Artykuły w czasopismach na temat „Rhamnolipides (RLs)”

Abstract Within the circular economy framework, our study aims to assess the rhamnolipid production from winery and olive oil residues as low-cost carbon sources by nonpathogenic strains. After evaluating various agricultural residues from those two sectors, Burkholderia thailandensis E264 was found to use the raw soluble fraction of nonfermented (white) grape marcs (NF), as the sole carbon and energy source, and simultaneously, reducing the surface tension to around 35 mN/m. Interestingly, this strain showed a rhamnolipid production up to 1070 mg/L (13.37 mg/g of NF), with a higher purity, on those grape marcs, predominately Rha-Rha C14-C14, in MSM medium. On olive oil residues, the rhamnolipid yield of using olive mill pomace (OMP) at 2% (w/v) was around 300 mg/L (15 mg/g of OMP) with a similar CMC of 500 mg/L. To the best of our knowledge, our study indicated for the first time that a nonpathogenic bacterium is able to produce long-chain rhamnolipids in MSM medium supplemented with winery residues, as sole carbon and energy source. Key points • Winery and olive oil residues are used for producing long-chain rhamnolipids (RLs). • Both higher RL yields and purity were obtained on nonfermented grape marcs as substrates. • Long-chain RLs revealed stabilities over a wide range of pH, temperatures, and salinities

Rhamnolipids (RLs) are the bacterial derived biosurfactants and known for a wide range of industrial and therapeutic applications. They exhibit potent anti-bacterial activity against various gram positive, gram negative and acid fast bacteria including Mycobacterium tuberculosis. Since, Pseudomonas is one of the largest known genuses containing a variety of rhamnolipid producing strains. Therefore, in this study, we selectively isolated the Pseudomonas aeruginosa strains from the rhizospheric soil of the Indian plants of medicinal value, e.g. Azadirachta Indica and Ficus spp., and evaluated them for their natural ability to produce antibacterial rhamnolipids. The bacteria were identified on the basis of 16s rRNA sequencing and biochemical characterization. Among 33 of P. aeruginosa isolates from different soil samples, four isolates showed potent inhibitory activity against methicillin resistant Staphylococcus aureus (MRSA) and fast grower mycobacterial spp. The inhibitory potential of the isolates was found to be correlated with their ability to produce RLs in the medium. The industrial viability of the strains was assessed on the basis of cytotoxicity determining alternative allele, exoS/exoU and cell mediated cytotoxicity against murine macrophages J774.1. The newly isolated strains harbor exoS allele and exhibits lower cell mediated cytotoxicity on macrophage cell line as compared to the clinical strains PA-BAA-427 and PA-27853 used as a control in this study.Evaluation of antimycobacterial rhamnolipid production from non-cytotoxic strains of Pseudomonas aeruginosa isolated from rhizospheric soil of medicinal plants

Guava is consumed in natura and is also of considerable importance to the food industry. The seeds and peel of this fruit are discarded, however, guava seeds yield oil (~13%) that can be used for the bioproducts synthesis. The use of a by-product as a carbon source is advantageous, as it reduces the environmental impact of possible harmful materials to nature, while adding value to products. In addition, the use of untested substrates can bring new yield and characterization results. Thus, this research sought to study rhamnolipids (RLs) production from guava seed oil, a by-product of the fructorefinery. The experiments were carried out using Pseudomonas aeruginosa LBI 2A1 and experimental design was used to optimize the variables Carbon and Nitrogen concentration. Characterization of RLs produced occurred by LC-MS. In this study, variables in the quadratic forms and the interaction between them influenced the response (p < 0.05). The most significant variable was N concentration. Maximum RLs yield achieved 39.97 g/L, predominantly of mono-RL. Characterization analysis revealed 9 homologues including the presence of RhaC10C14:2 (m/z 555) whose structure has not previously been observed. This research showed that guava seed oil is an alternative potential carbon source for rhamnolipid production with rare rhamnolipid homologues.

This present study aimed to investigate the effects of rhamnolipids (RLS) on the growth performance, intestinal morphology, immune function, short-chain fatty acid content, and microflora community in broiler chickens challenged with lipopolysaccharides (LPS). A total of 450 broiler chickens were randomly allocated into three groups: basal diet with no supplement (NCO), basal diet with bacitracin (ANT), and basal diet with rhamnolipids (RLS). After 56 d of feeding, 20 healthy broilers were selected from each group, with half being intraperitoneally injected with lipopolysaccharides (LPS) and the other half with normal saline. Treatments with LPS were labelled LPS-NCO, LPS-ANT, and LPS-RLS, whereas treatments with normal saline were labelled NS-NCO, NS-ANT, and NS-RLS. LPS-challenged birds had lower jejunal villus height and higher crypt depth than unchallenged birds. LPS-RLS broilers had increased jejunal villus height and villus height/crypt depth ratio (V/C) but lower crypt depth than LPS-NCO. Dietary supplementation with RLS reduced the LPS-induced immunological stress. Compared with LPS-NCO, birds in LPS-RLS had lower concentrations of IL-1β, IL-6, and TNF-α. In LPS-challenged broilers, RLS and ANT increased the concentrations of IgA, IgM, and IgY compared with LPS-NCO. In LPS treatments, RLS enhanced the contents of acetic acid, butyrate, isobutyric acid, isovalerate, and valerate more than LPS-NCO birds. High-throughput sequencing indicated that RLS supplementation led to changes in the cecal microbial community of broilers. At the species level, Clostridium-sp-Marseille-p3244 and Slakia_eqcsolifaciens were more abundant in NS-RLS than in NS-NCO broilers. In summary, RLS improved the growth performance and relative abundance of cecal microbiota and reduced the LPS-induced immunological stress in broiler chickens.

Background/Aim: A number of biologically active substances were proved as an alternative to conventional anticancer medicines. The aim of the study is in vitro investigation of the anticancer activity of mono- and di-Rhamnolipids (RL-1 and RL-2) against human breast cancer. Additionally, the combination with Cisplatin was analyzed. Materials and Methods: Breast cell lines (MCF-10A, MCF-7 and MDA-MB-231) were treated with RLs and in combination with Cisplatin. The viability was analyzed using MTT assay, and investigation of autophagy was performed via acridine orange staining. Results: In contrast to the healthy cells, both tested cancer lines exhibited sensitivity to RLs treatment. This effect was accompanied by an influence on the autophagy-related acidic formation process. Only for the triple-negative breast cancer cell line (MDA-MB-231) the synergistic effect of the combined treatment (10 µM Cisplatin and 1 µg/mL RL-2) was observed. Conclusion: Based on studies on the reorganization of membrane models in the presence of RL and the data about a higher amount of lipid rafts in cancer cell membranes than in non-tumorigenic, we suggest a possible mechanism of membrane remodelling by formation of endosomes. Shortly, in order to have a synergistic effect, it is necessary to have Cisplatin andRL-2 as RL2 is a molecule inducingpositive membrane curvature.

Rhamnolipids (RLs) production using coconut oil as a carbon source by the bacterium P. aeruginosa is studied. This bacterium was grown in media containing 1% carbon source (glucose/ coconut oil). The RLs were characterized by emulsification index (E24), thermal stability and oil spreading test. Further RLs quantification was carried out by the orcinol assay with L-rhamnose as the standard. The result showed that the highest production of RLs occurred in the presence of both coconut oil and glucose at 96 h (2.51 g/L). A stable emulsification index (E24) was observed using diesel with a maximum value of 57% at room temperature. Good stability to high temperature (120 oC) was observed when exposed at 55%. Oil displacement activity showed the presence of RLs with the highest value was at the highest RLs production. This study shows P. aeruginosa is able to produce RLs using coconut oil as the substrate and may potentially become a good source of biosurfactant for industry in the future.

