Journal articles on the topic 'Biosurfactants. eng'

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. Federation and W. E. Federation, 1920. Standard methods for the examination of water and wastewater. American Public Health Association.Baati, H., R. Amdouni, N. Gharsallah, A. Sghir and E. Ammar, 2010. Isolation and characterization of moderately halophilic bacteria from tunisian solar saltern. Current microbiology, 60(3): 157-161.Berridge, N., 1952. Some observations on the determination of the activity of rennet. Analyst, 77(911): 57b-62.DasSarma, S. and P. Arora, 2001. Halophiles. Encyclopedia of life sciences. Nature publishishing group: 1-9.Donio, M. B. S., F. A. Ronica, V. T. Viji, S. Velmurugan, J. S. C. A. Jenifer, M. Michaelbabu, P. Dhar and T. Citarasu, 2013. Halomonas sp. Bs4, a biosurfactant producing halophilic bacterium isolated from solar salt works in India and their biomedical importance. SpringerPlus, 2(1): 149.El-Sersy, N. A., 2012. Plackett-burman design to optimize biosurfactant production by marine Bacillus subtilis n10. Roman biotechnol lett, 17(2): 7049-7064.Elazzazy, A. M., T. Abdelmoneim and O. Almaghrabi, 2015. Isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from Saudi Arabia. Saudi journal of biological Sciences, 22(4): 466-475.Graham, J. E. and B. Wilkinson, 1992. Staphylococcus aureus osmoregulation: Roles for choline, glycine betaine, proline, and taurine. Journal of bacteriology, 174(8): 2711-2716.Gupta, S., P. Sharma, K. Dev and A. Sourirajan, 2016. Halophilic bacteria of lunsu produce an array of industrially important enzymes with salt tolerant activity. Biochemistry research international, 1: 1-10.Gupta, S., P. Sharma, K. Dev, M. Srivastava and A. Sourirajan, 2015. A diverse group of halophilic bacteria exist in lunsu, a natural salt water body of Himachal Pradesh, India. SpringerPlus 4(1): 274.Hacěne, H., F. Rafa, N. Chebhouni, S. Boutaiba, T. Bhatnagar, J. C. Baratti and B. Ollivier, 2004. Biodiversity of prokaryotic microflora in el golea salt lake, Algerian Sahara. Journal of arid environments, 58(3): 273-284.Jeffries, C. D., D. F. Holtman and D. G. Guse, 1957. Rapid method for determining the activity of microorgan-isms on nucleic acids. Journal of bacteriology, 73(4): 590.Karan, R. and S. Khare, 2010. Purification and characterization of a solvent‐stable protease from Geomicrobium sp. Emb2. Environmental technology, 31(10): 1061-1072.Khopade, A., R. Biao, X. Liu, K. Mahadik, L. Zhang and C. Kokare, 2012. Production and stability studies of the biosurfactant isolated from marine Nocardiopsis sp. B4. Desalination, 3: 198-204.Kim, K. K., J.-S. Lee and D. A. Stevens, 2013. Microbiology and epidemiology of Halomonas species. Future microbiology, 8(12): 1559-1573.Lane, D., 1991. 16s/23s rRNA sequencing in nucleic acid techniques in bacterial systematics. Stackebrandt e., editor;, and goodfellow m., editor. Chichester, UK: John Wiley & Sons.Morikawa, K., R. L. Ohniwa, T. Ohta, Y. Tanaka, K. Takeyasu and T. Msadek, 2009. Adaptation beyond the stress response: Cell structure dynamics and population heterogeneity in Staphylococcus aureus. Microbes environments, 25: 75-82.Morikawa, M., Y. Hirata and T. J. B. e. B. A.