Biosurfactant Activity Research Articles

Isolation, Characterization and Molecular Identification of Bacteria Associated with Green and Brown Seaweeds

Aims: The co-existence of bacterial communities along with macroalgae in marine environment develops a mutualistic relationship resulting in functional advantages for both the groups. The present research studied the composition of epiphytic bacteria associated with green (Ulva spp.) and brown seaweeds (Sargassum spp. and Padina spp.) sampled off Maharashtra coast, India, and their physiological and bioactive properties. Study Design: Green and brown seaweeds were collected from three locations along Maharashtra coast and analyzed for the associated bacteria. Place and Duration of Study: Seaweed samples analyzed in this study were from Kelwa, Manori and Ratnagiri along the Maharashtra coast. The analysis was performed during June to September 2021. Methodology: Bacteria from freshly collected seaweed samples were homogenized in saline, 10-fold serially diluted and plated on Zobell marine agar. Distinct colonies were selected and identified by a series of biochemical tests, followed by partial 16SrRNA gene sequencing. The physiological characteristics of the isolated bacteria were studied by screening for temperature and salinity tolerance, production of protease, lipase, agarose, amylase and biosurfactants. Results: The total seaweed bacterial count ranged from 4.5 ×103 to 2.9 × 104 CFU/g. Seventy-seven bacteria were isolated, 44 (57.14%) and 33 (42.85%) isolates from green and brown seaweeds respectively. The 16SrRNA sequencing of 20 representative isolates revealed the dominance of Bacillus spp., followed by Vibrio spp. Growth at 0°C was exhibited by all bacteria except Bacillus tequilensis, Bacillus altitudinis, Oceanobacillus iheyensis and one isolate of Vibrio spp. A majority of the isolates grew at 45°C. Vibrio spp. exhibited protease, amylase, gelatinase, and agarase activities, whereas the biosurfactant activity was commonly associated with Bacillus spp. Conclusion: The results of this study illustrate the occurrence of seaweed-associated beneficial bacteria exhibiting bioactive properties with potential biomedical applications.

Optimized Rhamnolipid Production by a Pseudomonas marginalis C9 Strain Isolated from a Biopurification System to Enhance Pesticide Solubilization

Biopurification systems designed for pesticide treatment are a source of diverse bacteria with environmental and biotechnological applications, including Pseudomonas marginalis C9, which has been reported as a biosurfactant-producing bacterium. The optimization of biosurfactant produced from P. marginalis C9 to enhance the solubility of a hydrophobic pesticide of environmental interest was investigated. The response surface methodology (RSM) was used to optimize the combined effect of the initial pH (5–9), agitation (100–300 rpm), and temperature (24–32 °C) on biosurfactant production. A DASbox® automated mini-bioreactor system was used to evaluate the critical factors in biosurfactant production using a full factorial design (FFD). The results showed that the optimal culture conditions using RSM were a pH of 8.5, a temperature of 25 °C, and agitation at 200 rpm. The extraction yield of the biosurfactant was 7.40 g L−1, the surface tension was reduced to 27.45 mN m−1, and the critical micelle concentration (CMC) was 48.9 mg L−1. The FFD analysis indicated that a high agitation rate (300 rpm) strongly influenced the biosurfactant activity, regardless of the inlet oxygen supply (0.5–1.5 vvm). The rhamnolipid increased the water solubility of chlorpyrifos by 11.2- and 21.7-fold at the CMC and twice the CMC, respectively.

Discovery and characterization of novel lipopeptides produced by Virgibacillus massiliensis with biosurfactant and antimicrobial activities

The study aimed to evaluate the biosurfactants (BSs) production by SM-23 strain of Virgibacillus identified by phenotypical and WGS analysis as Virgibacillus massiliensis. We first demonstrated the lipopeptides production by Virgibacillus massiliensis specie and studied their biochemical and molecular analysis as well as their biological potential. The GC–MS analysis indicated that methyl.2-hyroxydodecanoate was the major fatty acid compound with 33.22%. The maximum BSs production was obtained in LB medium supplemented by 1% olive oil (v/v) at 30 °C and 5% NaCl with 1.92 g/l. The obtained results revealed the significant biosurfactants/bioemulsifier potential compared to triton X100 with E24 of 100%, and an emulsification stability SE of 83%. The lipopeptides types were identified by FTIR analysis. A strong antimicrobial action was observed by the produced lipopeptides by the agar diffusion method against E.coli, K. pneumoniae, S. aureus, Fusarium sp, Alternaria sp, and Phytophtora sp. The complete genome sequencing showed genes involved in the synthesis of multiple compounds identified as amphipathic cyclic lipopeptides such as locillomycin/locillomycin B/locillomycin C and bacillibactin. Our results highlighted significant lipopeptides properties displayed by V. massiliensis that can be exploited to develop a novel strategy in the formulation of natural biocidal and fungicidal agents.