Finding new biological ways to control biofouling of the membrane in reverse osmosis (RO) is an important substitute for synthetic chemicals in the water industry. Here, the study was focused on the antimicrobial, biofilm formation, and biofilm dispersal potential of rhamnolipids (RLs) (biosurfactants). The MTT assay was also carried out to evaluate the effect of RLs on biofilm viability. Biofilm was qualitatively and quantitatively assessed by crystal violet assay, light microscopy, fluorescence microscopy (bacterial biomass (µm2), surface coverage (%)), and extracellular polymeric substances (EPSs). It was exhibited that RLs can reduce bacterial growth. The higher concentrations (≥100 mg/L) markedly reduced bacterial growth and biofilm formation, while RLs exhibited substantial dispersal effects (89.10% reduction) on preformed biofilms. Further, RLs exhibited 79.24% biomass reduction while polysaccharide was reduced to 60.55 µg/mL (p < 0.05) and protein to 4.67 µg/mL (p < 0.05). Light microscopy revealed biofilm reduction, which was confirmed using fluorescence microscopy. Microscopic images were processed with BioImageL software. It was revealed that biomass surface coverage was reduced to 1.1% at 1000 mg/L of RLs and that 43,245 µm2 of biomass was present for control, while biomass was reduced to 493 µm2 at 1000 mg/L of RLs. Thus, these data suggest that RLs have antimicrobial, biofilm control, and dispersal potential against membrane biofouling.

Biosurfactants are surface active compounds and amphiphatic in nature which consist of hydrophilic head and hydrophobic tail accumulating at the interphase of two immiscible liquid with different polarity. A study was conducted to investigate the effectiveness of sunflower oil in the production of rhamnolipids (RLs) by locally isolated Pseudomonas aeruginosa in shake flask fermentation. In this process, four different fermentation treatments were done for seven days at 30°C and 180 rpm. Sampling was carried out in time intervals of 24 h followed by monitoring of cell growth and biosurfactants production. Colorimetric Orcinol analysis was used for determination of RLs concentrations (g/L). The RLs were studied for emulsification activity using emulsification index (E24%) methods. In addition, oil displacement activity and thermal stability were also studied (4-120°C). All treatments allow the growth of P. aeruginosa and the utilization of sunflower oil as carbon source and glucose as growth initiator were observed to be the best strategy for maximum RLs production. The maximum RLs production was achieved after 120 h with 3.18 g/L of RLs. Diesel shows the highest emulsification activity among the substrate tested ranging from 55.56% - 60.00%. The oil displacement activity was corresponding to RLs concentration with stability up to 120°C (for 60 min). Therefore, from this research a good potential of RLs that may provide good application for industry were produced.

Emerging and re-emerging viruses represent a serious threat to human health at a global level. In particular, enveloped viruses are one of the main causes of viral outbreaks, as recently demonstrated by SARS-CoV-2. An effective strategy to counteract these viruses could be to target the envelope by using surface-active compounds. Rhamnolipids (RLs) are microbial biosurfactants displaying a wide range of bioactivities, such as antibacterial, antifungal and antibiofilm, among others. Being of microbial origin, they are environmentally-friendly, biodegradable, and less toxic than synthetic surfactants. In this work, we explored the antiviral activity of the rhamnolipids mixture (M15RL) produced by the Antarctic bacteria Pseudomonas gessardii M15 against viruses belonging to Coronaviridae and Herpesviridae families. In addition, we investigated the rhamnolipids’ mode of action and the possibility of inactivating viruses on treated surfaces. Our results show complete inactivation of HSV-1 and HSV-2 by M15RLs at 6 µg/mL, and of HCoV-229E and SARS-CoV-2 at 25 and 50 µg/mL, respectively. Concerning activity against HCoV-OC43, 80% inhibition of cytopathic effect was recorded, while no activity against naked Poliovirus Type 1 (PV-1) was detectable, suggesting that the antiviral action is mainly directed towards the envelope. In conclusion, we report a significant activity of M15RL against enveloped viruses and demonstrated for the first time the antiviral effect of rhamnolipids against SARS-CoV-2.

Biosurfactants have a biological origin, and are widely known as surface active agents. Different classes of biosurfactant have significant importance in both the biotechnological and microbiological arena. Pseudomonas aeruginosa, Bacillus subtilis and Candida sp. are important classes of microorganisms that are highly investigated for the production of rhamnolipids (RLs) biosurfactants. Rhamnolipids have unique surface activity and have gained interest in various industrial applications. Due to their high biodegradability, renewability and functionally maintenance at extreme conditions, microbial biosurfactants are more advantageous than chemical-based biosurfactants. Biosurfactants produced by microorganisms are a potential candidate for biodegradation, environmental cleanup of pollutants and also play a role in the heavy metal removal of metallurgical industries also many patents have been filed. Therefore, greater attention has been paid to biosurfactants and identifying their potential applications for further studies.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana. HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Staphylococcus aureus is a Gram-positive opportunistic human pathogen responsible for severe infections and thousands of deaths annually, mostly due to its multidrug-resistant (MDR) variants. The cell membrane has emerged as a promising new therapeutic target, and lipophilic molecules, such as biosurfactants, are currently being utilized. Herein, we evaluated the antimicrobial activity of a rhamnolipids mixture produced by the Antarctic marine bacterium Pseudomonas gessardii M15. We demonstrated that our mixture has bactericidal activity in the range of 12.5–50 µg/mL against a panel of clinical MDR isolates of S. aureus, and that the mixture eradicated the bacterial population in 30 min at MIC value, and in 5 min after doubling the concentration. We also tested abilities of RLs to interfere with biofilm at different stages and determined that RLs can penetrate biofilm and kill the bacteria at sub-MICs values. The mixture was then used to functionalize a cotton swab to evaluate the prevention of S. aureus proliferation. We showed that by using 8 µg of rhamnolipids per swab, the entire bacterial load is eradicated, and just 0.5 µg is sufficient to reduce the growth by 99.99%. Our results strongly indicate the possibility of using this mixture as an additive for wound dressings for chronic wounds.

Pseudomonas aeruginosa NCAIM(P), B001380, a propitious bacterial strain isolated from mineral cutting oil was identified to be chromium tolerant and a producer of biosurfactant rhamnolipid (RL) with potential application in heavy metal bioremediation. Culture growth, RL production and Cr(VI) removal capacity of the strain in the presence of 50 mg L-1 (I) and 100 mg L-1 of Cr(VI) (II) were studied. Maximum of RL production were found in the late-stationary phase at 72 h for both Cr(VI)-amended cultures: I (236 mg L-1) and II (160 mg L-1), as well as the maximum of Cr(VI) removal capacity: 70 % (I) and 57 % (II). The amount of Cr in RL preparation II was 22 mg mg-1 determined by flame atomic absorption spectroscopy (FAAS). Appearance of a new band at 914 cm-1 in infrared (IR) spectrum of RL (II) indicated a significant proof for possible coordination of CrO42-ion with RL. The effect of Cr(VI) on monorhamnolipids (RL1) and dirhamnolipids (RL2) distribution and its ratio were studied by electrospray ionization mass spectrometry (ESI-MS). An increase was observed in a RL2/RL1 ratio for II compared to control.

Rhamnolipids (RLs) are potential biocontrol agents for crop culture protection. Their mode of action has been proposed as dual, combining plant protection activation and antifungal activities. The present work focuses on the interaction of natural RLs with plant and fungi membrane models at the molecular scale. Representative models were constructed and the interaction with RLs was studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (2H NMR) spectroscopic measurements. Molecular dynamic (MD) simulations were performed to investigate RL insertion in lipid bilayers. Our results showed that the RLs fit into the membrane models and were located near the lipid phosphate group of the phospholipid bilayers, nearby phospholipid glycerol backbones. The results obtained with plant plasma membrane models suggest that the insertion of RLs inside the lipid bilayer did not significantly affect lipid dynamics. Oppositely, a clear fluidity increase of fungi membrane models was observed. This effect was related to the presence and the specific structure of ergosterol. The nature of the phytosterols could also influence the RL effect on plant plasma membrane destabilization. Subtle changes in lipid dynamics could then be linked with plant defense induction and the more drastic effects associated with fungal membrane destabilization.

Rhamnolipids (RLs) are surface-active molecules mainly produced by Pseudomonas spp. Antarctica is one of the less explored places on Earth and bioprospecting for novel RL producer strains represents a promising strategy for the discovery of novel structures. In the present study, 34 cultivable bacteria isolated from Edmonson Point Lake, Ross Sea, Antarctica were subjected to preliminary screening for the biosurfactant activity. The positive strains were identified by 16S rRNA gene sequencing and the produced RLs were characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRESIMS) and liquid chromatography coupled with tandem spectrometry (LC-MS/MS), resulting in a new mixture of 17 different RL congeners, with six previously undescribed RLs. We explored the influence of the carbon source on the RL composition using 12 different raw materials, such as monosaccharides, polysaccharides and petroleum industry derivatives, reporting for the first time the production of RLs using, as sole carbon source, anthracene and benzene. Moreover, we investigated the antimicrobial potential of the RL mixture, towards a panel of both Gram-positive and Gram-negative pathogens, reporting very interesting results towards Listeria monocytogenes with a minimum inhibitory concentration (MIC) value of 3.13 µg/mL. Finally, we report for the first time the antimicrobial activity of RLs towards three strains of the emerging multidrug resistant Stenotrophomonas maltophilia with MIC values of 12.5 µg/mL.