-M. Imanaka, 2000. A study on the structure–function relationship of lipopeptide biosurfactants. Biochimica et biophysica acta, 1488(3): 211-218.Oren, A., 2002. Diversity of halophilic microorganisms: Environments, phylogeny, physiology, and applications. Journal of industrial microbiology biotechnology, 28(1): 56-63.Oren, A., 2006. Halophilic microorganisms and their environments. Springer science & business media.Oren, A., R. Vreeland and L. Hochstein, 1993. Ecology of extremely halophilic microorganisms. The biology of halophilic bacteria, 2(1): 1-8.Phillips, K., F. Zaidan, O. R. Elizondo and K. L. Lowe, 2012. Phenotypic characterization and 16s rDNA identification of culturable non-obligate halophilic bacterial communities from a hypersaline lake, la sal del rey, in extreme south texas (USA). Aquatic biosystems, 8(1): 1-5.Post, F. and N. Collins, 1982. A preliminary investigation of the membrane lipid of Halobacterium halobium as a food additive 1. Journal of food biochemistry, 6(1): 25-38.Rocha, C., F. San-Blas, G. San-Blas and L. Vierma, 1992. Biosurfactant production by two isolates of Pseudomonas aeruginosa. World Journal of microbiology biotechnology, 8(2): 125-128.Rohban, R., M. A. Amoozegar and A. Ventosa, 2009. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from howz soltan lake, Iran. Journal of industrial microbiology biotechnology, 36(3): 333-340.Roohi, A., I. Ahmed, N. Khalid, M. Iqbal and M. Jamil, 2014. Isolation and phylogenetic identification of halotolerant/halophilic bacteria from the salt mines of Karak, Pakistan. International journal of agricultural and biology, 16: 564-570.Sambrook, J., E. F. Fritsch and T. Maniatis, 1989. Molecular cloning: A laboratory manual, 2nd edn. Cold spring harbor laboratory, cold spring harbor, New York.Sánchez‐Porro, C., S. Martin, E. Mellado and A. Ventosa, 2003. Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes. Journal of applied microbiology, 94(2): 295-300.Sarafin, Y., M. B. S. Donio, S. Velmurugan, M. Michaelbabu and T. Citarasu, 2014. Kocuria marina bs-15 a biosurfactant producing halophilic bacteria isolated from solar salt works in India. Saudi journal of biological sciences, 21(6): 511-519.Smibert, R., 1994. Phenotypic characterization. In methods for general and molecular bacteriology. American society for microbiology: 611-651.Solomon, E. and K. J. I. Viswalingam, 2013. Isolation, characterization of halotolerant bacteria and its biotechnological potentials. International journal scientific research paper publication sites, 4: 1-7.Spring, S., W. Ludwig, M. Marquez, A. Ventosa and K.-H. Schleifer, 1996. Halobacillus gen. Nov., with descriptions of Halobacillus litoralis sp. Nov. and Halobacillus trueperi sp. Nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. Nov. International journal of systematic evolutionary microbiology, 46(2): 492-496.Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar, 2011. Mega5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology evolution, 28(10): 2731-2739.Yakimov, M. M., K. N. Timmis, V. Wray and H. L. Fredrickson, 1995. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis bas50. Applied and environmental microbiology, 61(5): 1706-1713.Yarza, P., P. Yilmaz, E. Pruesse, F. O. Glöckner, W. Ludwig, K.-H. Schleifer, W. B. Whitman, J. Euzéby, R. Amann and R. Rosselló-Móra, 2014. Uniting the classification of cultured and uncultured bacteria and archaea using 16s rRNA gene sequences. Nature reviews microbiology, 12(9): 635-645

