Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi

Background & objectives: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India. Methods: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations. Results: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2. Interpretation & conclusion: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2 . The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria

Extraction and antimicrobial activity of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa UKMP14T

Aims@#Rhamnolipids are seeking utmost attention as a new class of biosurfactants having promising potential in diverse fields as they offer a wide range of advantages over chemically synthesised surfactants. However, the high extraction costs make large scale production face difficulty. In present study, hydrocarbon degrading bacteria Pseudomonas aeruginosa UKMP14T was exploited for its biosurfactant producing ability including a comparative study between different extraction procedures for its recovery. In addition to this, the recovered biosurfactant was explored for its potential application as an antimicrobial agent. @*Methodology and results@#The production of rhamnolipid biosurfactant was confirmed through various detection methods which are drop-collapse test, oil spreading assay, emulsification index, cetyltrimethylammonium bromide (CTAB) assay and hemolytic assay. The test strain P. aeruginosa UKMP14T showed positive results for all the detection assays. Following this, shake flask cultivation was carried out for several time intervals (1, 3, 5, 7 and 9 days) to discover the optimum time for rhamnolipid biosurfactant production. The results were evaluated by quantifying the rhamnolipid yield using Anthrone method and maximum yield was obtained on day 7. Then, three commonly employed rhamnolipid biosurfactant extraction methods (acid precipitation, solvent extraction and zinc sulphate precipitation) were incorporated for the extraction of rhamnolipid biosurfactant. Among these methods, organic solvent extraction (using methanol, chloroform and acetone in 1:1:1 ratio) gave the highest yield (7.37 ± 0.81 g/L) of biosurfactant, followed by zinc sulphate precipitation (5.83 ± 0.02 g/L), whereas acid precipitation gave the lowest yield (2.8 ± 0.12 g/L) and required longer time (30 days). Finally, the antimicrobial activity of several concentrations of rhamnolipid was tested using modified microdilution method and highest antibacterial activity (in the form of percent reduction in growth) of 95.05% and 91.89% was recorded for Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 11632, respectively, at 100 µg/mL concentration of rhamnolipid biosurfactant.@*Conclusion, significance and impact of study@#The ability of P. aeruginosa UKMP14T in producing rhamnolipid biosurfactant was confirmed. Despite the higher yield obtained by organic solvent extraction method, the recovery technique (involving the separation of solvent system) caused some loss in product. In addition, the transfer and storage of rhamnolipid was challenging using solvent extraction in comparison to acid precipitation and zinc sulphate precipitation. On the other hand, recovery using acid precipitation suffered from lowest yield of rhamnolipid. Therefore, zinc sulphate precipitation is prioritised over the other two methods. Furthermore, the antimicrobial potential of rhamnolipid biosurfactant was tested successfully for as low as 10 µg/mL concentration against E. coli ATCC 10536 and S. aureus ATCC 11632. Therefore, the recovery cost of a high value product like rhamnolipid can be reduced by incorporating the results of this study in the downstream processing and promote rhamnolipid biosurfactant as a potential antimicrobial agent.

Characterization and Production of Rhamnolipid Biosurfactant in Recombinant Escherichia coli Using Autoinduction Medium and Palm Oil Mill Effluent

Abstract Rhamnolipid is a potent biodegradable surfactant, which frequently used in pharmaceutical and environmental industries, such as enhanced oil recovery and bioremediation. This study aims to engineer Escherichia coli for the heterologous host production of rhamnolipid, to characterize the rhamnolipid product, and to optimize the production using autoinduction medium and POME (palm oil mill effluent). The construction of genes involved in rhamnolipid biosynthesis was designed in two plasmids, pPM RHLAB (mono-rhamnolipid production plasmid) and pPM RHLABC (di-rhamnolipid production plasmid). The characterization of rhamnolipid congeners and activity using high-resolution mass spectrometry (HRMS) and critical micelle concentration (CMC). In order to estimate rhamnolipid yield, an oil spreading test was performed. HRMS and CMC result show E. coli pPM RHLAB mainly produced mono-rhamnolipid (Rha-C14:2) with 900 mg/L and 35.4 mN/m of CMC and surface tension value, whereas E. coli pPM RHLABC mainly produced di-rhamnolipid (Rha-Rha-C10) with 300 mg/L and 34.3 mN/m of CMC and surface tension value, respectively. The optimum condition to produce rhamnolipid was at 20 h cultivation time, 37 oC, and pH 7. In this condition, the maximum rhamnolipid yield of 1245.68 mg/L using autoinduction medium and 318.42 mg/L using 20% (v/v) of POME. In conclusion, the characteristics of the rhamnolipid by recombinant E. coli is very promising to be used in industries as the most economical way of producing rhamnolipid.