The hemibiotrophic fungus Zymoseptoria tritici, responsible for Septoria tritici blotch, is currently the most devastating foliar disease on wheat crops worldwide. Here, we explored, for the first time, the ability of rhamnolipids (RLs) to control this pathogen, using a total of 19 RLs, including a natural RL mixture produced by Pseudomonas aeruginosa and 18 bioinspired RLs synthesized using green chemistry, as well as two related compounds (lauric acid and dodecanol). These compounds were assessed for in vitro antifungal effect, in planta defence elicitation (peroxidase and catalase enzyme activities), and protection efficacy on the wheat-Z. tritici pathosystem. Interestingly, a structure-activity relationship analysis revealed that synthetic RLs with a 12 carbon fatty acid tail were the most effective for all examined biological activities. This highlights the importance of the C12 chain in the bioactivity of RLs, likely by acting on the plasma membranes of both wheat and Z. tritici cells. The efficacy of the most active compound Rh-Est-C12 was 20-fold lower in planta than in vitro; an optimization of the formulation is thus required to increase its effectiveness. No Z. tritici strain-dependent activity was scored for Rh-Est-C12 that exhibited similar antifungal activity levels towards strains differing in their resistance patterns to demethylation inhibitor fungicides, including multi-drug resistance strains. This study reports new insights into the use of bio-inspired RLs to control Z. tritici.

Rhamnolipids (RLs) are one of the most promising eco-friendly green alternatives to commercially viable fossil fuel-based surfactants. However, the current bioprocess practices cannot meet the required affordability, quantity, and biocompatibility within an industrially relevant framework. To circumvent these issues, our study aims to develop a sustainable biorefinery approach using post-consumption food waste as a second-generation feedstock. In-depth substrate screening revealed that food waste hydrolysate (FWH) was rich in readily assimilable carbohydrates, volatile fatty acids, and amino acids. The fermentative valorization of FWH as a sole carbon and energy source with Burkholderis thailandensis E264 in a bioreactor showed active RLs biosynthesis of up to 0.6–0.8 g/L (34–40 mg/g FWH) in a short duration (72 h). In terms of the kinetic parameters, the FWH-RLs outperformed other supplemented pure/waste streams. Interestingly, the recovered RLs had a long chain length, with Rha-Rha-C12-C14 being the predominant isoform and exhibiting a strong emulsification ability (E24, 54.6%). To the best of our knowledge, this study is the first to prove bioreactor-level RLs production and their abundance in food waste. Moreover, the feasibility of this developed process could propel next-generation biosurfactants, lower waste burdens, and increase the industrial applicability of RLs, thereby significantly contributing to the development of a circular bioeconomy.

Alzheimer’s disease (AD) is increasingly affecting the aging population and the estimated prevalence reaches 50 million people worldwide. The need for the discovery of new biomarkers for AD diagnosis is urgent and especially in biological fluids other than cerebrospinal fluid (CSF), as its collection is invasive. Arguments are numerous that chronic bacterial infections might be considered as one of the possible causes of AD. Rhamnolipids (RLs) are bacterial virulence factors, suspicious for dysfunctions and disorders including AD. The aim of this pilot trial was to investigate RLs levels in saliva of Mild Cognitive Impairment (MCI) and AD patients with indirect ELISA. Specifically, salivary RLs were determined in 30 AD patients, 24 MCI patients and 15 cognitively healthy individuals and were found elevated in AD and MCI patients compared to those of the control group. The established biomarkers of AD, tau and Aβ42 amyloid, and the inflammatory markers cyclooxygenases (COX-1 and COX-2) were also determined, to evaluate their possible interdependence from RLs levels. Levels of RLs positively correlate with COX-2 levels and negatively with the mental state according to Mini–Mental State Examination (MMSE) score of donors. Multilinear regression verified the tight interrelation of RLs with COX-2 in saliva of MCI and AD patients. The results of this study stand by the hypothesis of inflammatory involvement in AD and indicate that RLs could be suggested as eventual biomarkers for AD diagnosis using saliva as biological fluid.

Silicone-based medical devices composed of polydimethylsiloxane (PDMS) are widely used all over the human body (e.g., urinary stents and catheters, central venous catheters stents) with extreme clinical success. Nevertheless, their abiotic surfaces, being prone to microorganism colonization, are often involved in infection occurrence. Improving PDMS antimicrobial properties by surface functionalization with biosurfactants to prevent related infections has been the goal of different works, but studies that mimic the clinical use of these novel surfaces are missing. This work aims at the biofunctional assessment of PDMS functionalized with rhamnolipids (RLs), using translational tests that more closely mimic the clinical microenvironment. Rhamnolipids were covalently bonded to PDMS, and the obtained surfaces were characterized by contact angle modification assessment, ATR-FTIR analysis and atomic force microscopy imaging. Moreover, a parallel flow chamber was used to assess the Staphylococcus aureus antibiofilm activity of the obtained surfaces under dynamic conditions, and an in vitro characterization with human dermal fibroblast cells in both direct and indirect characterization assays, along with an in vivo subcutaneous implantation assay in the translational rabbit model, was performed. A 1.2 log reduction in S. aureus biofilm was observed after 24 h under flow dynamic conditions. Additionally, functionalized PDMS lessened cell adhesion upon direct contact, while supporting a cytocompatible profile, within an indirect assay. The adequacy of the biological response was further validated upon in vivo subcutaneous tissue implantation. An important step was taken towards biofunctional assessment of RLs-functionalized PDMS, reinforcing their suitability for medical device usage and infection prevention.

The rapeseed crop is susceptible to many pathogens such as parasitic plants or fungi attacking aerial or root parts. Conventional plant protection products, used intensively in agriculture, have a negative impact on the environment as well as on human health. There is therefore a growing demand for the development of more planet-friendly alternative protection methods such as biocontrol compounds. Natural rhamnolipids (RLs) can be used as elicitors of plant defense mechanisms. These glycolipids, from bacteria secretome, are biodegradable, non-toxic and are known for their stimulating and protective effects, in particular on rapeseed against filamentous fungi. Characterizing the organ responsiveness to defense-stimulating compounds such as RLs is missing. This analysis is crucial in the frame of optimizing the effectiveness of RLs against various diseases. A Tandem Mass Tags (TMT) labeling of the proteins extracted from the shoots and roots of rapeseed has been performed and showed a differential pattern of protein abundance between them. Quantitative proteomic analysis highlighted the differential accumulation of parietal and cytoplasmic defense or stress proteins in response to RL treatments with a clear effect of the type of application (foliar spraying or root absorption). These results must be considered for further use of RLs to fight specific rapeseed pathogens.

To evaluate the efficacy of biochelators for phytoremediation of cadmium (Cd), effects of biochelators (humic acid, HA; polyaspartic acid, PASP; rhamnolipid, RLs) and synthetic chelator (sodium tripolyphosphate, STPP) on the growth, accumulation, and antioxidant index of Cd were investigated in Iris sibirica L. The results indicated that the addition of HA increased the dry weight of leaves and stems, and the addition of PASP, RLs, and STPP increased the growth of leaves but inhibited the growth of stems. The determination of Cd accumulation indicated that HA decreased the content of Cd in all tissues, while the addition of PASP, RLs, and STPP increased the accumulation of Cd in stems and roots. In addition, the determination of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities indicated that the treatment of PASP and RLs increased the activities of antioxidant enzymes, which might be beneficial to the resistance of Cd for Iris sibirica L. Overall, these results indicated that biochelators may improve the phytoremediation efficacy of Iris sibirica L. on Cd polluted paddy field. Among the tested chelators, PASP exhibited the best comprehensive efficacy and the highest commercial potential.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

The application of modified agricultural wastes for removing polycyclic aromatic hydrocarbons (PAHs) from water is gaining a growing interest. However, most modified methods using synthetic chemicals may cause secondary pollution. To overcome this limitation, in this study, a rhamnolipid modified corn stalk (RL-CS) for the removal of phenanthrene (PHE) from aqueous solution was prepared using a rhamnolipid-enhanced acid modification method. RL-CS with higher surface area and lower polarity exhibited higher PHE removal efficiency than that of raw corn stalk (RCS). The adsorption kinetics of RL-CS fitted well with pseudo-second-order kinetics (R2 &gt; 0.999). Sorption coefficients and carbon-normalized sorption coefficient of RL-CS were 4.68 and 2.86 times higher than that of RCS. Sorption process of RL-CS was nonlinear. Meanwhile, the sorption was an exothermic process and could occur spontaneously. The present study demonstrated that biosurfactant-modified biosorbent RL-CS may be of great potential for the removal of low concentrations of PAHs from the contaminated waters.