In winemaking, oenological tannins are used to preserve wine colour by enhancing the antioxidant activity, taking part in copigmentation, and forming polymeric pigments with anthocyanins. As a novel processing aid, in this study, a biosurfactant extract was evaluated as a solubilizing and stabilizing agent of anthocyanins in red wine. The biosurfactant extract under evaluation was obtained from a fermented residual stream of the corn milling industry named corn steep liquor (CSL). Two red winegrape varieties (Vitis vinifera L. cv. Aglianico and Cabernet sauvignon) were studied for anthocyanin content and profile, and colour traits, during simulated skin maceration for 7 days at 25 °C, as well as polymerization and copigmentation at the end of maceration. A model wine solution was used as a control, which was added either with the CSL biosurfactant or with four different oenological tannins (from grape skin, grape seed, quebracho, and acacia). The results showed that CSL biosurfactant addition improved the colour properties of skin extracts by the formation of more stable compounds mainly through copigmentation interactions. These preliminary results highlighted that the effectiveness of CSL biosurfactant is variety-dependent; however, there is no significant protection of individual anthocyanin compounds as observed for delphinidin and petunidin forms using quebracho tannin.

Eighty five local isolates of Lactobacillus sp. which were isolated from different sources and identified by biochemical test then subjected to the primary and secondary screening processes to select the active Lactobacillus sp. isolate for biosurfactant production. Among the isolates screened, twenty six isolates with maximum for tests in primary screening were selected for secondary screening. It has been found that Lactobacillus sp. ADK2 had the highest productivity of the biosurfactant. The selected isolate with highest level of biosurfactant activity was identified as Lactobacillus plantarum ADK2 according to PCR technique. The optimum conditions of biosurfactant production by isolate Lactobacillus plantarum ADK2 using submerged fermentation were obtained in the synthetic mineral salt medium (MSM) and natural BCDFTM medium the best production medium separately, 1.5% (lactose and egg) as the best carbon source, 2% meat extract and 3.5% Pease as nitrogen source, temperature 30 °C for two media and pH 5 with pH 3 in MSM and BCDFTM respectively, after 96 hr and 72 hr in MSM and BCDFTM respectively of incubation period.

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.

Surface active agents (SAAs) are molecules with the capacity to adsorb to solid surfaces and/or fluid interfaces, a property that allows them to act as multifunctional ingredients (e.g., wetting and dispersion agents, emulsifiers, foaming and anti-foaming agents, lubricants, etc.) in a widerange of the consumer products of various industrial sectors (e.g., pharmaceuticals, cosmetics, personal care, detergents, food, etc.). Given their widespread utilization, there is a continuously growing interest to explore their role in consumer products (relevant to promoting human health) and how such information can be utilized in order to synthesize better chemical derivatives. In this review article, weaimed to provide updated information on synthetic and biological (biosurfactants) SAAs and their health-promoting properties (e.g., anti-microbial, anti-oxidant, anti-viral, anti-inflammatory, anti-cancer and anti-aging) in an attempt to better define some of the underlying mechanism(s) by which they exert such properties.

Extracellular polymeric substances (EPSs) are involved in both detrimental and beneficial consequences of microbial aggregates such as biofilms, flocs and biological sludges. In biofouling, they are responsible for the increase of friction resistance, change of surface properties such as hydrophobicity, roughness, colour, etc. In biocorrosion of metals they are involved by their ability to bind metal ions. In bioweathering, they contribute by their complexing properties to the dissolution of minerals. The EPSs represent a sorption site for pollutants such as heavy metal ions and organic molecules. This can lead to a burden in wastewater sludge; on the other hand, the sorption properties can be used for water purification. Other biotechnological uses of EPS exploit their contribution to viscosity, e.g., in food, paints and oil-drilling ‘muds’; their hydrating properties are also used in cosmetics and pharmaceuticals. Furthermore, EPSs may have potential uses as biosurfactants, e.g., in tertiary oilproduction, and as biological glue. EPSs are an interesting component of all biofilm systems and still hold a large biotechnological potential.

Pseudomonas facilis and Pseudomonasspp., isolated on the basis of its ability to grow on polycyclic aromatic hydrocarbon, was assayed for biosurfactant production (BP) potentials by measuring the surface tension (ST) of the culture supernatant at different time intervals. The strains in three levels of initial inoculum size (OD600 nm = 0.5, 1, 1.5) were added to medium to determine if bacterial inoculum size affects solubilization of phenanthrene (PHE).The result showed that although the two strains reduced the mean ST to less than 34.12 mN m−1 at the end of day 6, mean solubilization activity of PHE reached 77.05 mg L−1 on the sixth day. There was a significant increase in BP over time (P = 0.008); reaching its peak, 157.84 mg L−1, at the end of the sixth day. Mean solubilization activity of PHE was not significantly different for the two strains (P = 0.216). The time-course study revealed that the ST reduction and BP potential was enhanced as inoculation size increased, leading to higher PHE solubility during the incubation time. However, the trend of increase in PHE solubility was not totally in the same way to cell growth and BP. It may be suggested that more bacterial density needs to be inoculated for practical application of effective bioremediation.