Modelling and impact of tensiometer plate geometry and sample volume on biosurfactant surface activity assessment

Biosurfactants are molecules with hydrophilic and hydrophobic moieties with the capacity to reduce the surface tension of water. Given the limited quantity of biosurfactant extracts in laboratories, it is recommended to use equipment that requires minimal sample quantities for detecting the presence of biosurfactants. In this work, commercial glycolipids biosurfactants (rhamnolipids or sophorolipids) were diluted in water and subjected to different analyses to obtain their minimum surface tension (ST) reduction and their critical micellar concentration (CMC). The independent variables of the study were: the geometry of platinum plate (rectangular or cylindrical), the sample volume (2, 4 and 20 mL) and the container material consisting of either glass or polytetrafluoroethylene (PTFE). The variation of ST with biosurfactant concentration was studied based on the isotherm model proposed by Li & Lu. It was observed that the profile of ST values did not vary so much using the different independent variables described, observing that platinum rectangular plate can be used for volumes of 4 mL biosurfactants instead of cylindrical plate usually recommended for volumes lower than 20 mL, the container material was also not significant based on the Pearson and Spearman statistical treatment. Moreover, well-fitting regression model results were obtained for a non-commercial biosurfactant extract obtained from a residual stream of the dairy industry, predicting values close to the observed data.

Metabolite profiling and molecular characterization of NBAIR BSWG1: A potential strain of Bacillus subtilis against Fusarium oxysporium f. sp. udum

To address the fungal wilt of pigeon pea caused by Fusarium oxysporium f. sp. udum, farmers currently rely on chemical fungicides, despite their harmful effects. However, there is a growing need for safer alternatives like green pesticides. Bacterial biocontrol agents and their derivatives serve as potential green pesticides in the management of plant pathogens. In the present study, we aimed to identify indigenous Bacillus subtilis strains effective against F. oxysporium f. sp. udum. We used PCR and MALDI-TOF analysis to identify the active components responsible for the efficiency of efficient strain. Biochemical studies of cell-free extracts extracted from B. subtilis strains demonstrated the highest biosurfactant activity in NBAIR BSWG1, with an oil displacement of 2 cm and an emulsification index of 60 %. Molecular characterization confirmed the presence of surfactin, fengycin, and iturin coding genes in the B. subtilis strains, among them, NBAIR BSWG1 showed the highest number of lipopeptide-producing genes. Meanwhile, NBAIR BSWG1 showed inhibition of 79.84 % against F. oxysporium f. sp. udum using cell-free extract. Further metabolite profiling of NBAIR BSWG1 using MALDI-TOF analysis further confirmed surfactin, fengycin, and iturin in the purified cell-free extract of NBAIR BSWG1. Two peaks with m/z of 923.77 and 1149.92 were identified as novel lipopeptide compounds which need further characterization. The present study identified NBAIR BSWG1 as an efficient bacterial strain for the inhibition of F. oxysporium f. sp. udum and its antifungal properties are mainly due to the production of cyclic lipopeptides.

Antibacterial and biosurfactant activity of endophytic bacteria isolated from mangrove plant in Lamongan, Indonesia

Abstract. Fatimah, Rahayuningtyas ND, Nastiti A, Alawiyah DD, Ramadhan E, Geraldi A, Junairiah. 2024. Antibacterial and biosurfactant activity of endophytic bacteria isolated from mangrove plant in Lamongan, Indonesia. Biodiversitas 25: 3035-3042. This study aims to determine the antibacterial and biosurfactant activity of 61 endophytic bacteria from mangrove plants in Kutang Lamongan Beach. Antibacterial screening of supernatant of endophytic bacteria was performed using the disc diffusion method against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. The most potent isolate was identified using 16s rRNA. The chemical compounds of the most potent isolate were identified using GC-MS analysis. The data were analyzed descriptively. Biosurfactant activity was also performed on the most potential isolate. All the isolates showed inhibitory zones against E. coli and S. aureus. Isolate LMG-2 produced the highest antimicrobial activity, averaging 9.57±0.4 mm against E. coli and 8.87±0.36 mm against S. aureus. The biosurfactant activity of LMG-2 isolate produced a surface tension of 39.09+0.49 mN/m and emulsification activity of 65.03+0.03% against kerosene, 47.63+0.10% against crude oil and 46.53+0.27 to diesel. The isolate LMG-2 had 98,41% similarity with the Bacillus amyloliquefaciens strain NBRC 15535 with a query cover of 100%. The biosurfactant extract contains 9-octadecenoic acid methyl ester, (E)-, 9,12-octadecadienoic acid (Z, Z) methyl ester, and 9,12,15-octadecadienoic acid methyl ester. These compounds, known as Fatty Acid Methyl Esters (FAMEs), were integral components of biosurfactants, surface-active agents produced by microorganisms.