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa PA 1 and its efficacy against Phytophtora infestans

Aim: The aim of the present study was to identify biosurfactant produced by Pseudomonas aeruginosa and to determine the effectiveness of biosurfactants (rhamnolipid) against P. infestans causing late blight of potato. Methodology: Pseudomonas aeruginosa strains were isolated from soil samples and the potential strain PA 1, selected because of its antagonistic ability, was used to optimize anti-metabolite production and its characterization using HPLC-Mass spectrometry. Rhamnolipid based formulation was developed and its efficacy was tested against late blight disease. Results: The results revealed that four rhamnolipids congeners were identified, among them three were mono-rhamno-di-lipidic congeners and one was di-rhamno-di-lipdic congeners, abundantly present in the crude biosurfactant obtained from P. aeruginosa PA1. The mass spectra of mono-rhamno-di-lipidic Rha-C12-C14 peak value (m/z 584), Rha-C12.1-C10.CH3 peak value (m/z 545), Rha-C12-C-12-CH3 peak value (m/z 575) and di-rhamno-di-lipidic. Rha-Rha-C10-C10 peak value (m/z 651) were also detected. Rhamnolipid-based formulation was developed and evaluated at different concentration ranging from 0.012 and 0.3% in detached leaf test. Significant reduction in lesion area was recorded at 0.2% concentration (lesion area 0.06 cm2 as against 9.8 cm2 on 5th day of inoculation). Interpretation: Microbial produced rhamnolipid based formulation at 0.2% concentration was found effective against late blight of potato in detached leaf test. Further, it could be used in field study as green chemical which would help in replacing application of chemicals in agriculture.

Rhamnolipid Biosurfactants Produced by Pseudomonas Species

ABSTRACT: Surfactants are chemical products widely used in our daily life in toothpaste and other personal hygiene and cosmetic products, and in several industries. Biosurfactants are surfactants of biological origin that can be produced by microorganisms and have many advantages, such as low toxicity and high biodegradability, compared to synthetic counterparts. Unfortunately, high production costs limit the use of biosurfactants. Low-cost production is the most important factor for biosurfactants to be able to compete in the global market place. This review presents general information on rhamnolipid biosurfactant produced by Pseudomonas species, as well as on their production and applications. In addition, industrial products and their wastes used for rhamnolipid production are reviewed in detail based on recent studies.

Biosurfactant production by Pseudomonas aeruginosain kefir and fish meal

The aim of this study was to increase rhamnolipid production by formulating media using kefir and fish meal for Pseudomonas aeruginosa strains isolated from different environmental resources. The strains, named as H1, SY1, and ST1, capable of rhamnolipid production were isolated from soil contaminated with wastes originating from olive and fish oil factories. Additionally, P. aeruginosa ATCC 9027 strain, which is known as rhamnolipid producer, was included in the study. Initially, rhamnolipid production by the strains was determined in Mineral Salt Medium (MSM) and then in media prepared by using kefir and fish meal. The obtained rhamnolipids were purified and quantified according to Dubois et al. (1956). The quantity of rhamnolipids of ATCC, H1 and SY1 strains in kefir media were determined as 11.7 g/L, 10.8 g/L and 3.2 g/L, respectively, and in fish meal media as 12.3 g/L, 9.3 g/L and 10.3 g/L, respectively. In addition, effect of UV light exposure on rhamnolipid production was also investigated but contrary a decrease was observed. The results indicate that P. aeruginosa strains isolated from various environmental resources used in this study can be important due to their rhamnolipid yield, and fish meal, which is obtained from waste of fish, can be an alternative source in low cost rhamnolipid production.

Screening, stability and antibacterial potential of rhamnolipids from Pseudomonas sp., isolated from hydrocarbon contaminated soil.