Sophorolipids (SLs), biosurfactants with antimicrobial properties, have been tried to address the problem of antibiotic resistance. The synergistic action of SL and antibiotics was checked using standard microdilution and spread plate methods. WithStaphylococcus aureus, SL-tetracycline combination achieved total inhibition before 4 h of exposure while tetracycline alone couldnot achieve total inhibition till the end of 6 h. The inhibition caused by exposure of bacterium to SL-tetracycline mixture was~25% more as compared to SL alone. In spite of known robustness of gram-negative bacteria, SL-cefaclor mixture proved to be efficient againstEscherichia coliwhich showed~48% more inhibition within 2 h of exposure as compared to cefaclor alone. Scanning electron microscopy of the cells treated with mixture revealed bacterial cell membrane damage and pore formation. Moreover, SLs being a type of asymmetric bola, they are expected to form self-assemblies with unique functionality. This led to the speculation that SLs being amphiphilic in nature can span through the structurally alike cell membrane and facilitate the entry of drug molecules.

ABSTRACT Surfactin is a cyclic lipopeptide biosurfactant. Transposon mutagenesis was performed in Bacillus subtilis strain 168, and a surfactin-susceptible mutant, strain 801, was isolated. Analysis of the region of insertion revealed that yerPwas the determinant of surfactin self-resistance. YerP had homology with the resistance, nodulation, and cell division (RND) family proton motive force-dependent efflux pumps only characterized in gram-negative strains. The yerP-deficient strain 802, in which the internal region of the yerP gene of B.subtilis strain 168 was deleted, showed susceptibility to acriflavine and ethidium bromide. When strain 802 was converted to a surfactin producer by introducing a functional sfp which encodes a 4′-phosphopantetheinyl transferase and is mutated inB. subtilis strain 168, thisyerP-deficient strain produced surfactin, although surfactin production was significantly reduced. The expression ofyerP was at its maximum at the end of the logarithmic growth phase and was not induced by surfactin. yerP is the first RND-like gene characterized in gram-positive strains and is supposed to be involved in the efflux of surfactin.

Peptides with specific affinities for various materials have been identified in the past three decades and utilized in materials science and engineering. A peptide’s capability to specifically interact with materials is not naturally derived but screened from a biologically constructed peptide library displayed on phages or cells. To date, due to limitations in the screening procedure, the function of screened peptides has been primarily limited to the affinity for target materials. Herein, we demonstrated the screening of surfactant-like peptides from a phage-displayed peptide library. A screened phage clone displaying a peptide showed high activity for accumulating at emulsion surfaces with certain assembled structures, resulting in stable emulsions. The surface tension for the solution of the chemically synthesized peptide decreased with increasing peptide concentration, demonstrating certain surface activity, which corresponded to the ability to decrease the surface tension of liquids (e.g., water), owing to the accumulation of molecules at the air–liquid or liquid–liquid interface. Peptides with a randomized sequence did not lower the surface tension, indicating the essential role of amino acid sequences in surface activity. Our strategy for identifying novel functional peptides from a phage-displayed peptide library can be used to expand the applicability of peptidyl materials and biosurfactants.

Surfactins are lipopeptide-type biosurfactants produced mainly by Bacillus species, consisting of a peptide loop of seven amino acids and a hydrophobic fatty acid chain (C12–C16). These molecules have been proven to exhibit various biological activities; thus, their therapeutic and environmental applications are considered. Within the surfactin lipopeptide family, there is a wide spectrum of different homologues and isomers; to date, more than 30 variants have been described. Since the newest members of these lipopeptides were described recently, there is no information that is available on their characteristic features, e.g., the dependence of their production from different cultivation parameters. This study examined the effects of both the different carbon sources and various metal ions on the surfactin production of a selected B. subtilis strain. Among the applied carbon sources, fructose and xylose had the highest impacts on the ratio of the different variants, regarding both the peptide sequences and the lengths of the fatty acids. Furthermore, the application of metal ions Mn2+, Cu2+ and Ni2+ in the media completely changed the surfactin variant compositions of the fermenting broths leading to the appearance of methyl esterified surfactin forms, and resulted in the appearance of novel surfactin variants with fatty acid chains containing no more than 11 carbon atoms.