The potential of indigenous Bacillus sp. BT3.1 as an antifungal biopesticide against the plant pathogen Fusarium oxysporum

Abstract. Nafidiastri FA, Lolita NT, Riqina HS, Aurora BZ, Geraldi A, Nurhariyati T, Supriyanto A, Ni’matuzahroh, Fatimah, Salamun. 2024. The potential of indigenous Bacillus sp. BT3.1 as an antifungal biopesticide against the plant pathogen Fusarium oxysporum. Biodiversitas 25: 2679-2686. Biosurfactants are green surfactants that bacteria can produce, one of which is Bacillus. This genus can produce lipopeptide-type biosurfactants, namely surfactin, that can inhibit the growth of fungal pathogens. This research aims to determine the biosurfactant activity on molasses substrates and the antifungal activity of isolate BT3.1 against Fusarium oxysporum. The biosurfactant activity test method tested hemolytic activity using the spot method, surface tension analysis, and emulsification activity. In addition, an antifungal activity test was also carried out using the swab method. Characterization of isolate BT3.1 was carried out macroscopically and microscopically. The macroscopic characteristics of isolate BT3.1 show that bacteria from the genus Bacillus are moderate in size, white, irregular with serrate colony edges, shiny raised elevations, and a shiny surface texture. Microscopic characteristics of the isolate showed Gram-positive bacteria with oval-shaped endospores in the center. Based on the results of the biosurfactant activity test, isolate BT3.1 showed beta-type hemolysis activity, reduced surface tension by 23.3 mN/m on distilled water and 12 mN/m on NB media, and produced an emulsion activity value of around 52%. The results of the production of crude extract of BT3.1 isolate on 2, 4, and 6% molasses substrates showed optimal biosurfactant activity at a concentration of 4% with an incubation time of 48 hours. The antifungal activity of isolate BT3.1 showed that 80% inhibited the growth of Fusarium oxysporum, so the indigenous bacteria Bacillus sp. BT-3.1 has the potential to be developed as a raw material candidate for antifungal biopesticides against the plant pathogen Fusarium oxysporum.

Bioelectrochemical synthesis of rhamnolipids and energy production and its correlation with nitrogen in air-cathode microbial fuel cells

Microbial fuel cells (MFCs) have been recently proven to synthesise biosurfactants from waste products. In classic bioreactors, the efficiency of biosynthesis process can be controlled by the concentration of nitrogen content in the electrolyte. However, it was not known whether a similar control mechanism could be applied in current-generating conditions. In this work, the effect of nitrogen concentration on biosurfactant production from waste cooking oil was investigated. The concentration of NH4Cl in the electrolyte ranged from 0 to 1 g L−1. The maximum power density equal to 17.5 W m−3 was achieved at a concentration of 0.5 g L−1 (C/N = 2.32) and was accompanied by the highest surface tension decrease (to 54.6 mN m−1) and an emulsification activity index of 95.4%. Characterisation of the biosurfactants produced by the LC-MS/MS method showed the presence of eleven compounds belonging to the mono- and di-rhamnolipids group, most likely produced by P. aeruginosa, which was the most abundant (19.6%) in the community. Importantly, we have found a strong correlation (R = −0.96) of power and biosurfactant activity in response to C/N ratio. This study shows that nitrogen plays an important role in the current-generating metabolism of waste cooking oil. To the best of our knowledge, this is the first study where the nitrogen optimisation was investigated to improve the synthesis of biosurfactants and power generation in a bioelectrochemical system.

