Rhamnolipids Induced by Glycerol Enhance Dibenzothiophene Biodegradation in Burkholderia sp. C3

In highly urbanized areas,pollution from anthropogenic activities has compromised the integrity of the land,decreasing soil availability for agricultural practices.Dibenzothiophene(DBT)is a heterocyclic aromatic hydrocarbon frequently found in urbanized areas,and is often used as a model chemical to study the microbial transformation of pollutants.The potential for human exposure and its health risk makes DBT a chemical of concern;thus,it needs to be environmentally managed.We utilized glycerol to stimulate Burkholderia sp.C3 in order to degrade DBT in respect to①DBT biodegradation kinetics,②bacterial growth,③rhamnolipid(RL)biosynthesis,and④RL secretion.Under an optimum glycerol-to-DBT molar ratio,the DBT biodegradation rate constants increased up to 18-fold and enhanced DBT biodegradation by 25%–30%at day 1 relative to cultivation with DBT alone.This enhancement was correlated with an increase in bacterial growth and RL biosynthesis.Proteomics studies revealed the enzymes involved in the upper and main steps of RL biosynthesis.The RL congeners Rha-C10-C10,Rha-Rha-C10-C10,Rha-Rha-C10-C12,and Rha-Rha-C12-C12 were identified in the medium supplemented with glycerol and DBT,whereas only Rha-C12-C12 was identified in cultures without glycerol or with RL inhibitors.The studies indicated that glycerol enhances DBT biodegradation via increased RL synthesis and bacterial growth.The results warrant further studies of environmental biostimulation with glycerol to advance bioremediation technologies and increase soil availability for agricultural purposes.

Physicochemical Properties of Rhamnolipid Biosurfactant from <i>Pseudomonas aeruginosa</i>PA1 to Applications in Microemulsions

Interfacial properties rhamnolipids from an extract produced by a strain of Pseudomonas aeruginosa were analyzed in this study. The extract of rhamnolipid was characterized by surface tension in different conditions;interfacial tension with different hydrocarbons;critical micelle concentration under different pH and temperatures;particle size and emulsification capacity using laser light profiling. It was observed that the rhamnolipids extract are sensitive to variations in pH, thermostable and function as good emulsificant for emulsification of methyl methacrylate. The emulsion stability order in function of the oil phase was methyl methacrylate > emulsions of castor oil > emulsion n-heptane > emulsion toluene > emulsion hexadecane > octane emulsion. The data presented show that rhamnolipid extracts may be used to formulate stable emulsions of methyl methacrylate. This process can be used to do nano/microsphere of polymethyl methacrylate.

Simultaneous Desorption of Polycyclic Aromatic Hydrocarbons and Heavy Metals from Contaminated Soils by Rhamnolipid Biosurfactants

Hydrophobic organic compounds(HOCs)and heavy metals(HMs)are toxins that usually coexist in natural environments.Due to the differences in their properties,it remains challenging to simultaneously remove HMs and HOCs.In this study,the removal of phenanthrene(Phe)and lead(Pb)from co-contaminated soils by single rhamnolipid(RL)and mixed RL-sophorolipid(SL)biosurfactants were evaluated via soil column experiments.Biosurfactant micelle sizes were determined by dynamic light scattering,and the mechanisms of micelle solubilization were studied.The effects of biosurfactant concentrations,pH,washing agent salinity and the ageing time of polluted soils on Phe and Pb desorption efficiencies were also assessed.The substantial removal of Phe and Pb using mixed RL-SL systems,when molar fractions of RLs were 0.7,was attributed to large mixed micelle formation and lower sorption losses of these systems.The optimal pH value was 6.0,while Phe desorption was favoured at high RLs and low ionic strengths.However,the RLs concentration and ionic strength had no obvious influence on Pb removal.In addition,both Phe and Pb desorption decreased with increased ageing of the polluted soils.Combined RL-SL biosurfactants can be effective for simultaneously removing HOCs and HMs from polluted soils.

Reuse of waste frying oil for production of rhamnolipids using Pseudomonas aeruginosa zju.u1M

In this work,rhamnolipid production was investigated using waste frying oil as the sole carbon source. By culture in shaking flasks,a naturally isolated strain synthesized rhamnolipid at concentration of 12.47 g/L and its mutant after treatment by UV light increased this productivity to 24.61 g/L. Fermentation was also conducted in a 50 L bioreactor and the productivity reached over 20 g/L. Hence,with a stable and high productive mutant strain,it could be feasible to reuse waste frying oil for rhamnolipid production on industrial scale.

Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids

The potential biodegradation of crude oil was assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa which produced 15.4 g/L rhamnolipids when cultured in a basal mineral medium using glycerol as a sole carbon source. However, neither cell growth nor rhamnolipid production was observed in the comparative culture system using crude oil as the sole carbon source instead. As rhamnolipid, an effective biosurfactant, has been reported to stimulate the biodegradation of hydrocarbons, 1 g/L glycerol or 0.22 g/L rhamnolipid was initially added into the medium to facilitate the biodegradation of crude oil. In both situations, more than 58% of crude oil was degraded and further converted into accumulated cell biomass and rhamnolipids. These results suggest that Pseudomonas aeruginosa could degrade most of crude oil with direct or indirect addition of rhamnolipid. And this conclusion was further supported by another adsorption experiment, where the ad-sorption capacity of crude oil by killed cell biomass was negligible in comparison with the biologic activities of live cell biomass.

Optimizing rhamnolipid production by Pseudomonas aeruginosa ATCC 9027 grown on waste frying oil using response surface method and batch-fed fermentation

Rhamnolipid production by Pseudomonas aeruginosa ATCC 9027 with waste frying oil as sole carbon source was studied using response surface method. Cultures were incubated in shaking flask with temperature, NO3- and Mg2+ concentrations as the variables. Meanwhile, fed-batch fermentation experiments were conducted. The results show that the three variables are closely related to rhamnolipid production. The optimal cultivation conditions are of 6.4 g/L NaNO3 , 3.1 g/L MgSO4 at 32 ℃, with the maximum rhamnolipid production of 6.6 g/L. The results of fed-batch fermentation experiments show that feeding the oil in two batches can enhance rhamnolipid production. The best time interval is 72 h with the maximum rhamnolipid production of 8.5 g/L. The data are potentially useful for mass production of rhamnolipid on oil waste with this bacterium.

Enzymatic reaction of ethanol and oleic acid by lipase and lignin peroxidase in rhamnolipid(RL) reversed micelles

An environment friendly bio-surfactant of rhamnolipid(RL) was used as a solvent. The enzymatic reaction of oleic acid catalyzed by lipase and lignin peroxidase(lip) was evaluated. The optimum conditions of enzymatic reaction catalyzed by lipase(lip) were water to amphiphile molar ratio of 30(20), RL of 60(60) critical micelle concentration(CMC), pH of 7.0(3.0) and temperature of 40(30) °C, respectively. The change of enzyme conformation indicates that, for catalytic of lipase, water content is the most important factor of the enzymatic reaction of oleic acid, and p H for lip. With individual optimum conditions, the enzymatic efficiency of oleic acid catalyzed by lipase is higher than that by lip. In the presence of ethanol, the enzymatic reaction of oleic acid catalyzed by lipase suits Ping-Pong Bi-Bi mechanism. As an alternative to chemical reversed micelles, the RL reversed micelles are promising methods to enzymatic reaction of oleic acid.

鼠李糖脂抑制芽孢态蜡样芽孢杆菌的活性及机制

以蜡样芽孢杆菌(Bacillus cereus)芽孢为研究对象,从表观、形态和胞膜状态等方面解析鼠李糖脂(rhamnolipids,RLs)对芽孢态B.cereus的抑制活性和作用机制。结果表明,RLs的最低抑菌浓度和最低杀菌浓度分别为80.0 mg/L和160.0 mg/L。两个浓度的RLs均可以引起芽孢表面出现严重的皱缩和凹陷,破坏芽孢细胞膜完整性和通透性,导致胞内电解质、DNA和蛋白质等生物大分子外泄。同时,RLs可以通过降低芽孢的耐热性、产生胞内氧化应激反应、降低表面黏附能力、结合并干扰DNA分子等多种抑菌机制对B.cereus芽孢起到抑制和灭活作用。研究结果有利于推动RLs在食品安全领域的开发和应用。

鼠李糖脂对污泥自热高温微氧体系有机碳源释放的影响

为探究表面活性剂在自热高温微氧(ATMAD)发酵体系中对有机碳源的释放会产生何种影响,选取鼠李糖脂(RL)这一无毒无害且易于获取的典型生物表面活性剂,依托ATMAD发酵体系,展开实验探究.结果表明,投加0.02~0.07g/gTSS鼠李糖脂对反应体系中污泥的溶解,可溶性有机物的释放及挥发性脂肪酸的积累有较为明显的促进作用,相关指标均有明显提高,产酸相关功能菌的丰度也有所增加,当投加量为0.07g/gTSS时,VFA累积浓度最高,为空白组的1.63倍,判断此时为最佳投放浓度;当投加浓度高于0.10g/gTSS时,会对有机碳源的释放产生抑制.在ATMAD体系中添加适量鼠李糖脂对于开发优质外加碳源这一工作具有较高的可行性.

