Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces.

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters (BGCs), albeit only a limited number have been analyzed to date. In this study, we described the activation of a cryptic polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene cluster (lsh) in Lysobacter sp. DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp. S001. As a result of this methodology, we were able to isolate two novel linear lipopeptides, lysohexaenetides A (1) and B (2), from the recombinant strain S001-lsh. Furthermore, we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs. This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes, particularly in the absence of genetic manipulation tools.

Safety assessment of surfactin-producing Bacillus strains and their lipopeptides extracts in vitro and in vivo.

Beneficial Bacillus strains can be administered to livestock as probiotics to improve animal health. Cyclic lipopeptides produced by Bacillus such as surfactins may be responsible for some of the beneficial effects due to their anti-inflammatory and immunomodulatory activity. The aim of the present study was to isolate and evaluate the biocompatibility of native Bacillus spp. strains and their surfactin-like lipopeptides in vitro and in vivo to determine their potential to be used on animals. Biocompatibility of endospore suspensions (108 UFC/mL), and different dilutions (1:10; 1:50; 1:100; 1:500, and 1:1000) of Bacillus lipopeptide extracts containing surfactin was tested on Caco-2 cells by microculture tetrazolium-based colorimetric assay. Genotoxicity was tested on BALB/c mice (n = 6) administered 0.2 mL of endospore suspensions by the bone marrow erythrocyte micronuclei assay. All the isolates tested produced between 26.96 and 239.97 µg mL- 1 of surfactin. The lipopeptide extract (LPE) from isolate MFF1.11 demonstrated significant cytotoxicity in vitro. In contrast, LPE from MFF 2.2; MFF 2.7, TL1.11, TL 2.5, and TC12 had no cytotoxic effect (V% > 70%) on Caco-2 cells, not affecting cell viability signifficantly in most treatments. Similarly, none of the endospore suspensions affected cell viability (V% > 80%). Likewise, endospores did not cause genotoxicity on BALB/c mice. This study was elementary as a first step for a new line of research, since it allowed us to choose the safest isolates to keep working on the search of new potentially probiotic strains destined to production animals to improve their performance and health.

Antimicrobial and antioxidant activities of Bacillus mojavensis I4 lipopeptides and their potential application against the potato dry rot causative Fusarium solani.

Lipopeptides are diverse metabolites produced by various bacterial and fungal genera. They are known for their antimicrobial and surfactant activities with diverse environmental, pharmaceutical, and also agronomic applications as biocontrol agents. In this study, a PCR was used to confirm the presence of NRPS genes in Bacillus mojavensis I4. This bacterial strain could produce diverse lipopeptides which belong to the fengycin, and surfactin families. The antioxidant activity of I4 biosurfactants was determined through four different in vitro assays. Furthermore, antimicrobial activity assays indicated that I4 lipopeptides exhibited marked inhibitory activity against several bacterial and fungal strains. Further treatment of potato dry rot causative pathogen Fusarium solani with I4 lipopeptides demonstrated a remarkable reduction in the fungal penetration by almost 80% after 15 days of incubation. The findings suggest that I4 lipopeptide is a potential biocontrol agent during potato tuber storage.

Disease Inhibiting Effect of Strain Bacillus subtilis EG21 and Its Metabolites Against Potato Pathogens Phytophthora infestans and Rhizoctonia solani.