Isolation and Identification of Bacterial Biosurfactant Activity from Mangrove Sediments

Background: Ujung Pangkah mangroves are reported to have been contaminated with heavy metals. Such heavy metals can induce microorganisms to produce biosurfactants. Biosurfactants with amphiphilic characteristics can lower surface tension. Biosurfactants can be used as antibacterial, antifungal, and antiviral for biomedical purposes. Purpose: This study aimed to identify and test the activity of biosurfactant isolates of bacteria from Ujung Pangkah Mangrove sediments, Gresik Regency. Methods: Biosurfactant activity test methods include emulsification index, oil spreading, drop collapse, and parafilm test. Results: The results of the identification of bacterial isolates in this study obtained the genus Bacillus sp. because bacterial isolates show rod shape, Gram-positive, aerobic, and have ellipse-shaped endospores on the subterminal. The results of the biosurfactant activity test with the Emulsification index method showed an average result of 54.39% and the results of the biosurfactant activity test with the oil spreading method showed that there was a clear zone. The average result of the clear zone obtained is 54.83 mm. The average result in the parafilm test was 8.02 mm and the drop collapse test showed positive results characterized by falling and spreading of bacterial isolate fermentation broth supernatants.

Valorization and kinetic modeling of biosurfactant produced by Kocuria marina DAGII using waste cooking oil as a substrate

Microbial biosurfactant has wide applications in industrial sectors owing to their eco-friendly and biocompatible nature giving, an alternative to chemically synthesized surfactants. In our study, Kocuria marina DAG II, an isolated bacterium was observed to produce a biosurfactant. The production of biosurfactant was established by oil-spreading (3.72 ± 0.15 cm), drop collapse, and CTAB (Cetyltrimethyl ammonium bromide) assay. Biosurfactant activity was re-confirmed by the reduction in surface tension of uninoculated broth from 68.52 mN/m to 47.40 ± 0.57 mN/m. Chemical characterization study of the biosurfactant was done by TLC (thin-layer chromatography) indicating the presence of proteins (Rf = 0.78 ± 0.08), lipids (Rf = 0.79 ± 0.09), and polysaccharides (Rf = 0.81 ± 0.06). FTIR and NMR (1HNMR and 13CNMR) studies confirmed the presence of different functional groups and revalidated the presence of amino acids, lipids, and polysaccharides in the biosurfactant. From the optimization study, the highest production of biosurfactant having a yield of 3.05 ± 0.05 g/L was obtained at a temperature of 31 °C and pH 8, when kept for an incubation period of 88 hr. The addition of rancid and waste-frying mustard oil and used engine oil could enhance the production of biosurfactant by Kocuria marinaDAGII hence substantiating the concept of value-added biosurfactant production from waste. Kinetic modeling showed a good fit between experimental and predicted values using modified Gompertz model with R2 values of 0.99 and 0.99. The biosurfactant exhibited radical scavenging activity through DPPHand ABTS assays with IC50 values of 1.29 mg/ml and 0.56 mg/ml respectively. The biosurfactant produced by Kocuria marina DAG II could be utilized for various applications, bypassing the need for a synthetic surfactant.

Biochemical characterization of immobilized recombinant subtilisin and synthesis and functional characterization of recombinant subtilisin capped silver and zinc oxide nanoparticles

This pioneering research explores the transformative potential of recombinant subtilisin, emphasizing its strategic immobilization and nanoparticle synthesis to elevate both stability and therapeutic efficacy. Achieving an impressive 95.25 % immobilization yield with 3 % alginate composed of sodium along with 0.2 M CaCl2 indicates heightened pH levels and thermal resistance, with optimal action around pH 10 as well as 80 °C temperature. Notably, the Ca-alginate-immobilized subtilisin exhibits exceptional storage longevity and recyclability, affirming its practical viability. Comprehensive analyses of the recombinant subtilisin under diverse conditions underscore its adaptability, reflected in kinetic enhancements with increased Vmax (10.7 ± 15 × 103 U/mg) and decreased Km (0.19 ± 0.3 mM) values post-immobilization using N-Suc-F-A-A-F-pNA. UV–visible spectroscopy confirms the successful capping of nanoparticles made of Ag and ZnO by recombinant subtilisin, imparting profound antibacterial efficacy against diverse organisms and compelling antioxidant properties. Cytotoxicity was detected against the MCF-7 breast cancer line of cells, exhibiting IC50 concentrations at 8.87 as well as 14.52 µg/mL of AgNP as well as ZnONP, correspondingly, indicating promising anticancer potential. Rigorous characterization, including FTIR, SEM-EDS, TGA and AFM robustly validate the properties of the capped nanoparticles. Beyond therapeutic implications, the investigation explores industrial applications, revealing the versatility of recombinant subtilisin in dehairing, blood clot dissolution, biosurfactant activity, and blood stain removal. In summary, this research unfolds the exceptional promise of recombinant subtilisin and its nanoparticles, presenting compelling opportunities for diverse therapeutic applications in medicine. These findings contribute substantively to biotechnology and healthcare and stimulate avenues for further innovation and exploration.