Bacillus cereus is a Gram-positive, endospore-forming bacterium well-known as a food pathogen that causes great losses in the food industry, especially in dairy. In this study, rhamnolipid (RL) biosurfactants were evaluated as a bio-based alternative for controlling the growth of vegetative cells and endospores of B. cereus. RLs were tested against 14 B. cereus strains isolated from different types of foodstuffs. The antimicrobial activity against vegetative cells and endospores revealed minimal inhibitory concentration (MIC) values of 0.098 mg/mL for almost all strains tested and minimal bactericidal concentration (MBC) varying between 0.098 and >25 mg/mL. The presence of RLs inhibited endospore germination by more than 99%, reducing by 5.5 log the outgrowth of strain 0426. Scanning and transmission electron microscopy confirmed that exposure to RL causes damage to the structure of endospores. When skim milk was utilized as a food model, RL inhibited the growth of vegetative cells and endospores of B. cereus, showing MBC of 3.13 mg/mL for the vegetative cells of strain 0426. The surfactant also reduced bacterial growth in milk at refrigerator temperature. The results suggest that RLs are promising candidates for the development of novel strategies to control B. cereus in the food industry.

The production of biosurfactants is often hampered by excessive foaming in the bioreactor, impacting system scale-up and downstream processing. Foam fractionation was proposed to tackle this challenge by combining in situ product removal with a pre-purification step. In previous studies, foam fractionation was coupled to bioreactor operation, hence it was operated at suboptimal parameters. Here, we use an external fractionation column to decouple biosurfactant production from foam fractionation, enabling continuous surfactant separation, which is especially suited for system scale-up. As a subsequent product recovery step, continuous foam adsorption was integrated into the process. The configuration is evaluated for rhamnolipid (RL) or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., RL precursor) production by recombinant non-pathogenic Pseudomonas putida KT2440. Surfactant concentrations of 7.5 gRL/L and 2.0 gHAA/L were obtained in the fractionated foam. 4.7 g RLs and 2.8 g HAAs could be separated in the 2-stage recovery process within 36 h from a 2 L culture volume. With a culture volume scale-up to 9 L, 16 g RLs were adsorbed, and the space-time yield (STY) increased by 31% to 0.21 gRL/L·h. We demonstrate a well-performing process design for biosurfactant production and recovery as a contribution to a vital bioeconomy.

Despite the extremely high activity of researchers in the direction of green synthesis of nano-particles of silver, copper, as well as bimetallic nanoparticles Ag/Cu-NPs, the many aspects of the synthesis of nanoparticles based on silver and copper remain unexplored, in particular, the studies on the influence of the nature of biosurfactants on the characteristics are relevant obtained nano¬particles, in particular their size, shape, etc., as well as the influence of the morphology and compo¬sition of nanoparticles on their antimicrobial properties. Therefore, the purpose of this work was to synthesize colloidal solutions of bicomponent Ag/CuO-NPs nanoparticles of different composition and to investigate their antimicrobial activity. Bicomponent Ag/CuO nanoparticles of different composition were obtained by the method of coprecipitation of Ag+ and Cu2+ cations in solution of rhamnolipid. It was found that the process of formation of monocomponent CuO nanoparticles is completed within 2.5 hours but the reduction of silver ions in aqueous solutions of RL occurs within a few minutes. The obtained Ag/CuO-NPs were studied using UV-visible spectroscopy and the method of powder diffraction of X-rays. It was found that the absorption spectra of Ag/CuO-NPs solutions are characterized by two maxima at 280 and 410 nm, which correspond to the surface plasmon resonance bands of CuO and Ag-NPs, res-pectively. Based on the obtained data, it is reasonable to assume that the obtained Ag/CuO-NPs can form core-shell structures in which the role of the core will be played by silver surrounded by a CuO shell. Taking into account the prospects of using the nanoparticles based on silver and copper as antimicrobial agents for the plant protection, the antimicrobial activity of the synthesized Ag/CuO-NPs was investigated. The gram-negative bacteria-phytopathogens were used as test microorganisms, namely Agrobacterium tumefaciens, which is the causative agent of crown gall disease of a wide range of agricultural crops and Xanthomonas campestris, which is the causative agent of the "black rot" disease in cruciferous vegetables. The antimicrobial activity of the synthesized colloidal solutions of Ag/CuO was evaluated by the minimum inhibitory concentration values. The results of the studies of antimicrobial activity of the synthesized Ag/CuO colloidal solutions showed that the obtained preparations effectively inhibit the growth of phytopathogenic bacteria Agrobacterium tumefaciens and Xanthomonas campestris and it was found that the obtained drugs are more active against the Xanthomonas campestris.

Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane). Biosurfactants can reduce the stress between solids and liquids on the surface and at the end. Biosurfactants have several properties, i.e. they are stable, less harmful, as well as readily degradable, and extremely eco-friendly. Biosurfactants also have a wide range of industrial uses because they are a versatile category of chemical substances. The principal justification for conducting such research was the isolation of possible biosurfactants containing bacteria. Sampling was performed for the isolation of bacteria producing biosurfactants from different oil-polluted sites That is to say, experiment for emulsification, test for oil spreading, test for drop collapse, and measure for hemolysis. The capability to produce biosurfactants was seen in 22 different isolates from polluted sites B1, B2, and B3. Through different biochemical tests and Gram staining, it was identified that isolated bacterial strains are Pseudomonas spp and that is Pseudomonas aeruginosa. The procedure used as characterizing biosurfactants was the TLC plate’s procedure, by using TLC plates process yellow dots emerged after spraying on silica gel plates with an throne and ninhydrin reagents. These yellow spots confirmed the presence and production of rhamnolipid in the biosurfactant. Hence, it was concluded that identified strains in the study can be helpful in the heavy metals, pesticides, and hydrocarbons bio-degradation and bioremediation. These may also be used as biological control agents to protect plants from various pathogens, resulting in improved crop yields. Introduction Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane) (Chen et al., 2007; Ghayyomiet al., 2012). Main purpose of the bio-surfactantsgeneration or production is a consequence of financial availability (Van Dyke et al., 1993 It is reported that almost 50 percent of the world's surfactants are used because of the need for cleaning agents as well as the rate of growth grows every day (Deleu and Paquot, 2004). Appropriate use of bio-surfactants will control environmental emissions what these are the most dangerous, constantly rising gradually and disrupting the routine maintenance of life every day. Awareness campaign initiatives have been introduced and also increase for environmental laws, various innovative approaches need to be implemented and even the issue of pollution focused entirely. Developing appropriate advanced technologies to help clear up chemicals and toxins from the ecosystem, like hydrocarbons (both inorganic and organic). Studies on biosurfactants are being launched by scholars and researchers with significant health issues like adverse environmental effects, air contamination, environmental change, and waste management (Makkar and Cameotra, 2002 Biosurfactants contribute to expanded demand for such microbial products as alternatives to chemical surfactants (Benatet al., 2000). Microbes seem to have the capability to degrade contaminants, but their biodegradation is limited leading to hydrophobicity, low solubility in water, and inadequate bioavailability, of such pollutants (Patil, et al., 2012). GhayyomiJazeh, Mishraet. al (2001) those bacteria that produce biosurfactants were isolated from the site of petroleum spills and afterward, 160 strains and as well as 59 strains were able to produce biosurfactants have shown better performance in a test for hemolysis of blood, and 45 strains with positive findings within oil spread experiment were applied in the laboratory to isolate and segregate the media cultured Banat process (Rahman et al., 2002) These were observed and researched that biosurfactants of Pseudomonas aeruginosa spp are most likely to disrupt the bonding of hydrocarbons like nonadecane, octa, Hexa, and hepta, in marine Water contaminated with oil spills up To approximately 47%, 53%, 73% and 60%(Abrar et al., 2020). Current study concluded that the isolated strain having the ability to degrade hydrocarbon as well as the ability to degrade the heavy metal. The strain also can protect the plant from various diseases. The present research found that the isolated strain is capable of degrading hydrocarbon while also being capable of degrading the heavy metal. As well as the strain does have the capability to defend plants from different diseases. Material And Methods Area of Study The investigation was conducted at HazaraUniversity(HU) Microbiology Laboratory, MansehraPakistan. Assemblage of Samples Thehomestay area of the city Mansehra Pakistan which is named as a township, where oil spills arose, oil spills soil samples were obtained as well as sampling from various Mansehra automobile workshops were also done. Sterilized bags of polythene were being used to collect samples of the soil, after thatthe sample was taken towards the Hazara University (HU) Mansehra Microbiology Laboratory to examine and extract bacterial strains that could develop biosurfactants. The soil temperature at the time of sample selection was around 30 ° C. The pH was also verified by Galvano science companies at the time of selection by pH meter, and the pH being reported was 7. Preparation of Media 15 x 100 mm Petri dishes were being used to prepare the media. Agar plates were thoroughly cleaned with water from the tap and then carefully covered in aluminum foil following cleaning then placed within autoclave at 121°C for about 15 min at 15 psi for sterilization. The nutrient agar which contains 0.5% NaCl, 0.3% beef extract, 0.5% peptone, and 1.5% agar, in 500 ml of distilled water, 14 g of the nutrient agar media (Merck) were dissolved. The nutrient level used mainly for the production of non-fastidious species. Nutrient agar is widely known as it's capable of growing a variety of bacteria types and provides nutrients required for the growth of bacteria. Upon sufficient dissolution of such nutrient agar in distilled water, these were then sterilized by autoclaving for 15 min at 15 psi in the autoclave and held at 121 °C Upon autoclaving, pouring of the media was done in laminar flow hood, and then packed and placed for yet more use in a fridge at 4°C. 2.4 Preparation of serial dilution The bacteria are isolated using the serial dilution process. During this process, 10 test tubes were taken and distilled water (9ml) was added in each tube. After that tubes were put for 15 minutes in the autoclave machine at 121°C. After that 1gm of a crude oil sample from the soil was added in a test tube containing distilled water. Further, 1 ml of the solution was taken from the first test tube and poured to the adjoining tubes for the preparation dilution as under . Afterward, 10μl of the solution was pipetted from both the dilution of and shifted for spread culture techniques, then incubated the plates at 37°C for 48hrs. Biosurfactants extraction Firstly, in nutrient broth solution theculture of bacteria was added and inoculated with oil, the bacterial colony was then incubated at the temperature of 25°C in a shaking incubator just for 7 days. Incubation after seven days of trembling. Thebacterial Crop was then taken and centrifuged at 5000rpm at temperature 4°C for 20minutes. Following centrifugation, the supernatant was collected and then mixed in the equivalent amount in Methanol: Chloroform. White sediment was then retained and collected for further use . Bacterial Colonies Isolation 1 g of the soil polluted with oil was diluted serially up to 106 dilutions.10 μl of 104 and 106 dilutions for spread culture were transferred to the MSM agar plates and nutrient agar. The plates were then incubated at 37°C for 48hrs. Twenty-two morphologically separate colonies were separated for further specific examination just after the incubation and processed by using the technique of streak plate. Screening of Isolates’ Biosurfactants Behavior To check the activity of biosurfactants produced by the bacterial species the following methods of screening were done. Hemolytic Activity of Biosurfactants for Erythrocytes Blood agar containing 5% of blood was prepared as after the fresh isolates were added and inoculated on blood agar plates, then the plates were taken and placed in the incubator at temperature 37°C for 48hrs (Rashediet al., 2005). Thereafter the observation of clear zone in the colonies indicated the existence of bacterial species that produce biosurfactants. This experiment was undertaken to control the ability of isolated bacteria to induce blood agar hemolysis. Three forms of hemolysis usually involve; alpha, beta, and hemolysis of the gamma. The agar underneath the species is dark greenish, then it is Alpha, the yellowish color produced in beta hemolysis and gamma hemolysis does not affect the bacterial sppwhichadded on the plates (Anandaraj and Thivakaran, 2010). Bio-surfactant identification with process of CTAB MSM (Mineral salt agar medium) with (2%) of glucose serving both as carbon source, (0.5 mg / ml) acetyl-tri-methyl-ammonium-bromide (CTAB), and methylene blue (MB: 0.2 mg/ml) are used to detect anionic bio-surfactants (Satpute et al., 2008). For this method, thirty microliters (30μl) of cell-free supernatant were added to each of the wells of the methylene blue agar plate that comprises of borer (4 mm in diameter). after that, the incubation of the plates was done for 48-72 hrs at 37°C. Just after incubation in each of the wells, a dark blue halo zone was being used to show the successful anionic bio-surfactant production. Table 1: Composition of MSM Media S. No Ingredients Amount (gm/L) I Potassium dihydrogen phosphate (KH2PO4) II Magnesium Sulfate (MgSO4) III Iron Sulfate (FeSO4) IV Sodium Nitrate (NaNO3) V Calcium Chloride (CaCl2) VI Ammonium Sulfate (NH4)2SO4 Technique for Spreading of Oil A sufficient number of isolated bacteria were inoculated into a solution of 100ml nutrient broth. Over 3 days, the culture was incubated at 37 ° C in a rotating shaker incubator (150 rpm). After that biosurfactants synthesis was checked in culture suspensions (Priya and Usharani, 2009; Anandaraj and Thivakaran, 2010). For this process, thirty milliliters (30ml) of distilled water was added in a Petri dish. In the middle of the distilled water, 1 milliliter (1ml) of diesel oil was added, and then a centrifuged twenty microliter (20μl) culture was introduced to the middle of a plate, which was isolated from oil spilled soil or local oily groundwater. The species producing the bio-surfactant displace the hydrocarbons and disperse it even in the water. Then it was calculated and analyzed within 1 mint (Ali et al., 2013). Technique for Drop collapse In this process, 96-wellsformed in each of the plates of nutrient agar. Afterwards, all the 96-wells of microliter plates was then filled withmineral oil of about 2ml. Then stabilized the plate at 37oC for 1 hour, after which the oil surface was filled with 5μl of supernatant culture. Therefore, the drop shape was taken to be observed on the oil surface after 1min. The drop which was collapsed, generated by the supernatant culture which is used to signify positive(+ive) outcome and the drops which stayed the same and displayed no changeindicates negative(-ive) outcome. And was taking distilled water as a control(Plaza et al., 2006). Emulsification index The emulsification index was calculated, as stated by the process followed by Cooper and Goldenberg (1981) In this process, 2 ml of kerosene oil was taken and inserted in each of the test tubes to the same amount of cell-free supernatant, and then homogenized for 2 min in a vortex at high speed and allowed for 24 hours to stand. The emulsification steadiness was then determined after the 24 hours, and the emulsification value was estimated by measuring the emulsified layer height by the total liquid layer height, then multiplied by 100. Quantification for the Dry weight of Biosurfactants The bacterial colony was inserted and inoculated in the nutrient broth medium, followed by oil and centrifuged at 5000rpm and after that, the supernatant was clutched and treated with chloroform and methanol and mixed. The white colored deposits were taken and used for the furtherprocess of dry weight. Afterwards, took the clean Petri plate and determined the empty plate weight. Next, the sediment was poured onto Petri plates. Now, for the drying process the hot air oven was used and set the 100ºC of temperature for 30minutes and the plates were put in the oven. After the drying process, the plates were weighted again. The dry weight was calculated for the biosurfactants using the formula which described below: Selected strains Identification and their characterization Instead, various basic biochemical methods were used to identify the isolated bacterial strains. Various biochemical tests, such as Gram staining, Oxidase test, Urease test.Catalase test, Methyl red test, Motility test, Indole test, Starch hydrolysis, Citrate test, Spore staining, Gelatin hydrolysis. Then afterwards, for the preliminary characterization of the biosurfactant, the thin layer chromatography process was used. Physical characterization of the strains selected Gram staining First, on the slide, using the wire loop the bacterial pure culture was taken, and smear was prepared on the slide, and then a drop of purified water was applied. Then, the sterile loop or needle was correctly mixed the bacterial colony and purified water, then mixed up until it is somewhat turbid. Then, spirit lamp was used to fixed the bacterial smear on slide and cooled to room temperature. With this glass slide was loaded with solution of crystal violet and stood for 1minute anddistilled water was applied on slide. Meanwhile the slide was submerged for 1 minute with the iodine solution, and then flushed and rinsed with water. Therefore, decolorizer of about 1 to 2 drops(5 percent acetone and 95 percent alcohol) were added to the slide’s smear and stand for 30seconds, and then treated with water. After then slide was rinsed with safranin for 60seconds, and then treated with water anddry in air. Microscopic analysis was done with 100x objective lenses using emersion oil on smear. Cell morphology The isolates of the bacterial cell were gram stained on slides and then the slides were observed under the light microscope, showing the shape and color of the cells. Biochemical characterization of the selected strains Catalase test Aim of this study is to identify, evaluate and examine that, whether or not the microbes are capable of producing catalase enzymes, while catalase is a protective enzyme, i.e. catalase has the potential to protect against the lethal chemicals known as (H2O2). In this study a bacterial culture that was clarified overnight was used. This culture has been smeared on a glass slide, and 3 percent hydrogen peroxide (H2O2) has been applied and observed on smear. Effects have been observed for bubble formation. Citrate test This study was performed to check the amount or ingest the citrate as the carbon and energy supply for growth and metabolism. Medium containing bromothymol blue and sodium citrate as pH indicator, bacterial was introduced. Ammonium chloride is also present in this medium used as a nitrogen source. Results were noted with variations of color from green to blue. Urease test The capability of urease enzyme for degrading urea was calculated in this bacterial capacity test. Bacterial culture was taken and inoculated for 48 hours at 37 ° C in urease broth, and then color was observed. Methyl red test Through using the process known as mixed acid fermentation which is used to evaluate the bacteria's acid production. The bacterial culture was taken and introduced in the broth of MR-VP and then incubated for 3days at a temperature of 37°C. Two (2) to three (3) drops of Methyl red were added in the broth medium after the incubation period. The change in broth color was observed for final results after a few seconds. Indole test Through using the process to assess the bacteria 's capability to crash indole from tryptophane molecules. After the 24 hours of incubated, taken the fresh inoculum of bacteria and then inserted into the tryptone medium, 24 hours of incubation of about 30oC, 2ml of the tryptone broth medium was added into a sterile test tube. Kovac's reagent was taken to be added (few drops) in sterile test tube and stimulated for a few minutes, and variations of color were detected. Gelatin test It is the approach assess to figure out the use of enzymes known as gelatins from bacterial organisms that precipitate the gelatin. Fresh inoculum of bacteria was taken after 24 hours, and inserted into the media of gelatin agar. This was incubated for around 24 hours, so the temperature did not exceed 30 ° C. Media was observed after incubation time. Starch hydrolysis Several of the micro-organisms that use the starch as a carbon energysource. Therefore, this method has been used to assess whether or not bacteria may use starch as a source of carbon. The bacterial fresh inoculum was spread on the petri starch agar plates, and after that the plate was incubated for 24 hours andmaintained the temperature at 30 to 35 ° C, then gradually applying the supplements of iodine to the plates to flow the change, and then examining the plates. Preliminary characterization of the strains selected Experimental characterization of the bio-surfactant was performed by using the process of TLC (Anandaraj et al., 2010). On a silica gel plate, crude portion of the rudimentary bio-surfactant was separated using Methanol: Chloroform: water (CH3OH: CHCl3: H2O) in the ratio of as an eluent with a different color producing reagents. Ninhydrin reagent (0.5 g ninhydrin in 100ml anhydrous acetone) was used to find bio-surfactant lipopeptide as red spots and anthrone reagent (1 g anthrone in 5ml sulfuric acid combined with 95ml ethanol) as yellow spots to identify rhamnolipid bio-surfactant (Yin et al., 2008). Results and Discussion Isolation of bacteria At first, twenty-two (22) strains from a polluted soil sample were isolated from nutrient agar media.Mixed culture provided by these colonies, so they were taken and smeared on the plates of nutrient agar and then fresh inoculum was collected and stored at temperature of 4oC for the further analysis. Bio-surfactants (surface-active compounds)are formed by a variety of amphiphilic bacterial and fungal organisms that are extracellular (a part of the cellular membrane) (Chen et al., 2007). Screening of Isolated strains for biosurfactant producing colonies Different experiments were carried out to identify, isolate and screen bacteria that are capable of generating bio-surfactants and that is Oil spreading technique(OST), blood hemolysis test(BHT), CTAB test, Emulsification operation. There were twenty-two distinct isolates observed in the current research. And the B1, B2 and B3culture were taken and selected from the twenty-two (22) strains isolated from the polluted spot, which were found to produce biosurfactant. And the oil spreading technique showed promising results for these strains. And strain B2 showed a greater displacement of oil and this is 4 mm. Oil spreading method is quick and often easy to handle, and this technique requires no particular equipment, only a very small amount of sample is used. This approach can be applied when the production and quantity of biosurfactant is small (Plaza et al., 2006) and (Youssef et al., 2004) Only bacterial cultures have been allocated and screened for bacterial species that can generate or use biosurfactants. Just three (3) strainsamong them presented the best results.Those 3 strain,s (B1, B2 and B3) were selected as an additional analysis. Blood hemolysis test On the petri plates of blood agar, the . Isolated bacteriaof B1, B2 and B3 were taken andstreak at the temperature about 37°C for 48 hours. Strain B1 demonstrated β (Beta) hemolysis after the incubation cycle and B2 and B3strains demonstrated γ (Gamma) hemolysis. The B1 strain had an emulsification index of about 74 percent and that was very high as compared to 70 percent for B2 and about 53 percent for B3 respectively. Around the same time, B1 strain showed β (Beta) hemolysis and γ (Gamma) hemolysis was shown bystrains B2 and B3 on the platesof bloodagar. The β hemolysisshowed by the strain B1 in the blood agar test, and the strain B2 and B3 showed γ (Gamma) hemolysis. It is determined that 20 percent strains that are the bestproducer of rhamnolipid have not fully lysed the blood, because the ability of the producer strains capacity not be responsible for the hemolytic activity. According to many researchers, who have shown that this is not such an effective tool for biosurfactant detection due to many bioproducts that may also induce red blood cell lysis, that is not so sufficient to be the surface-active molecule (Youssef et al., 2004). (Rashedi and others, 2005). Table2 Blood Hemolysis Test CTAB agar plate test This test confirms the anionic biosurfactants development. After plate incubation at a temperature of 37 ° C for 72 hours, dark blue hollow zone was existedaround each of the B1 strains wells, which clearly indicated the positive (+ive) development of anionic Biofactant. In addition, the B1 and B2 strains showed positive (+ I ve) results and, in the CTAB analysis, the B3 strain was found to be negative (-ive). The growing microorganisms when secreted the anionic biosurfactants on the plates of CTAB (cetyl-tri-methyl-ammonium-bromide) and methylene blue, then as a result the dark blue-purple insoluble ion pairs formed on the plates. The halo zone around each of the colonies was developed that can recognize rhamnolipid production and that was dark blue in colour, and could correlate with production of rhamnolipid (Siegmund et al., 1991). As indicated in (Fig1) Fig1: B1 positive on CTAB agar plate Oil Spreading Technique The oil was displaced by B1, B2and B3 strains in this test strain and showed a zone that was so clear. The bacterial strains capable of developing biosurfactant were tested and separated from the sample of soil which was oil spilled and brought from the District of Mansehra, Pakistan and from automobile workshops of Mansehra. As shown in (Fig.2). Fig.2: Results of Oil Spreading by B1, B2 and B3Table 3;.Test for oil spreads Bacterial culture Formation of zone (mm) Readings B,1 B,2 B,3 Drop-collapse technique During this process the drop shape was observed at the oil surface. As seen in Fig 3, the collapsed drop was provided by the supernatant culture B1 , B2 and B3.. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Fig 3: Result of Drop-collapse test Table 4: The activity of Biosurfactant emulsification Dry weight of bio-surfactants In this examination, white-colored sediment was collected. Then measured the weight of the sterile Petri plate which was empty in the first step. Then, the sediment was poured into plates. The plates were taken and weighted after 30 minutes of drying on a hot air oven, following the process of drying. The weight of biosurfactants (dry weight) was measured using the following formulations: Fig 4: Dry weight of biosurfactants Table: 5: Dry weight of the biosurfactants Bacterial Culture Weight of the plate (g) biosurfactant in The plate after drying (g) Dry weight of Biosurfactant (g) B,1 B,2 B,3 Identification of selected strains and their characterization Gram staining For structural applications, and stroke analysis gram staining method was used.(Fig.5) shows findings from the process of gram staining. Fig 5: Microscopic view of Gram staining Biochemical identification of bacterial strains and their characterization Specific biochemical studies were performed to identify the species for further recognition and characterization. The bio-surfactant producing microorganism was found to be Pseudomonas aeruginosa after conducting various characterizations and the biochemical tests(Eric Deziel et al., 1996), Which can be used to further analyze and study the industrial development of the biosurfactant. Rhamnolipid is also isolated and produced from the Pseudomonas aeruginosa species on the silica gel plate (Rashedi et al., 2005), a form of biosurfactants highly recommended for processes of bioremediation. All the findings collected from biochemical testing were labeled as Berge 's Manual and it revealed that the protected microorganism was (Pseudomonas aeruginosa). Results of biochemical test were tabulated in (Table.5) Table 6: Bacterial strain identification Tests B1 B2 B3 Gram staining Negative Negative Negative Oxidases Positive e Positive Positive Catalase Positive Positive Positive Indole Positive Negative Negative Citrate Positive Negative Negative Urease Negative Positive Negative Nitrate Positive Positive Positive Motility Positive Positive Positive Gelatin hydrolysis Positive Negative Negative Lactose Negative Positive Positive Methyl red Negative Positive Positive Voges Proskauer Negative Negative Negative Fig 6: Results of biochemical tests(A) Methyl red and Voges Proskauer tests (b) catalase tests (c) oxidase tests (d) indole tests (e) citrate tests (g) lactose tests (h) urease tests Preliminary bacterial strain’s characterization The plates showed yellow dots, when sprayed with anthrone reagent. It indicated the existence of biosurfactants of rhamnolipid in the organism on the plate of TLC as seen in theFig.7 Fig 7: Biosurfactant characterization by TLC Conclusion Biosurfactant development is exciting and perceptible across industries to clean up oil waste and pollutants, particularly in the ecosystem.Compared with chemical surfactants, the biosurfactants are less harmful. It plays an important role in defining the advantages and the importance of industrial applications. Therefore, it is not possible to disregard the growing role and importance of biosurfactants in environmental sustainability.Biosurfactant formulations which can be used for bacterial, fungal, and viral organisms as growth inhibitors. Such biosurfactant inhibition properties can make them components that are applicable to Numerous illnesses that are used as medicinal agents. Therefore it was decided that the described strain could be used as a potential source for heavy metal bioremediation pesticide and hydrocarbon polluted sites. And also used as shielding the plant from different pathogens, contributing to improved crop yields. There is no doubt that the biosurfactants are a multifunctional, advanced, versatile, long-lasting and updated type not only for the twenty-first century but beyond. Conflict of interest The authors declared that they have no conflict of interest and the paper presents their own work which does not been infringe any third-party rights, especially authorship of any part of the article is an original contribution, not published before and not being under consideration for publication elsewhere. References Ali, S.R.; Chowdhury, B.R.; Mondal, P. and Rajak, S. “Screening and characterization of biosurfactants producing microorganism from natural environment (Whey spilled soil)”. Nat. Sci. Res. 2013, 3(13), 34–64. 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Abstract Rhamnolipids are important biosurfactants for application in bioremediation, enhanced oil recovery, pharmaceutical, and detergent industry. In this study, rhamnolipids extracted from P. aeruginosa P6 were characterized to determine their potential fields of application. Thin-layer chromatographic analysis of the produced rhamnolipids indicated the production of two homologues: mono- and di-rhamnolipids, whose structures were verified by 1H and 13C nuclear magnetic resonance spectroscopy. Additionally, high performance liquid chromatography-mass spectrometry identified seven different rhamnolipid congeners, of which a significantly high proportion was di-rhamnolipids reaching 80.16%. Rha-Rha-C10-C10 was confirmed as the principal compound of the rhamnolipid mixture (24.30%). The rhamnolipids were capable of lowering surface tension of water to 36 mN/m at a critical micelle concentration of 0.2 g/L, and exhibited a great emulsifying activity (E24 = 63%). In addition, they showed excellent stability at pH ranges 4–8, NaCl concentrations up to 9% (w/v) and temperatures ranging from 20 to 100 °C and even after autoclaving. These results suggest that rhamnolipids, produced by P. aeruginosa P6 using the cheap substrate glycerol, are propitious for biotechnology use in extreme and complex environments, like oil reservoirs and hydrocarbon contaminated soil. Moreover, P. aeruginosa P6 may be considered, in its wild type form, as a promising industrial producer of di-RLs, which have superior characteristics for potential applications and offer outstanding commercial benefits.