Potato production worldwide is plagued by several disease-causing pathogens that result in crop and economic losses estimated to billions of dollars each year. To this day, synthetic chemical applications remain the most widespread control strategy despite their negative effects on human and environmental health. Therefore, obtainment of superior biocontrol agents or their naturally produced metabolites to replace fungicides or to be integrated into practical pest management strategies has become one of the main targets in modern agriculture. Our main focus in the present study was to elucidate the antagonistic potential of a new strain identified as Bacillus subtilis EG21 against potato pathogens Phytophthora infestans and Rhizoctonia solani using several in vitro screening assays. Microscopic examination of the interaction between EG21 and R. solani showed extended damage in fungal mycelium, while EG21 metabolites displayed strong anti-oomycete and zoosporecidal effect on P. infestans. Mass spectrometry (MS) analysis revealed that EG21 produced antifungal and anti-oomycete cyclic lipopeptides surfactins (C12 to C19). Further characterization of EG21 confirmed its ability to produce siderophores and the extracellular lytic enzymes cellulase, pectinase and chitinase. The antifungal activity of EG21 cell-free culture filtrate (CF) was found to be stable at high-temperature/pressure treatment and extreme pH values and was not affected by proteinase K treatment. Disease-inhibiting effect of EG21 CF against P. infestans and R. solani infection was confirmed using potato leaves and tubers, respectively. Biotechnological applications of using microbial agents and their bioproducts for crop protection hold great promise to develop into effective, environment-friendly and sustainable biocontrol strategies. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition.

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

Potato scab and blight are two major diseases which can cause heavy crop losses. They are caused, respectively, by the bacterium Streptomyces scabies and an oomycete (a fungus-like organism) known as Phytophthora infestans. Fighting these disease-causing microorganisms can involve crop management techniques ­ for example, ensuring that a field is well irrigated helps to keep S. scabies at bay. Harnessing biological control agents can also offer ways to control disease while respecting the environment. Biocontrol bacteria, such as Pseudomonas, can produce compounds that keep S. scabies and P. infestans in check. However, the identity of these molecules and how irrigation can influence Pseudomonas population remains unknown. To examine these questions, Pacheco-Moreno et al. sampled and isolated hundreds of Pseudomonas strains from a commercial potato field, closely examining the genomes of 69 of these. Comparing the genetic information of strains based on whether they could control the growth of S. scabies revealed that compounds known as cyclic lipopeptides are key to controlling the growth of S. scabies and P. infestans. Whether the field was irrigated also had a large impact on the strains forming the Pseudomonas population. Working out how Pseudomonas bacteria block disease could speed up the search for biological control agents. The approach developed by Pacheco-Moreno et al. could help to predict which strains might be most effective based on their genetic features. Similar experiments could also work for other combinations of plants and diseases.

Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

A gelatinized lipopeptide diet effectively modulates immune response, disease resistance and gut microbiome in Penaeus vannamei challenged with Vibrio parahaemolyticus.

Penaeus vannamei is one of the most economically vital shrimp globally, but infectious diseases have hampered its proper production and supply. As antibiotics pose a huge threat to the environment and humankind, it is essential to seek an alternative strategy to overcome infection and ensure proper culture and production. The present study investigates the effect of an anti-infective biosurfactant derivative lipopeptide MSA31 produced by a marine bacterium on the growth performance, disease resistance, and the gut microbiome of P. vannamei when challenged with pathogenic Vibrio parahaemolyticus SF14. The shrimp were fed with a commercial and lipopeptide formulated diet for 60 days and the growth performance was analyzed. The lipopeptide fed shrimp group showed enhanced growth performance and specific growth rate with improved weight gain than the control group. The challenge experiment showed that the survival rate was significant in the lipopeptide fed group compared to the control group. The results revealed 100% mortality in the control group at the end of 12 h of challenge, while 50% of the lipopeptide diet-fed group survived 24 h, which indicates the enhanced disease resistance in shrimp fed with a lipopeptide diet. The test group also showed higher levels of digestive and immune enzymes, which suggests that the lipopeptide diet could positively modulate the digestive and immune activity of the shrimp. The gut microbiome profiling by Illumina high-throughput sequencing revealed that the most abundant genera in the lipopeptide diet-fed group were Adhaeribacter, Acidothermus, Brevibacillus, Candidatus, Mycobacterium, Rodopila, and Streptomyces, while opportunistic pathogens such as Streptococcus, Escherichia, Klebsiella, Neisseria, Rhizobium, and Salmonella were abundant in the control diet-fed shrimp. Also, lipopeptide diet-fed shrimp were found to have a high abundance of ammonia and nitrogen oxidizing bacteria, which are essential pollutant degraders. Therefore, the study reveals that the dietary supplementation of lipopeptide in shrimp aquaculture could positively modulate the gut microbiome and enhance the shrimp's overall health and immunity in an eco-friendly manner.