Combination of rhamnolipid and biochar in assisting phytoremediation of petroleum hydrocarbon contaminated soil using Spartina anglica

Biochar (BC) and rhamnolipid (RL) is used in bioremediation of petroleum hydrocarbons,however,the combined effect of BC and RL in phytoremediation has not been studied until now.In this paper,the phytoremediation of petroleum hydrocarbon-contaminated soil using novel plant Spartina anglica was enhanced by the combination of biochar (BC) and rhamnolipid (RL).Samples of petroleum-contaminated soil (10,30 and 50 g/kg) were amended by BC,BC+ RL and rhamnolipid modified biochar (RMB),respectively.After 60 day's cultivation,the removal rate of total petroleum hydrocarbons (TPHs) for unplanted soil (UP),planted soil (P),planted soil with BC addition (P-BC),planted soil with BC and RL addition (P-BC + RL) and planted soil with addition ofRMB (P-RMB) were 8.6%,19.1%,27.7%,32.4% and 35.1% in soil with TPHs concentration of 30 g/kg,respectively.Compared with UP,the plantation of Spartina anglica significantly decreased the concentration of C8-14 and tricyclic PAHs.Furthermore,the application of BC and RMB alleviated the toxicity of petroleum hydrocarbons to Spartina anglica via improving plant growth with increasing plant height,root vitality and total chlorophyll content.High-throughput sequencing result indicated that rhizosphere microbial community of Spartina anglica was regulated by the application of BC and RMB,with increase of bacteria and plant mycorrhizal symbiotic fungus in biochar and RMB amended soil.

RhlA、RhlB基因表达对微生物采油的影响

为了探究RhlA、RhlB基因表达在微生物采油中对降黏效果的影响,建立了RhlA和RhlB基因实时荧光绝对定量(Q-PCR)方法,测定不同培养时期的铜绿假单胞菌X7发酵液和克拉玛依油田不同地层水的RhlA、RhlB基因表达量,并测定其鼠李糖脂产量、表面活性、乳化活性、乳化稳定性及原油降黏率。结果表明,RhlA、RhlB基因表达量与鼠李糖脂产量、表面活性、乳化活性及原油降黏率呈正相关。随着发酵时间的延长,发酵液中RhlA、RhlB基因表达量不断升高并在24 h时达到最大值,随后下降并于60 h趋于平稳,RhlA、RhlB基因表达量最大值分别为2.51×106和3.25×105 copies/µL,发酵液中12~60 h期间鼠李糖脂产量快速上升,并在60 h时达到最大值(824.08 mg/L);发酵液的表面活性、原油乳化活性及稠油降黏率也随着发酵时间的增长而升高,并分别在24、48、60 h时达到最大。发酵液中的鼠李糖脂粗提物有良好的稳定性,能在一定的极端条件下稳定发挥作用。RhlA、RhlB基因很可能通过影响鼠李糖脂产量、表面活性、乳化活性及原油降黏率对微生物采油产生影响。本研究初步揭示了生物表面活性相关功能基因表达对稠油降黏效果的影响,对推进微生物采油的工业化应用具有重要的意义。

Rhamnolipid synthesis and production with diverse resources

Rhamnolipids are one of the most effective biosurfactants that are of great interest in industrial applications such as enhancing oil recovery, health care, cosmetics, pharmaceutical processes, food processing, detergents for protein folding, and bioremediation due to their unique characteristics such as low toxicity, surface active property to reduce surface/interfacial tensions, and excellent biodegradability. The genes and metabolic path- ways for rhamnolipid synthesis have been well elucidated, but its cost-effective production is still challenging. Pseudomonas aeruginosa, the most powerful rhamnolipid producer, is an opportunistic pathogen, which limits its large scale production and applications. Rhamnolipid production using engineered strains other than Pseudomo- nas aeruginosa such as E. coli and Pseudomonas putida has received much attention. The highest yield of rhamnolipids is achieved when oil-type carbon sources are used, but using cheaper and renewable carbon sources such as lignocellulose would be an attractive strategy to reduce the production cost of rhamnolipids for various industrial applications.