Antibiotics are used to combat bacterial infections by slowing down and preventing the proliferation of bacteria. Antibiotic resistance is a threat to human health, attributed to its overuse and misuse. Altering the membrane permeability to induce antibiotic uptake may be an effective strategy used against both Gram-positive and Gram-negative infectious bacteria. Rhamnolipids (RLs) are biosurfactants produced by Pseudomonas aeruginosa. RLs surface-active properties operate by creating holes in bacterial cell membranes, increasing target cell permeability; allowing antibiotics to penetrate the cell. Rhamnolipids enhance the effect of antibiotics by targeting the intracellular machinery of bacteria. This project tested the susceptibility of bacteria when exposed to antibiotics with and without the addition of RLs, to quantitatively determine if RLs increase antibiotic potency. By analyzing the zones of inhibition data, the results demonstrated that RLs potentiated the antibiotics. Notably, kanamycin coupled with RLs had the most effect inhibiting bacterial growth. To further assess rhamnolipid biosynthesis, a BLAST search was performed exclusively on two genes, rhlA and rhlB. These genes code for the production of two proteins necessary for rhamnolipids. The search indicated a 48% correlation with putative proteins found in Burkholderia pseudomallei. Therefore, based on the experimental results and the BLAST analysis, further research should be conducted to explore the possible role of using rhamnolipids as antibiotic enhancers. Specifically, future experiments could focus on isolating the putative proteins of B.pseudomallei to genetically modify E.coli. Furthermore, isolated studies analyzing the genes of proteins to determine their role in the pathogenicity of Burkholderia species.