Sensitization of avian pathogenic Escherichia coli to amoxicillin in vitro and in vivo in the presence of surfactin.

The purpose of this study is to assess the antibiotics adjuvant effect of surfactin for boosting the treatment effect of amoxicillin. Surfactin is used as a surfactant to mediate flux of mono-and divalent cations, such as calcium, across lipid bilayer membranes. In this study, we demonstrated that surfactin can increase the activity of amoxicillin against avian pathogenic Escherichia coli (APEC) in vitro with antimicrobial assays such as minimum inhibitory concentrations (MIC) and fractional inhibitory concentration (FIC). Additionally in the model of chick infection, surfactin exerted adjuvant effects with amoxicillin against APEC by lowering the numerical value of mortality and liver bacterial loads, and regulating the expression of inflammatory cytokines et al. We concluded that surfactin can act as a novel antimicrobial adjuvant with amoxicillin against AEPC infection in chicken.

Biological control of Pseudomonas syringae pv. aptata on sugar beet with Bacillus pumilus SS-10.7 and Bacillus amyloliquefaciens (SS-12.6 and SS-38.4) strains.

AIM: Assessment of biological control of Pseudomonas syringae pv. aptata using crude lipopeptide extracts (CLEs) of two Bacillus amyloliquefaciens strains (SS-12.6 and SS-38.4) and one Bacillus pumilus strain (SS-10.7). METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of CLEs and their combinations against the pathogen and potential interaction between the extracts were determined in vitro. The most effective antibacterial activity was achieved with the CLE from B. amyloliquefaciens SS-12.6, with an MIC value of 0·63 mg ml-1 . Interactions between CLE combinations were mostly indifferent. The biocontrol potential of CLEs, mixtures of CLEs, and cell culture of B. amyloliquefaciens SS-12.6 was tested on sugar beet plants inoculated with P. syringae pv. aptata P53. The best result in inhibiting the appearance of tissue necrosis (up to 92%) was achieved with B. amyloliquefaciens SS-12.6 cell culture. CONCLUSION: This work demonstrated significant biocontrol potential of the CLE and cell culture of B. amyloliquefaciens SS-12.6 which successfully suppress leaf spot disease severity on sugar beet plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of biocontrol of sugar beet emerging pathogen will contribute to growers in terms of alternative disease control management. This study represents first assessment of biological control of P. syringae pv. aptata.

High-throughput optimization of medium components and culture conditions for the efficient production of a lipopeptide pseudofactin by Pseudomonas fluorescens BD5.

BACKGROUND: Lipopeptides are a promising group of surface-active compounds of microbial origin (biosurfactants). These diverse molecules are produced mainly by Bacillus and Pseudomonas strains. Because of their attractive physiochemical and biological properties, biosurfactants are considered to be "green and versatile molecules of the future". The main obstacles in widespread use of biosurfactants are mainly their low yields and high production costs. Pseudofactin (PF) is a lipopeptide produced by Pseudomonas fluorescens BD5. Recently, we identified two analogues, PF1 (C16-Val) and PF2 (C16-Leu), and reported that PF2 has good emulsification and foaming activities, as well as antibacterial, antifungal, anticancer, and antiadhesive properties. Reported production of PF in a mineral salt medium was approximately 10 mg/L. RESULTS: Here, we report successful high-throughput optimization of culture medium and conditions for efficient PF production using P. fluorescens BD5. Compared with production in minimal medium, PF yield increased almost 120-fold, up to 1187 ± 13.0 mg/L. Using Plackett-Burman and central composite design methodologies we identified critical factors that are important for efficient PF production, mainly high glycerol concentration, supplementation with amino acids (leucine or valine) and complex additives (e.g. tryptone), as well as high culture aeration. We also detected the shift in a ratio of produced PF analogues in response to supplementation with different amino acids. Leucine strongly induces PF2 production, while valine addition supports PF1 production. We also reported the identification of two new PF analogues: PF3 (C18-Val) and PF4 (C18-Leu). CONCLUSIONS: Identification of critical culture parameters that are important for lipopeptide production and their high yields can result in reduction of the production costs of these molecules. This may lead to the industrial-scale production of biosurfactants and their widespread use. Moreover, we produced new lipopeptide pure analogues that can be used to investigate the relationship between the structure and biological activity of lipopeptides.