Glycolipids have become an ecofriendly alternative to chemically obtained surfactants, mainly for the cosmetic, pharmaceutical, and food industries. However, the sustainable production of these compounds is still challenging, because: (i) water is a recognized inhibitor, (ii) multiphases make the use of cosolvent reaction medium necessary, and (iii) there are difficulties in finding a source for both starting materials. This study used sugars and lipids from peach palm fruit shells or model compounds as substrates to synthesize glycolipids on five different renewable deep eutectic solvents (Re-DES) alone or with a cosolvent system. Substrate conversions up to 24.84% (so far, the highest reported for this reaction on DES), showing (1) the non-precipitation of glucose in the solvent, (2) emulsification and (3) low viscosity (e.g., more favorable mass transfer) as the main limiting factors for these heterogeneous enzymatic processes. The resulting conversion was reached using a cosolvent system Re-DES:DMSO:t-butanol that was robust enough to allow conversions in the range 19–25%, using either model compounds or sugar and fatty acid extracts, with free or immobilized enzymes. Finally, the characterization of the in-house synthesized glycolipids by surface tension demonstrated their potential as biosurfactants, for instance, as an alternative to alcohol ethoxylates, industrially produced using less sustainable methods.

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

The chemical synthesis of surface active compounds is economically inefficient. It requires much energy expense, raw materials and harmful reagents. Biological biosynthesis of surface active substances happens in milder conditions without the use of dangerous chemical reagents. The main goal of this work was to select a microorganism strain capable of producing a bioemulsifier with an ability to create a stable water / fuel-oil emulsion that could be used to design a new ecological heating technology. To this end, 3 microorganism strains displaying a high emulsification activity were used. The new discovered surface active substance (SAS) was investigated with different methods (hydrocarbon overlay agar method, emulsification activity determination, microscopic observation). The production of bioemulsifier (BE) was studied by using soluble and insoluble carbon sources. It was found that Arthrobacter sp. Pr82 is the best bioemulsifier producer. Oleic acid was ascertained as the best carbon source for the production of discovered BE.

Members of the Bacillus genus, particularly the “Bacillus subtilis group”, are known to produce amphipathic lipopeptides with biosurfactant activity. This includes the surfactins, fengycins and iturins that have been associated with antibacterial, antifungal, and anti-viral properties. We have screened a large collection of Bacillus, isolated from human, animal, estuarine water and soil samples and found that the most potent lipopeptide producers are members of the species Bacillus velezensis. B. velezensis lipopeptides exhibited anti-bacterial activity which was localised on the surface of both vegetative cells and spores. Interestingly, lipopeptide micelles (6–10 nm diameter) were detectable in strains exhibiting the highest levels of activity. Micelles were stable (heat and gastric stable) and shown to entrap other antimicrobials produced by the host bacterium (exampled here was the dipeptide antibiotic chlorotetaine). Commercially acquired lipopeptides did not exhibit similar levels of inhibitory activity and we suspect that micelle formation may relate to the particular isomeric forms produced by individual bacteria. Using naturally produced micelle formulations we demonstrated that they could entrap antimicrobial compounds (e.g., clindamycin, vancomycin and resveratrol). Micellar incorporation of antibiotics increased activity. Bacillus is a prolific producer of antimicrobials, and this phenomenon could be exploited naturally to augment antimicrobial activity. From an applied perspective, the ability to readily produce Bacillus micelles and formulate with drugs enables a possible strategy for enhanced drug delivery.

