. This culture could utilize diesel as the sole source of carbon and energy. Under aerobic conditions, 39% loss of diesel oil was observed over 8 days where 80% of the loss was due to aliphatic constituents. Under anoxic nitrate reducing conditions the rate and extent of degradation was significantly lower, i.e., 18% over 50 days. Salt acclimatized cultures could tolerate salinities up to 3.5% and demonstrated optimal performance at a salinity of 0.5%. The optimum N/P ratio for these cultures was found to be in the range of 2:1-5:1. Addition of two trace elemental substance formulations exhibited a significant inhibitory effect on culture growth. This culture has good potential for decontamination of oil-contaminated marine and subsurface environments.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield

Abstract

In this study, a diesel oil-degrading bacterium was isolated from an oilfield water injection (water-bearing formations, 1,205?m depth) in Algeria. The bacterial strain, designated NL1, was cultivated on diesel oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene sequence (KY397882) placed NL1 strain closely related to distinct cultivated species of the Delftia genus. Optimal diesel oil biodegradation by Delftia sp NL1 strain occurred at pH 11, 40?°C, 2?M NaCl and initial hydrocarbon concentration of 5% (v/v) as sole carbon source. GC-MS analyses evidenced that strain Delftia sp NL1 was able to degrade more than 66.76% of diesel oil within only 7?days. On the other hand, and in the same conditions, biosurfactant production by Delftia sp NL1 was also evaluated evidencing high emulsifying capacity (E₂₄ = 81%), ability to lower the surface tension of growing media (with the value of 25.7?mN m^?1), and production of glycolipids (8.7?g L^?1) as biosurfactants. This research presents indigenous strain Delftia sp NL1 for diesel degradation and synthesis of biosurfactant in extreme conditions. In this sense, strain NL1 is a good candidate for possible in situ oil recovery and in wastewater treatment in refineries and oil terminals in petroleum industry.     

Biocatalytic desulfurization of diesel oil in an air-lift reactor with immobilized Gordonia nitida CYKS1 cells

A new type of air-lift reactor with immobilized Gordonia nitida CYKS1 cells on a fibrous support was designed and used for the biocatalytic desulfurization (BDS) of diesel oil. Its performance was evaluated at different phase ratios of the oil to the aqueous medium (or oil phase fractions) and different sucrose concentrations. When the reaction mixture contained 10% diesel oil (v/v), 61-67% of sulfur was removed as the sulfur content decreased from 202-250 to 76-90 mg L(-1) in 72 h. The sulfur content did not decrease any further because the remaining sulfur compounds were recalcitrant to BDS. During the desulfurization, the strain CYKS1 consumed hydrocarbons in the diesel oil, mainly n-alkanes with 10-26 carbons, as carbon source even though an easily available carbon source, sucrose, was supplied.     

Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8

The potential production of rhamnolipids was demonstrated using the thermophilic eubacterium Thermus thermophilus HB8 and sunflower seed oil or oleic acid as carbon sources. Sunflower seed oil was directly hydrolyzed by secretion of lipase and became a favorable carbon source for rhamnolipids production. Rhamnolipids levels were attainted high values, comparable to those produced by Pseudomonas strains from similar sources. Rhamnolipids synthesis in oleic acid exhibited a long period of induction, while in sunflower seed oil, the synthesis is more rapid. Glucose resulted in a more protracted period of rhamnolipids production after exhaustion of each or both carbon sources. Both mono- and di-rhamnolipids were identified by thin-layer chromatography (TLC) in the total rhamnolipids extract. The molecular composition of the produced biosurfactant was evaluated by Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and LC-MS analysis. Furthermore, secretion of rhamnolipids was confirmed on agar plates. The antimicrobial activity of rhamnolipids was detected against the bacterium Micrococcus lysodeikticus using a lysoplate assay. These results demonstrate that rhamnolipids produced in these substrates can be useful in both environmental and food industry applications by using cheap oil wastes. The alternative use of this thermophilic microorganism opens a new perspective concerning the valorization of wastes containing plant oils or frying oils to reduce the cost of rhamnolipids production.     