The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells.

The TLR2 agonist, dipalmitoyl lipopeptide (Pam2LP), has been used as an immune adjuvant without much success. Pam2LP is recognised by TLR2/6 receptors in humans and in mice. This study examined the proliferative activity of cytotoxic T lymphocytes (CTL) using mouse Ag-presenting dendritic cells (DCs) and OT-I assay system, where a library of synthetic Pam2LP was utilised from the Staphylococcus aureus database. Ag-specific CTL expansion and IFN-γ levels largely depended on the Pam2LP peptide sequence. The first Aa is cysteine (Cys), which has an active SH residue to bridge fatty acids, and the second and third Aa are hydrophilic or non-polar. The sequence structurally adapted to the residual constitution of the reported TLR2/6 pocket. The inactive sequence contained proline or leucine/isoleucine after the first Cys. Notably, no direct activation of OT-I cells was detected without DCs by stimulation with the active Pam2LP having the Cys-Ser sequence. MyD88, but not TICAM-1 or IFN pathways, in DCs participates in DC maturation characterised by upregulation of CD40, CD80 and CD86. Hence, the active Pam2LPs appear suitable for dimeric TLR2/6 on DCs, resulting in induction of DC maturation.

Identification and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry characterization of biosurfactants, including a new surfactin, isolated from oil-contaminated environments.

Biosurfactant-producing bacteria were isolated from samples collected in areas contaminated with crude oil. The isolates were screened for biosurfactant production using qualitative drop-collapse test, oil-spreading and emulsification assays, and measurement of their tensoactive properties. Five isolates tested positive for in the screening experiments and displayed decrease in the surface tension below 30 mN m-1 . The biosurfactants produced by these isolates were further investigated and their molecular identification revealed that they are bacteria related to the Bacillus genus. Additionally, the biosurfactants produced were chemically characterized via UHPLC-HRMS experiments, indicating the production of surfactin homologues, including a new class of these molecules.

Biological control of potato common scab by Bacillus amyloliquefaciens Ba01.

Potato common scab, which is caused by soil-borne Streptomyces species, is a severe plant disease that results in a significant reduction in the economic value of potatoes worldwide. Due to the lack of efficacious pesticides, crop rotations, and resistant potato cultivars against the disease, we investigated whether biological control can serve as an alternative approach. In this study, multiple Bacillus species were isolated from healthy potato tubers, and Bacillus amyloliquefaciens Ba01 was chosen for further analyses based on its potency against the potato common scab pathogen Streptomyces scabies. Ba01 inhibited the growth and sporulation of S. scabies and secreted secondary metabolites such as surfactin, iturin A, and fengycin with potential activity against S. scabies as determined by imaging mass spectrometry. In pot assays, the disease severity of potato common scab decreased from 55.6 ± 11.1% (inoculated with S. scabies only) to 4.2 ± 1.4% (inoculated with S. scabies and Ba01). In the field trial, the disease severity of potato common scab was reduced from 14.4 ± 2.9% (naturally occurring) to 5.6 ± 1.1% after Ba01 treatment, representing evidence that Bacillus species control potato common scab in nature.

Effect of exogenous inoculants on enhancing oil recovery and indigenous bacterial community dynamics in long-term field pilot of low permeability reservoir.