It is well known that during biodegradation of oil in natural geological conditions, or oil pollutants in the environment, a degradation of hydrocarbons occurs according to the well defined sequence. For example, the major changes during the degradation process of n-alkanes occur in the second, slight and third, moderate level (on the biodegradation scale from 1 to 10). According to previous research, in the fourth, heavy level, when intensive changes of phenanthrene and its methyl isomers begin, n-alkanes have already been completely removed. In this paper, the ex situ natural bioremediation (unstimulated bioremediation, without addition of biomass, nutrient substances and biosurfactant) of soil contaminated with heavy residual fuel oil (mazut) was conducted during the period of 6 months. Low abundance of n-alkanes in the fraction of total saturated hydrocarbons in the initial sample (identification was possible only after concentration by urea adduction technique) showed that the investigated oil pollutant was at the boundary between the third and the fourth biodegradation level. During the experiment, an intense degradation of phenanthrene and its methyl-, dimethyl-and trimethyl-isomers was not followed by the removal of the remaining n-alkanes. The abundance of n-alkanes remained at the initial low level, even at end of the experiment when the pollutant reached one of the highest biodegradation levels. These results showed that the unstimulated biodegradation of some hydrocarbons, despite of their high biodegradability, do not proceed completely to the end, even at final degradation stages. In the condition of the reduced availability of some hydrocarbons, microorganisms tend to opt for less biodegradable but more accessible hydrocarbons.

ABSTRACTThe bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of twoArthrobacterstrains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO4solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulatorAlyssum serpyllifoliumsubsp.malacitanumwas grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.

This review discusses recent progress in physicochemical understanding of the action of the saponin β -aescin (also called β -escin), the biologically active component in the seeds of the horse chestnut tree Aesculus hippocastanum. β -Aescin is used in pharmacological and cosmetic applications showing strong surface activity. In this review, we outline the most important findings describing the behavior of β -aescin in solution (e.g., critical micelle concentration ( c m c ) and micelle shape) and special physicochemical properties of adsorbed β -aescin monolayers at the air–water and oil–water interface. Such monolayers were found to posses very special viscoelastic properties. The presentation of the experimental findings is complemented by discussing recent molecular dynamics simulations. These simulations do not only quantify the predominant interactions in adsorbed monolayers but also highlight the different behavior of neutral and ionized β -aescin molecules. The review concludes on the interaction of β -aescin with phospholipid model membranes in the form of bilayers and Langmuir monolayers. The interaction of β -aescin with lipid bilayers was found to strongly depend on its c m c . At concentrations below the c m c , membrane parameters are modified whereas above the c m c , complete solubilization of the bilayers occurs, depending on lipid phase state and concentration. In the presence of gel-phase phospholipids, discoidal bicelles form; these are tunable in size by composition. The phase behavior of β -aescin with lipid membranes can also be modified by addition of other molecules such as cholesterol or drug molecules. The lipid phase state also determines the penetration rate of β -aescin molecules into lipid monolayers. The strongest interaction was always found in the presence of gel-phase phospholipid molecules.

While geological sequestration of anthropogenic carbon dioxide is a technically and economically viable option for reducing emissions to the level required to avoid the predicted 2 degrees Celsius increase of atmospheric temperature by the end of this century, efficient sequestration planning is vital for the achievement of this goal.The petroleum industry has used conventional surfactants in enhance oil recovery projects aimed at prolonging the life span of a field, thereby increasing ultimate reserves. Notable among these is the use of surfactants for injected gas relative mobility control. Therefore, the potential for carbon dioxide mobility control in saline aquifers using surfactant alternating gas injection is huge, given the rich experience that can be tapped from the petroleum industry practice.Considering the expected surfactant loses in surfactant-enhanced geological sequestration similar to that encountered in the petroleum industry, this paper has used the analytical solution to advective diffusive equation that exists in the literature with a linear adsorption model where, adsorption has been used to predict trends in minimum pressure drop required for foam generation. The greatest utility of this work lies in the fact that the analytical solution is related a linear adsorption model related to a novel surfactant found from biological and hydrocarbon sources of geologic origin. This paper, therefore, extends the work of linear adsorption models for this novel surfactant aimed at exploring improved oil recovery potentials; in addition to exploring its potential for efficient geological carbon storage in saline aquifers.