An Approach for Developing a Method for Biosurfactant Activity Detection

An Approach for Developing a Method for Biosurfactant Activity Detection

Biosurfactant Activity of Bacillus sp. Strain LP04 Isolate and Its Antifungal Potency against Ganoderma boninense and Fusarium sp.

Biosurfactants are a class of amphipathic molecules that various microorganisms can produce. Biosurfactants are used as biopesticides and biocontrol agents because they have antimicrobial activity, especially as antifungal agents in several species of fungal pathogens such as Ganoderma boninense and Fusarium sp. that attack crops. This study aims to detect the biosurfactant activity of Bacillus sp. and its potential as an antifungal agent against the fungi Ganoderma boninense and Fusarium sp. Biosurfactants were produced in mineral salt medium (MSM) by harvesting cell-free supernatants. Screening of biosurfactant-producing isolates was carried out using an oil-spreading assay, a hemolysis assay, and an emulsification index. The antifungal activity of the isolates was then tested using the agar diffusion method. The LP04 isolate was closely related to Bacillus thuringiensis with a 99% similarity level. It has the potential to have biosurfactant activity, which is characterized by a positive result on the oil spreading assay test and has an emulsification index of 48.33±2.87%. The cell-free supernatants of the bacterial isolate were able to inhibit the growth of Ganoderma boninense and Fusarium sp. with growth inhibition rates of 51.11% and 56.92%, respectively.

Antibacterial and Anticancer activity of Glycolipid Biosurfactant from Manglicolous Yeast Geotrichum candidum PV 37

Antibacterial and Anticancer activity of Glycolipid Biosurfactant from Manglicolous Yeast Geotrichum candidum PV 37

Diversity and enzymatic, biosurfactant and phytotoxic activities of culturable Ascomycota fungi present in marine sediments obtained near the South Shetland Islands, maritime Antarctica.

We studied the culturable fungal community recovered from deep marine sediments in themaritime Antarctic, and assessed their capabilities to produce exoenzymes, emulsifiers and metabolites with phytotoxic activity. Sixty-eight Ascomycota fungal isolates were recovered and identified. The most abundant taxon recovered was the yeast Meyerozyma guilliermondii, followed by the filamentous fungi Penicillium chrysogenum, P. cf. palitans, Pseudeurotium cf. bakeri, Thelebolus balaustiformis, Antarctomyces psychrotrophicus and Cladosporium sp. Diversity indices displayed low values overall, with the highest values obtained at shallow depth, decreasing to the deepest location sampled. Only M. guilliermondii and P. cf. palitans were detected in the sediments at all depths sampled, and were the most abundanttaxa at all sample sites. The most abundant enzymes detected were proteases, followed by invertases, cellulases, lipases, carrageenases, agarases, pectinases and esterases. Four isolates showed good biosurfactant activity, particularly the endemic species A. psychrotrophicus. Twenty-four isolates of P. cf. palitans displayed strong phytotoxic activities against the models Lactuca sativa and Allium schoenoprasum. The cultivable fungi recovered demonstrated good biosynthetic activity in the production of hydrolytic exoenzymes, biosurfactant molecules and metabolites with phytotoxic activity, reinforcing the importance of documenting the taxonomic, ecological and biotechnological properties of fungi present in deep oceanic sediments of the Southern Ocean.