Leaching of rare earth elements from contaminated soils using saponin and rhamnolipid bio-surfactant

The effective leaching of rare earth elements（La, Ce, Y and Eu） from simulated contaminated soil using bio-surfactant was investigated in a lab-scale column leaching experiment, where anionic biosurfactant rhamnolipid and non-ionic biosurfactant saponin were used as washing solutions. Soil properties and the rare earth element fractions were analysed to define the effect of leaching on soil and elemental speciation. Column leaching results showed that saponin solution was more effective than rhamnolipid in the removal of the four rare earth elements tested, with the accumulative removal efficiency of La, Ce, Y and Eu following flushing with 400 mL of 25 g/L saponin, reaching 35.258%, 26.072%, 31.476% and 30.849%, respectively. The change in REE speciation showed that REE removed from soils were mainly derived from the acid-soluble and residual fractions released when rhamnolipid solution was used as a leaching agent. However, for saponin leaching, removed REE amounts were derived from acid-soluble and reducible fractions. Complexation interactions were identified between saponin and REEs, according to infrared spectroscopy and ion exchange data, with saponin complexing with La, Ce, Y, and Eu at a complex ratio of 1：1.

Enhanced biodegradation of chlorobenzene via combined Fe^(3+) and Zn^(2+) based on rhamnolipid solubilisation

Biotrickling filters(BTFs) for hydrophobic chlorobenzene(CB) purification are limited by mass transfer and biodegradation. The CB mass transfer rate could be improved by 150 mg/L rhamnolipids. This study evaluated the combined use of Fe^(3+) and Zn^(2+) to enhance biodegradation in a BTF over 35 day. The effects of these trace elements were analysed under different inlet concentrations(250, 600, 900, and 1200 mg/L) and empty bed residence times(EBRTs;60, 45, and 32 sec). Batch experiments showed that the promoting effects of Fe^(3+)/Zn^(2+) on microbial growth and metabolism were highest for 3 mg/L Fe^(3+) and 2 mg/L Zn^(2+), followed by 2 mg/L Zn^(2+), and lowest at 3 mg/L Fe^(3+). Compared to BTF in the absence of Fe^(3+) and Zn^(2+), the average CB elimination capacity and removal efficiency in the presence of Fe^(3+) and Zn^(2+) increased from 61.54 to 65.79 g/(m 3 hr) and from 80.93% to 89.37%, respectively, at an EBRT of 60 sec. The average removal efficiency at EBRTs of 60, 45, and 32 sec increased by 2.89%, 5.63%, and 11.61%, respectively. The chemical composition(proteins(PN), polysaccharides(PS)) and functional groups of the biofilm were analysed at 60, 81, and 95 day. Fe^(3+) and Zn^(2+) significantly enhanced PN and PS secretion, which may have promoted CB adsorption and biodegradation. High-throughput sequencing revealed the promoting effect of Fe^(3+) and Zn^(2+) on bacterial populations. The combination of Fe^(3+) and Zn^(2+) with rhamnolipids was an efficient method for improving CB biodegradation in BTFs.

Milk phospholipids-based nanostructures functionalized with rhamnolipids and bacteriocin:Intrinsic and synergistic antimicrobial activity for cheese preservation

Milk fat globule membrane (MFGM) phospholipids-based nanostructures were developed and their functionalization with rhamnolipids (RLs) was carried out to enhance the preservation of cheese against resistant foodborne pathogens i.e.,Listeria monocytogenes and Escherichia coli .Void (without nisin) and nisin-loaded RLs functionalized MFGM nanostructures (RLs-MFGM-NS) were fabricated by ultrasonication-assisted self-assembly method.Cubic morphology of void and loaded RLs-MFGM-NS and pristine MFGM nanostructures (MFGM-NS) was observed under scanning electron microscopy (SEM),which indicated uniform size ranging from 43 nm (void RLs-MFGM-NS) to 194 nm (loaded RLs-MFGM-NS).FTIR analyses confirmed the electrostatic interaction,predominantly H-bonding and linkage of carboxyl ester group of MFGM with C–H group in RLs after functionalization of NS.Furthermore,quantitative antimicrobial assay on cheese slices confirmed the broad-spectrum potential of intrinsically active nanostructures (due to RLs) having synergistic activity with nisin against L.monocytogenes and E.coli .Hence,nisin-loaded RLs-MFGM-NS can be applied as promising bioactive additives for sustained preservation of cheese.