Pseudomonas aeruginosa DN1 strain and Bacillus subtilis QHQ110 strain were chosen as rhamnolipid and lipopeptide producer respectively, to evaluate the efficiency of exogenous inoculants on enhancing oil recovery (EOR) and to explore the relationship between injected bacteria and indigenous bacterial community dynamics in long-term filed pilot of Hujianshan low permeability water-flooded reservoir for 26 months. Core-flooding tests showed that the oil displacement efficiency increased by 18.46% with addition of exogenous consortia. Bacterial community dynamics using quantitative PCR and high-throughput sequencing revealed that the exogenous inoculants survived and could live together with indigenous bacterial populations. They gradually became the dominant community after the initial activation, while their comparative advantage weakened continually after 3 months of the first injection. The bacterial populations did not exert an observable change in the process of the second injection of exogenous inoculants. On account of facilitating oil emulsification and accelerating bacterial growth with oil as the carbon source by the injection of exogenous consortia, γ-proteobacteria was finally the prominent bacterial community at class level varying from 25.55 to 32.67%, and the dominant bacterial populations were increased by 2-3 orders of magnitude during the whole processes. The content of organic acids and rhamnolipids in reservoir were promoted with the change of bacterial community diversity, respectively. Cumulative oil increments reached 26,190 barrels for 13 months after the first injection, and 55,947 barrels of oil had been accumulated in all of A20 wells block through two rounds of bacterial consortia injection. The performance of EOR has a cumulative improvement by the injection of exogenous inoculants without observable inhibitory effect on the indigenous bacterial populations, demonstrating the application potential in low permeability water-flooded reservoirs.

Biosurfactants production potential of native strains of Bacillus cereus and their antimicrobial, cytotoxic and antioxidant activities.

Present study was designed to evaluate the biosurfactant production potential by native strains of Bacillus cereus as well as determine their antimicrobial and antioxidant activities. The strains isolated from garden soil were characterized as B. cereus MMIC 1, MMIC 2 and MMIC 3. Biosurfactants were extracted as grey white precipitates. Optimum conditions for biosurfactant production were 37°C, the 7th day of incubation, 0.5% NaCl, pH 7.0. Moreover, corn steep liquor was the best carbon source. Biuret test, Thin Layer Chromatography (TLC), agar double diffusion and Fourier Transform Infrared Spectroscopy (FTIR) characterized the biosurfactants as cationic lipopeptides. Biosurfactants exhibited significant antibacterial and antifungal activity against S. aureus, E. coli, P. aeruginosa, K. pneumoniae, A. niger and C. albicans at 30 mg/ml. Moreover, they also possessed antiviral activity against NDV at 10 mg/ml. Cytotoxicity assay in BHK-21 cell lines revealed 63% cell survival at 10 mg/ml of biosurfactants and thus considered as safe. They also showed very good antioxidant activity by ferric-reducing activity and DPPH scavenging activity at 2 mg/ml. Consequently, the study offers an insight for the exploration of new bioactive molecules from the soil. It was concluded that lipopeptide biosurfactants produced from native strains of B. cereus may be recommended as safe antimicrobial, emulsifier and antioxidant agent.

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles.

Protein delivery into the cytosol of cells is a challenging topic in the field of nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipopeptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.

Evaluation of Lipopeptides as Toll-like Receptor 2 Ligands.

BACKGROUND: Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. OBJECTIVE: The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay. METHOD: All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction. RESULTS: Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2. CONCLUSION: Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery.

BACKGROUND: Lipopeptides are known as promising microbial surfactants and have been successfully used in enhancing oil recovery in extreme environmental conditions. A biosurfactant-producing strain, Bacillus atrophaeus 5-2a, was recently isolated from an oil-contaminated soil in the Ansai oilfield, Northwest China. In this study, we evaluated the crude oil removal efficiency of lipopeptide biosurfactants produced by B. atrophaeus 5-2a and their feasibility for use in microbial enhanced oil recovery. RESULTS: The production of biosurfactants by B. atrophaeus 5-2a was tested in culture media containing eight carbon sources and nitrogen sources. The production of a crude biosurfactant was 0.77 g L-1 and its surface tension was 26.52 ± 0.057 mN m-1 in a basal medium containing brown sugar (carbon source) and urea (nitrogen source). The biosurfactants produced by the strain 5-2a demonstrated excellent oil spreading activity and created a stable emulsion with paraffin oil. The stability of the biosurfactants was assessed under a wide range of environmental conditions, including temperature (up to 120 °C), pH (2-13), and salinity (0-50 %, w/v). The biosurfactants were found to retain surface-active properties under the extreme conditions. Additionally, the biosurfactants were successful in a test to simulate microbial enhanced oil recovery, removing 90.0 and 93.9 % of crude oil adsorbed on sand and filter paper, respectively. Fourier transform infrared spectroscopy showed that the biosurfactants were a mixture of lipopeptides, which are powerful biosurfactants commonly produced by Bacillus species. CONCLUSIONS: The study highlights the usefulness of optimization of carbon and nitrogen sources and their effects on the biosurfactants production and further emphasizes on the potential of lipopeptide biosurfactants produced by B. atrophaeus 5-2a for crude oil removal. The favorable properties of the lipopeptide biosurfactants make them good candidates for application in the bioremediation of oil-contaminated sites and microbial enhanced oil recovery process.