Rhamnolipid (RL) is a glycolipid biosurfactant and exhibits the following outstanding characteristics: strong antibacterial properties, low toxicity, and high biodegradability. The present research was conducted to explore the protective effects and mechanisms of rhamnolipids as an alternative to antibiotics in LPS (lipopolysaccharide)-challenged broilers. 16S rRNA gene sequencing and metabolomics were used for analyzing the cecal microbial composition and serum metabolites. Dietary antibiotics and RLS supplementation decreased the weight loss rate, enhanced serum immunoglobulin levels, reduced serum diamine oxidase and D-lactate acid concentration, and improved the symptoms of intestinal bleeding and villus height, when broilers were challenged with LPS. The addition of RLS in the diet enhanced serum interleukin-4 and interleukin-10 contents and reduced serum interleukin-6 and tumor necrosis factor-α levels in LPS-challenged broilers compared with the antibiotics group. Spearman’s correlation analysis revealed that RLS may alleviate LPS-induced inflammatory responses through altering the 6-methoxymellein level in broilers. The genus Bacteroides may contribute to the decreased weight loss rate via regulating the serum lysoPC [20:5(5Z,8Z,11Z,14Z,17Z)] secretion. RLS alleviates LPS-induced intestinal injury, enhances the growth and immunity, ameliorates intestinal microflora, and improves serum metabolites in LPS-challenged broilers. RLS exhibited better protective effect than antibiotic supplementation in the diet of LPS-challenged broilers. These findings provide potential regulation strategies and novel insights for RLS enhancing its protective effect in LPS-challenged broilers.

Abstract Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (&lt;0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared with the parent strain, solely from CH4. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH4.