Biosurfactant production by Rhodococcus ALDO1 isolated from olive mill wastes

Rhodococcus is a genus of Gram-positive bacteria that are widely distributed in soil, water, and plants. They have a versatile metabolism that produces different kinds of compounds like biosurfactants, lipids and carotenoids, and therefore are promising for biotechnological applications. In this work, we aimed to characterize the ability of the strain Rhodococcus ALDO1 isolated from alperujo, a complex residue from the olive oil industry, to produce biosurfactants. Growing was determined by counting the number of cells and by dry weight determination Biosurfactant production was followed by xylose equivalents (μg/ml Xylose Eq.), the perimeter obtained in the oil spreading assay and the emulsification index at 24 h (E24). We found that this strain grew in different media like glucose, oil, and glycerol, and produced a biosurfactant of a glycolipid nature as suggested by FT-IR spectrum. The maximal amount of biosurfactant (18.8 ± 2.0 μg/ml Xylose Eq., perimeter = 4.17 ± 0.17 cm, and E24 = 63.1 ± 4.1 %) was obtained when Rhodococcus ALDO1 grew in the media with oil 3% + glucose 1 %. Glycerol was also a suitable substrate for biosurfactant production (8.3 ± 1.0 μg/ml Xylose Eq., perimeter = 2.84 ± 0.07 cm, and E24 = 35.7 ± 2.9 %). The biosurfactant activity is stable at salt concentrations up to 5 g/L, temperatures up to 60 °C and pHs above 9. The thermal degradation started at 220 °C which suggested that the biosurfactant is suitable for various industrial applications. These results indicated that Rhodococcus ALDO1 is a strain to be considered as a source of biocompounds with possible industrial applications obtained from cheap substrates.

Tuning the antimicrobial activity of microbial glycolipid biosurfactants through chemical modification.

Sophorolipids, glycolipid biosurfactants derived from microorganisms such as Starmerella bombicola, possess distinctive surface-active and bioactive properties, holding potential applications in cosmetics, pharmaceuticals and bioremediation. However, the limited structural variability in wild-type sophorolipids restricts their properties and applications. To address this, metabolic engineering efforts have allowed to create a portfolio of molecules. In this study, we went one step further by chemically modifying microbially produced sophorosides, produced by an engineered S. bombicola. Twenty-four new sophoroside derivatives were synthesized, including sophoroside amines with varying alkyl chain lengths (ethyl to octadecyl) on the nitrogen atom and their corresponding quaternary ammonium salts. Additionally, six different microbially produced glycolipid biosurfactants were hydrogenated to achieve fully saturated lipid tails. These derivatives, along with microbially produced glycolipids and three benchmark biosurfactants (di-rhamnolipids, alkyl polyglucosides, cocamidopropyl betaine), were assessed for antimicrobial activity against bacteria (Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa) and yeast (Candida albicans). Results indicated that microbially produced glycolipids, such as bola sophorosides, acidic sophorolipids and acidic glucolipids exhibit selective antimicrobial activity against the test organisms. Conversely, lactonic sophorolipids, sophoroside amines and quaternary ammonium salts display a broad antimicrobial activity. N-octyl, N-dodecyl and N-octadecyl derivatives exhibit the lowest minimal inhibitory concentrations, ranging from 0.014 to 20.0mgmL-1. This study demonstrates the potential synergy of thoughtful biotechnology and targeted chemistry to precisely tailor glycolipid biosurfactants to meet specific requirements across applications.

Characterization of a biosurfactant produced by Peruvian strain Pseudomonas aeruginosa 6K‐11

AbstractBACKGROUNDThe oil industry has a big impact on the environment by itself and by using chemically synthesized surfactants. Biosurfactants are an ecofriendly alternative, especially rhamnolipids produced by Pseudomonas aeruginosa. Rhamnolipids in a determined concentration decrease the liquid surface tension, and this allows access oil hydrocarbon to be degraded. The objective of this research is to characterize the biosurfactant produced by the native strain P. aeruginosa 6K‐11 by determining its critical micelle concentration (CMC), Krafft point, and emulsifying activity.RESULTSExperiments were initially performed with rhamnolipid produced by P. aeruginosa 6K‐11 growing on Siegmund‐Wagner medium with 1% of waste oil as carbon source and at 31.26 °C, 186 rpm after 42 h of incubation. After that, the biosurfactant was purified according to acid precipitation methodology. The first conductivity measurement, 34 μS/cm, was obtained with 12.5 g/L of rhamnolipid by using an APERA conductivity meter. The rhamnolipid CMC was 5.86 g/L due to the conductivity measurements graph and 0.49 was the dissociation degree. The equations of both lines were found by the method of least squares. The rhamnolipid has produced 1.97 Units of Emulsifying Activity (UEA) and 50% stability was observed based on the E24 Index. The highest conductivity achieved under 39 °C, as calculated from a conductivity against temperature graph, was the Krafft point.CONCLUSIONThese findings give important parameters to characterize the P. aeruginosa 6K‐11 biosurfactant activity that can be used to accelerate oil biodegradation in bioremediation processes. © 2024 Society of Chemical Industry (SCI).

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

Up to Date of Cyanobacterial Natural Products

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.