Bioelement Status with Oral Administration of Fish Oil Methyl Ester and Diesel Fuel in Male Rats

This paper is a study on the effects on the amounts of trace elements in case of possible repeat accidental or environmental exposure with fish oil biodiesel. For this purpose, 35 male Wistar albino rats were used in the study. Rats were divided into five groups. The first group was determined as the control group. The rats in this group were gavaged orally with 250?mg/kg sunflower oil. The rats in the second and third groups were administered by oral gavage of 250?mg/kg (D1) and 500?mg/kg (D2) diesel fuel mixed with equal amounts of sunflower oil, respectively. The rats in the fourth group were administered by oral gavage of 250?mg/kg fish oil biodiesel (F1) and the rats in the fifth group were administered by oral gavage of 500?mg/kg fish oil biodiesel (F2), both mixed with equal amounts of sunflower oil. At the end of the study, bioelement concentrations in the serum and the kidney, lung, and liver tissues were measured using inductively coupled plasma-optical emission spectroscopy. It was observed that serum Ca, Mg, and Sr concentrations were significantly (p?相似文献   

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.     

Prepared Bed Land Treatment of Soils Containing Diesel and Crude Oil Hydrocarbons

An ex situ, field-scale, prepared bed land treatment unit (LTU) was used to bio-remediate soils containing petroleum hydrocarbons. Two soils were treated in side-by-side units to compare performance: (1) a clayey silt containing crude oil hydrocarbons from releases 30 to 40 years ago and (2) a silty sand containing diesel fuel hydrocarbons from a leak about three years prior to the bioremediation. The effectiveness of the bioremediation in the LTU was evaluated over a period of 18 months. The results indicated that: (1) prepared bed bioremediation reduced the hydrocarbon concentration, mobility, and relative toxicity in the soil with the diesel fuel, and (2) chemical bioavailability appeared to limit bioremediation of the soil containing the crude oil hydrocarbons. Although the soils containing the crude oil hydrocarbons contained an average of 10,000?mg TPH/kg dry soil, these soils had limited hydrocarbon availability, nontoxic conditions, and low potential for chemical migration. For the soils containing the diesel fuel, active prepared bed bioremediation of about 15 weeks was adequate to reach an environmentally acceptable endpoint. At that time, there was little further TPH loss, no Microtox^TM toxicity, and limited hydrocarbon mobility.     

Microbial synthesis of rhamnolipids by Pseudomonas aeruginosa (ATCC 10145) on waste frying oil as low cost carbon source

Vegetable edible oils and fats are mainly used for frying purposes in households and the food industry. The oil undergoes degradation during frying and hence has to be replaced from time to time. Rhamnolipids are produced by microbial cultivation using refined vegetable oils as a carbon source and Pseudomonas aeruginosa (ATCC 10145). The raw material cost accounts for 10-30% of the overall cost of biosurfactant production and can be reduced by using low-cost substrates. In this research, attention was focused on the preparation of rhamnolipids, which are biosurfactants, using potential frying edible oils as a carbon source via a microbial fermentation technique. The use of low-cost substrates as a carbon source was emphasized to tilt the cost of production for rhamnolipids. The yield was 2.8 g/L and 7.5 g/L from waste frying oil before and after activated earth treatment, respectively. The crude product contained mainly dirhamnolipids, confirmed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LC-MS), and (1)H-nuclear magnetic resonance (NMR). Hence, the treatment can be used to convert waste frying oil as a low-cost substrate into a cost-effective carbon source.     

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.     

Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: effect of the type of blend and the addition of biosurfactants

Biodegradation experiments for diesel/biodiesel blends in liquid cultures by-petroleum degrading microbial consortium showed that for low amendments of biodiesel (10%) the overall biodegradation efficiency of the mixture after seven days was lower than for petroleum diesel fuel. Preferential usage of methyl esters in the broad biodiesel concentration range and diminished biodegradation of petroleum hydrocarbons for 10% biodiesel blend was confirmed. Rhamnolipids improved biodegradation efficiency only for blends with low content of biodiesel. Emulsion formation experiments showed that biodiesel amendments significantly affected dispersion of fuel mixtures in water. The presence of rhamnolipids biosurfactant affected stability of such emulsions and altered cell surface properties of tested consortium.     

Enhanced Biodegradation of Casablanca Crude Oil by A Microbial Consortium in Presence of a Rhamnolipid Produced by Pseudomonas Aeruginosa AT10

The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate. Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium. The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures. The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation. Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction. The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.     

Characterization of rhamnolipid biosurfactants produced by recombinant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain DAB with removal of crude oil

Objectives

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.

Results

Strain DAB had a higher yield of 17.3 g rhamnolipids l^?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l^?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m^?1 with CMC of 90 mg l^?1. The predominant rhamnolipid congeners were Rha–C₁₀–C₁₀ and Rha–Rha–C₁₀–C₁₀ detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.

Conclusion

Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.