Rhamnolipids (RLs) and fengycins (FGs) are amphiphilic lipid compounds from bacteria secretomes proposed to replace synthetic pesticides for crop protection. They both display plant defense triggering properties and direct antimicrobial activities. In particular, they have well reported antifungal effects against phytopathogenic fungi. RLs and FGs are considered to act through a direct interaction with membrane lipids and a destabilization of microorganism plasma membrane, thereby limiting the risk of resistance emergence. The main objective of this work was to gain insights in the antimycelial mode of action of these metabolites to promote them as environment and human health friendly biocontrol solutions. Their biocidal effects were studied on two Sclerotiniaceae fungi responsible for diseases in numerous plant species worldwide. We show here that different strains of Botrytis cinerea and Sclerotinia sclerotiorum have opposite sensitivities to RLs and FGs on plate experiments. Overall, B. cinerea is more sensitive to FGs while S. sclerotiorum is more sensitive to RLs. Electron microscopy observations demonstrated that RLs induce mycelial destructuring by asperities emergence and hyphal fusions whereas FGs promote swelling and formation of vesicle-like structures due to vacuole fusions and autophagy. Permeability studies, phosphatidylserine externalization and reactive oxygen species production assessments showed a programmed cell death triggering by RLs at medium concentrations (until 50 μg mL−1) and necrosis characteristics at higher concentration. Programmed cell death was always observed on hyphae treated with FGs. Quantifications of mycelial ergosterol content indicated that a higher ergosterol rate in S. sclerotiorum correlates with increasing sensitivity to RLs. Oppositely, a lower ergosterol rate in B. cinerea correlates with increasing sensitivity to FGs, which was confirmed by ergosterol biosynthesis inhibition with tebuconazole. This gain of knowledge will help to better understand the mode of action of RLs and FGs to fight specific plant fungal diseases.

AbstractRhamnolipids (RLs) are surface-active compounds and belong to the class of glycolipid biosurfactants, mainly produced from Pseudomonas aeruginosa. Due to their non-toxicity, high biodegradability, low surface tension and minimum inhibitory concentration values, they have gained attention in various sectors like food, healthcare, pharmaceutical and petrochemicals. The ecofriendly biological properties of rhamnolipids make them potent materials to be used in therapeutic applications. RLs are also known to induce apoptosis and thus, able to inhibit proliferation of cancer cells. RLs can also act as immunomodulators to regulate the humoral and cellular immune systems. Regarding their antimicrobial property, they lower the surface hydrophobicity, destruct the cytoplasmic membrane and lower the critical micelle concentration to kill the bacterial cells either alone or in combination with nisin possibly due to their role in modulating outer membrane protein. RLs are also involved in the synthesis of nanoparticles for in vivo drug delivery. In relation to economic benefits, the post-harvest decay of food can be decreased by RLs because they prevent the mycelium growth, spore germination of fungi and inhibit the emergence of biofilm formation on food. The present review focuses on the potential uses of RLs in cosmetic, pharmaceutical, food and health-care industries as the potent therapeutic agents.

Abstract Background Oral administration offered a painless way and improved compliance for diabetics. However, the emerging GLP-1 analog peptide drugs for diabetes primarily rely on the injection route, and the development of oral dosage forms was hampered by the low oral bioavailability due to the structural vulnerability to digestive enzymes and molecule impermeability in the gastrointestinal tract. Results In this study, the non-covalent interaction between cholic acid (CA) and liraglutide (LIRA) was found and theoretically explained by molecular docking simulation. Formation of this physical complex of liraglutide and cholic acid (LIRA/CA Complex) reduced the self-aggregation of LIRA and accelerated intestinal epithelium penetration. By the anti-solvent method, LIRA/CA Complex was loaded into zein/rhamnolipids nanoparticles (LIRA/CA@Zein/RLs) with a loading efficiency of 76.8%. LIRA was protected from fast enzymatic degradation by the hydrophobic zein component. Meanwhile, Rhamnolipids, a glycolipid with surface activity, promoted endocytosis while also stabilizing the nanoparticles. The two components worked synergistically to ensure the delivery of LIRA/CA Complex to intestinal villi and improved oral absorption without disrupting tight junctions. LIRA/CA@Zein/RLs demonstrated a considerable intestinal epithelium absorption in mouse gastrointestinal section and a retention in vivo over 24 h, resulting in a significant and long-lasting hypoglycemic effect in Type 2 diabetes mice. Conclusion This study provided a promising oral delivery approach for LIRA and exhibited the potential for further translation into clinical application.

Plant pathogens and pests can cause significant losses in crop yields, affecting food security and the global economy. Many traditional chemical pesticides are used to combat these organisms. This can lead to the development of pesticide-resistant strains of pathogens/insects and negatively impact the environment. The development of new bioprotectants, which are less harmful to the environment and less likely to lead to pesticide-resistance, appears as a sustainable strategy to increase plant immunity. Natural Rhamnolipids (RL-Nat) are a class of biosurfactants with bioprotectant properties that are produced by an opportunistic human pathogen bacterium. RL-Nat can act as plant resistance inducers against a wide variety of pathogens. Recently, a series of bioinspired synthetic mono-RLs produced by green chemistry were also reported as phytoprotectants. Here, we explored their capacity to generate novel colloidal systems that might be used to encapsulate bioactive hydrophobic compounds to enhance their performance as plant bioprotectants. The synthetic mono-RLs showed good surfactant properties and emulsification power providing stable nanoemulsions capable of acting as bio-carriers with good wettability. Synthetic RLs-stabilized nanoemulsions were more effective than RLs suspensions at inducing plant immunity, without causing deleterious effects. These nanoemulsions were innocuous to native substrate microbiota and beneficial soil-borne microbes, making them promising safe bio-carriers for crop protection.

Introduction: Rhamnolipids (RLs) are secondary metabolites naturally produced by bacteria of the genera Pseudomonas and Burkholderia with biosurfactant properties. A specific interest raised from their potential as biocontrol agents for crop culture protection in regard to direct antifungal and elicitor activities. As for other amphiphilic compounds, a direct interaction with membrane lipids has been suggested as the key feature for the perception and subsequent activity of RLs.Methods: Molecular Dynamics (MD) simulations are used in this work to provide an atomistic description of their interactions with different membranous lipids and focusing on their antifungal properties.Results and discussion: Our results suggest the insertion of RLs into the modelled bilayers just below the plane drawn by lipid phosphate groups, a placement that is effective in promoting significant membrane fluidification of the hydrophobic core. This localization is promoted by the formation of ionic bonds between the carboxylate group of RLs and the amino group of the phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups. Moreover, RL acyl chains adhere to the ergosterol structure, forming a significantly higher number of van der Waals contact with respect to what is observed for phospholipid acyl chains. All these interactions might be essential for the membranotropic-driven biological actions of RLs.

Rhamnolipids (RLs), glycolipids biosynthesized by the Pseudomonas and Burkholderia genera, are known to display various activities against a wide range of pathogens. Most previous studies on RLs focused on their direct antimicrobial activity, while only a few reports described the mechanisms by which RLs induce resistance against phytopathogens and the related fitness cost on plant physiology. Here, we combined transcriptomic and metabolomic approaches to unravel the mechanisms underlying RL-induced resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici, a major pathogen of this crop. Investigations were carried out by treating wheat plants with a bioinspired synthetic mono-RL with a 12-carbon fatty acid tail, dodecanoyl α/β-L-rhamnopyranoside (Rh-Est-C12), under both infectious and non-infectious conditions to examine its potential wheat defense-eliciting and priming bioactivities. Whereas, Rh-Est-C12 conferred to wheat a significant protection against Z. tritici (41% disease severity reduction), only a slight effect of this RL on wheat leaf gene expression and metabolite accumulation was observed. A subset of 24 differentially expressed genes (DEGs) and 11 differentially accumulated metabolites (DAMs) was scored in elicitation modalities 2, 5, and 15 days post-treatment (dpt), and 25 DEGs and 17 DAMs were recorded in priming modalities 5 and 15 dpt. Most changes were down-regulations, and only a few DEGs and DAMs associated with resistance to pathogens were identified. Nevertheless, a transient early regulation in gene expression was highlighted at 2 dpt (e.g., genes involved in signaling, transcription, translation, cell-wall structure, and function), suggesting a perception of the RL by the plant upon treatment. Further in vitro and in planta bioassays showed that Rh-Est-C12 displays a significant direct antimicrobial activity toward Z. tritici. Taken together, our results suggest that Rh-Est-C12 confers protection to wheat against Z. tritici through direct antifungal activity and, to a lesser extent, by induction of plant defenses without causing major alterations in plant metabolism. This study provides new insights into the modes of action of RLs on the wheat-Z. tritici pathosystem and highlights the potential interest in Rh-Est-C12, a low-fitness cost molecule, to control this pathogen.