鼠李糖脂的分离鉴定及其在食品中的应用研究进展

鼠李糖脂(rhamnolipids,RLs)是一种天然的具有多种生物活性的脂类物质,由于其具有促进发酵起泡、抑制细菌生物膜形成和调节免疫等功能,可广泛应用于食品、医药和农业等领域。目前工业上已经采用微生物发酵法对RLs进行生产制备。RLs作为最有前途的商业化生物表面活性剂,高效、准确的鉴定和表征RLs对其生产、应用至关重要。本文介绍了RLs的生产方法 ,重点概述RLs的分离纯化工艺和鉴定技术,并总结了RLs在食品领域的应用现状,为今后RLs活性开发及应用提供参考,推动其在食品领域的工业化进程。

Production of rhamnolipids-producing enzymes of Pseudomonas in E. coli and structural characterization

Rhamnolipids are a class ofbiosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in Pseudomonas aeruginosa. Four of the 5 proteins except RhlC were successfully over-expressed in E. coli and three of them （RhlA, RhlB and RhlI） were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins in vitro to favor the construction of super strains for rhamnolipids production.

鼠李糖脂润湿煤尘表面特性研究

本文以鼠李糖脂为例,测试生物表面活性剂在煤尘治理方面的应用。从表面张力、接触角、沉降时间试验着手,分析不同质量浓度下鼠李糖脂溶液的表面性能与润湿性能,确定鼠李糖脂溶液的最佳润湿浓度,并结合傅里叶红外光谱试验分析鼠李糖脂对煤体微观结构特性的影响,最终借助自行搭建的模拟巷道平台测定鼠李糖脂的降尘效率。试验结果表明:生物表面活性剂——鼠李糖脂具备用作煤尘抑尘剂的基本性能。鼠李糖脂的加入不仅降低了清水的表面张力、接触角及沉降时间,且全尘、呼尘降尘效率分别达到了79.11%,74.93%,同比清水提升了41.5%、42.48%,抑尘效果显著。综合成本因素,确定鼠李糖脂溶液的最佳润湿浓度为0.04%,红外光谱分析表明,羟基及含氧官能团含量变化是影响煤体润湿的关键因素。

Ultrasonic-assisted Biodegradation of Endocrine DisruptingCompounds in Soil by Pseudomonas putida： the Importance of Rhamnolipid for Intermediate Product Degradation

The present study aimed to completely remove estrogens, including oestrone（E1）, oestradiol（E2）, oes-triol（E3）, 17a-ethinylestradiol（EE2） and bisphenol-A（BPA）, from soil using Pseudomonas putida（P., putida）. A centralcomposite design was developed to determine the optimal conditions of three variables（ultrasonication time, quantityof P. putida, and concentration of added rhamnolipid） for the removal of the estrogens, and the biodegradation ratesof the estrogens were investigated under the optimum conditions. Moreover, a quantitative structure-biedegradationrelationship（QSBR） was used to analyze the effect of the estrogenic physicochemical properties on the enhancementof the biological degradation. The optimal conditions were an ultrasonication time of 3 min, a P. putida quantity of 8mL, and a rhamnolipid concentration of 100 mg/L. These conditions resulted in removal of 100%, 94.86%, 94.90%,96.56% and 94.56% of El, E2, EE2, BPA and E3, respectively after 7 d. The degradations were more rapid and com-plete than those reported in previous studies, indicating the suitability of the adaptation of P. putida to estrogen de-gradation under conditions of ultrasonic-assistance and adding rhamnolipid, improvement was particularly apparentfrom the complete degradation of E3. Based on a Pearson correlation analysis, the estrogen molecule polar surfacearea（PSA） and surface tension were significantly related to the biodegradation effect. An analysis of the QSBR modelwith the estrogen biodegradation rates as a dependent variable and the PSA and surface tension as independent va-riables indicated that larger PSA caused decreased estrogen biodegradation, while the biodegradation progress wasdominated by the surface tension of the estrogens. The interaction of PSA and surface tension had an antagonistic ef-fect on the biodegradation of estrogens. Therefore, rhamnolipid/ultrasonication can significantly improve the biode-gradation rates of oestrogens in soil, while simultaneously adjusting other environmental conditions would influenceand control the biodegradation processes of estrogens.