Surfactant plays an important role in industrial application such as oil recovery, lubricants and emulsifier. But chemical surfactants are toxic to human and other small animals. In recent years, biological based surfactants have gained increasing attention due to their ecofriendly in nature. The present study was focused to isolate biosurfactant producing bacteria, their stability and antibacterial ability from hydrocarbon contaminated and uncontaminated soil collected from different locations in Kanchipuram, Tamil Nadu, India. Biosurfactant producing bacteria were screened by following the haemolytic activity, drop collapsing test, emulsion against kerosene and was further confirmed through surface activity. The stability of the biosurfactant was determined by different physic-chemical conditions like pH, temperature and salinity. A total of 37 strains were selected in three different samples based on cultural characters and finally only 7 strains were confirmed as positive for biosurfactant. Among these strain H11 was considered as potential based on emulsification index (44%), surface activity (34.45 x 10-3 nm-1) and surface tension (23.17 x 10-3 nm-1) and was identified as Pseudomonas sp. The emulsification activity was stable at broad range of pH (4-12), temperature (4-120°C) and salt concentration (0-10%). The biosurfactant was further characterized in HPLC and one major peak was observed at a retention time of 2.033. The antibacterial activity of biosurfactant was high against gram positive pathogenic bacteria than gram negative bacteria. The rhamnolipid produced Pseudomonas sp. may be used as a tool to manage the oil pollution and to control the disease causing bacteria.

Rhamnolipid but not motility is associated with the initiation of biofilm seeding dispersal of Pseudomonas aeruginosa strain PA17.

Seeding dispersal is an active detachment exhibit in aging Pseudomonas aeruginosa biofilm. Yet, effect factors of this process in the biofilm of clinical isolated mucoid P. aeruginosa strain remain to be better characterized. In our previous work, one mucoid P. earuginosa strain PA17 was isolated from a patient with recurrent pulmonary infection. In this study, confocal scanning laser microscope combined with LIVE/DEAD viability staining revealed that PA17 biofilm exhibited earlier seeding dispersal than non-mucoid PAO1. We further compared the motility and the expression of motility-associated gene during biofilm development between PA17 and PAO1. PA17 was found to be impaired in all three kinds of motility compared to PAO1. Moreover, we investigated the expression of rhamnolipid-associated genes in PA17 and PAO1 biofilm. The expression of these genes was in accordance with the process of seeding dispersal. Our results indicated that rhamnolipid but not motility is associated with the initiation of seeding dispersal of PA17 biofilm.

Biosurfactant production in sugar beet molasses by some Pseudomonas spp.

In this study, rhamnolipid biosurfactant production was investigated in eighteen strains of Pseudomonas spp.. Rhamnolipid by these strains was determined by a spectrophotometric method in nutrient broth medium (NB). From the 18 strains screened, two Pseudomonas strains (Pseudomonas luteola B17 and Pseudomonas putida B12) which had produced the highest percentage yield of rhamnolipid were examined for rhamnolipid production at different incubation times (24, 48 and 72 hr) and different sugar beet molasses concentrations [1-5 % w/v concentration (1-5 g molasses/100 ml water)]. The rhamnolipid production increased with the increase in the concentration of molasses and maximum production occurred when 5 % (w/v) of molasses were used. At the same time, maximum rhamnolipid production occurred after 72 hr of incubation. When the amount of rhamnolipid produced at different incubation times (24, 48 and 72 hr) and with different concentrations of molasses [1-5 % w/v concentration (1-5 g molasses/100 ml water)] by Pseudomonas spp.; was compared, no significant difference in amount of production was seen. These studies show that the waste product from sugar industry may be suggested for important biotechnological processes such as rhamnolipid production.

Mass spectrometry analysis of surface tension reducing substances produced by a pah-degrading Pseudomonas citronellolis strain / Análise por espectrometria de massa de substâncias redutoras da tensão superficial produzidas por uma cepa de Pseudomonas citronellolis degradadora de hidrocarbonetos aromáticos policíclicos

In this work we investigated the structure of the iron-stimulated surface tension reducing substances produced by P. citronellolis 222A isolated from a 17-years old landfarming used for sludge treatment in petrochemical industries and oil refinery. Its mass spectrum differs from P. aeruginosa spectrum, indicating that the surface tension reducing substances produced by P. citronellolis can be a new kind of biosurfactant.

Neste trabalho é apresentado um estudo a respeito da análise da estrutura de substâncias redutoras de tensão superficial produzidas por Pseudomonas citronellolis 222A estimulado pela presença de ferro. Esta bactéria foi isolada de um solo que há 17 anos vem sendo utilizado para o tratamento de borra oleosa proveniente da indústria petroquímica e de refinaria de petróleo. O espectro de massa difere do espectro de P. aeruginosa, indicando que as substâncias redutoras de tensão superficial produzidas por P. citronellolis podem ser um novo tipo de biosurfactante.