Morphological Variation and Recovery Mechanism of Residual Crude Oil by Biosurfactant from Indigenous Bacteria: Macro- and Pore-Scale Experimental Investigations.

Microbial enhanced oil recovery (MEOR) is being used more widely, and the biological contributions involved in MEOR need to be identified and quantified for the improvement of field applications. Owing to the excellent interfacial activity and the wide distribution of producing strains in oil reservoirs, lipopeptides have proved to be an essential part of the complex mechanisms in MEOR. In this study, crude lipopeptides were produced by a strain isolated from an indigenous community in an oil reservoir. It was found that crude lipopeptides can effectively reduce the IFT (interfacial tension) to 10(-1)~10(-2) mN/m under high salinity without forming stable emulsions, and the wettability of natural sandstone can be enhanced (Amott index, from 0.36 to 0.48). The results of core flooding experiments indicate that an additional 5.2% of original oil in place can be recovered with a 9.5% reduction of injection pressure. After the shut-in period, the wettability of the core, the reduction of injection pressure, and the oil recovery can be improved to 0.63, 16.2% and 9.6%, respectively. In the microscopic flooding experiments, the crude oil in membrane, cluster, and throat states contribute nearly 90% in total of the additional oil recovery, and the recovery of membranestate oil was significantly enhanced by 93.3% after shut in. Based on the results in macro and pore scale, the IFT reduction and the wettability alteration are considered primary contributors to oil recovery, while the latter was more dominant after one shut-in period.

Computational insight in the role of fusogenic lipopeptides at the onset of liposome fusion.

We performed an extensive computational study to obtain insight in the molecular mechanisms that take place prior to membrane fusion. We focused on membrane-anchored hybrid macromolecules (lipid-polymer-oligopeptide) that mimic biological SNARE proteins in terms of liposome fusion characteristics [H. Robson Marsden et al., 2009]; efficient micro-second simulation was enabled by combining validated MARTINI force fields for the molecular building blocks in coarse-grained molecular dynamics (CGMD). We find that individual peptide domains in the hybrid macromolecules bind and partially integrate parallel to the membrane surface, in agreement with experimental findings. By varying several experimental design parameters, we observe that peptide domains remain in the solvent phase only in two cases: (1) for solitary lipopeptides (low concentration), below a threshold area per lipid in the membrane, and (2) when the lipopeptide concentration is high enough for the peptide domains to self-assemble into tetrameric homo-complexes. The peptide-membrane binding is not affected by solvent-induced peptide unfolding, which we mimicked by relaxing the usual MARTINI helix constraints. Remarkably, in this case, a reverse transition to a helical secondary structure is observed after binding, highlighting the role of the membrane as a template (partitioning-folding coupling). Our findings undermine the current view of the initial stages towards fusion, in which membranes are thought to be kept in close apposition via dimerization of individual complementary peptides in the solvent phase. Although we did not study actual fusion, our simulations show that the formation of homomers, which is suppressed in experimental peptide-pair design and therefore believed to be insignificant for fusion, by peptides anchored to the same membrane does play a key role in this locking mechanism and potentially also in membrane destabilization that precede fusion.