Recent Progress on Rhamnolipid Produced from Fermentation of Waste Edible Oils / 微生物学通报

Although it is widely studied as a promising bio-surfactant,biosynthesis of Rhamnolipid has not been applied in large-scale due to its high production cost.As a cheap alternative carbon source,waste edible oils have been extensively studied for the production of rhamnolipid.This paper reviewed the recent re-search in this field,including the influence of various waste edible oils and production,chemical structure and properties of the produced rhamnolipids.With waste edible oils,the maximum production of rham-nolipids was reported to be 24.61 g/L.The lowest surface tension was 24 mN/m and the lowest CMC of the produced rhamnolipids was 40.19 mg/L.In addition,this paper also summarized the effect of various factors on the rhamnolipids biosynthesis,such as bacteria strains,nitrogen sources,trace minerals,dissolved oxygen,pH and fermentation conditions.Based on this,the key points of the mass production of rhamnolipids with waste edible oils were also discussed.

Experimental Study on the Effects of Surfactants on Cellulase From Trichoderma viride / 中国生物工程杂志

The effects of surfactants on the production of cellulase by Trichoderma viride in liquid substrate fermentation process were investigated. Straw was used as the sole carbon source and the surfactants were biosurfactant rhamnolipid from Pseudomonas aeruginosa and Tween 80. The changes of FPA,CMCase,Avicelase and surface tension with time were analyzed under different concentrations of the two surfactants. The results showed that the surfactants can enhance the enzyme activity of Trichoderma viride. The FPA,CMCase,Avicelase were promoted 1.08,1.6 and 1.03 times higher than the controls by rhamnolipid. The enhancement of the enzyme activity by rhamnolipid was much higher than that of Tween 80. At the same time,rhamnolipid was not degraded prior to other substrate.

Recent Progress on Rhamnolipid Biosurfactant / 微生物学通报

Rhamnolipid,an important biosurfactant,is reviewed with respect to chemical strocture,properties,physiological role and their fermentation production,especially focusing on the production with inexpensive raw materials,such as vegetable oils and residues from agro- industrial wastes.This can not only reduce the production costs,but also contribute to the reduction of environmental impact generated by the discard of residues,and the treatment costs.

Study on Rapid Methods for Quantitative Analysis Rhamnolipid and Its Influence Factors / 微生物学通报

In order to find an easy and rapid quantitative analytical method to detect rhamnolipid produced by Pseudomonas aeruginosa, three methods, H_ 2 SO_ 4 -anthrone analysis method, L-cysteine-H_ 2 SO_ 4 method and phenol-H_ 2 SO_ 4 method, were compared in the present paper, and the influence factors were also considered.The results showed that H_ 2 SO_ 4 -Anthrone analysis method was better than the others and its optimal reaction condition was obtained.The influence to the quantitative analysis of rhamnolipid from the residual glucose and the top clean liquid layer in the ferment solution could be ignored.But the influence from the bacterial body and the middle layer of the ferment solution reached a certain degree.Thus, the bacterial body should be removed before measuring.However, the influence from the middle layer of the ferment solution could be avoided by making a standard curve which was made by using a rhamnose mixed with the middle layer ferment solution.

Mode of Long-chain Alkane Uptake by a Pseudomonas aeruginosa / 微生物学通报

The mode of long-chain alkane uptake by Pseudomonas aeruginosa (CGMCC 1.1785) was studied. P. aeruginosa 1.1785 is capable of using solid long-chain alkane as sole carbon source and producing surface active compound as metabolite. The mass transfer limitation in uptake of alkane was confirmed from the observation that interfacial area of eicosane with water dominates the growth rate of this strain. The enhancement of eicosane uptake by rhamnolipid was mainly caused by increase of interfacial area, since the pseudosolubilized alkane can not support the growth of P. aeruginosa 1.1785. Cell surface hydrophobicity was increased dramatically at the initial phase of growth and followed by a gradual decrease, which indicates that different modes are employed by P. aeruginosa 1.1785 at different growth phase. Therefore, the surfactant mediated mode can be negligible in the uptake process, while the directly attachment mode may not work throughout the growth of P. aeruginosa 1.1785. We proposed a novel uptake mode, in which the chemotaxis of this strain plays an important role.