ABSTRACT Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Introduction: This chapter addresses chemical aspects of occupational health and marine oil-spill response and is restricted to exposures to crude oil in its various forms. Thus in-situ burning of oil is included, but ancillary chemicals such as surfactants or bioremediation agents are not. The content of this chapter is largely based on the literature published after 1985, the date of a comprehensive review conducted by Politzer et al. [1985] for the American Petroleum Institute, and on a review carried out for the Marine Spill Response Corporation early in 1993 [Holliday and Park, 1993].Concern about health and safety is a normal part of every oil spill. In general, safety is easier to understand and address than are concerns about exposure to crude oil and other chemicals which might be used in the response. At one level, human exposure can be addressed through the enforcement of very conservative requirements for the use of personal protective equipment (PPE). In the real world, however, conditions at a spill site make the use of such equipment inconvenient or even hazardous, and so the goal becomes to balance the risk from exposure with the appropriate level of PPE.While oil-spill cleanup is a comparatively new aspect of occupational-health practice, and dates from the formalization of response measures by companies and national and international agencies (something that occurred over the last 30 years), exposure to crude oil itself is a "mature" occupational-health matter. Workers have been exposed, both by inhalation and dermally, to the effects of crude oil for the past century. The exposure of response workers during the early phases of the oil-spill response can be likened to that experienced by oil-well-drilling crews and, to a lesser extent, by oil-well-maintenance personnel or fighters of oil-well fires. In contrast, exposures in the later stages of the cleanup are less clearly related to occupations within the oil industry. The crude oil will have been altered by weathering, and exposure to cleanup chemicals (e.g., dispersants, bioremediation agents) will become relatively more prominent. Such substances are beyond the scope of this chapter, and in any event, few data are available on the compositions or mammalian toxicity of dispersants. Although there are frequent references to toxicity in connection with dispersants, these invariably seem to refer to ecotoxicity. Human hazard does not appear to be an issue. For example, in a recently published paper entitled, "Effectiveness and safety of biosurfactants as agents of oil spill response" [Lepo et al., 1997], "safety" refers to possible toxicity to crustaceans and fish.

The attention towards the bacteria associated with human health is growing more and more, above all regarding the bacteria that inhabit the niches offered by the human body, i.e., the gastrointestinal tract, skin, vaginal environment, and lungs. Among the secondary metabolites released by microorganisms associated with human health, little consideration is given to the biosurfactants, molecules with both hydrophobic and hydrophilic nature. Their role in the complex human environment is not only the mere biosurfactant function, but they could also control the microbiota through the quorum sensing system and the antimicrobial activity. These functions protect them and, accordingly, the human body principally from microbial and fungal pathogens. Consequently, nowadays, biosurfactants are emerging as promising bioactive molecules due to their very different structures, biological functions, low toxicity, higher biodegradability, and versatility. Therefore, this review provides a comprehensive perspective of biosurfactants with antimicrobial activity produced by bacteria associated with the human body and related to everything human beings are in contact with, e.g., food, beverages, and food-waste dumping sites. For the first time, the role of an “-omic” approach is highlighted to predict gene products for biosurfactant production, and an overview of the available gene sequences is reported. Besides, antimicrobial biosurfactants’ features, challenges, and potential applications in the biomedical, food, and nutraceutical industries are discussed.

Food spoilage organisms were isolated using standard procedures on Nutrient Agar, Cetrimide Agar and Pseudomonas Agar Base (supplemented with CFC). The samples were categorized as animal products (raw fish, egg, raw chicken, corned beef, pasteurized milk) and plant products (vegetable salad, water leaf (Talinium triangulare), boiled rice, tomatoes and pumpkin leaf (Teifairia occidentalis).They were characterised as Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas stutzeri, Burkholderia pseudomallei, Serratia rubidaea, Corynebacterium pilosum, Bacillus subtilis, Bacillus mycoides, Bacillus laterosporus, Bacillus laterosporus, Serratia marcescens, Bacillus cereus, Bacillus macerans, Alcaligenes faecalis and Alcaligenes eutrophus. Preliminary screening for biosurfactant production was done using red blood haemolysis test and confirmed by slide test, drop collapse and oil spreading assay. The biosurfactant produced was purified using acetone and the composition determined initially using Molisch’s test, thin layer chromatography and gas chromatography mass spectrometry. The components were found to be ethanol, amino acids, butoxyacetic acid, hexadecanoic acid, oleic acid, lauryl peroxide, octadecanoic acid and phthalic acid. The producing organisms grew readily on several hydrocarbons such as crude oil, diesel oil and aviation fuel when used as sole carbon sources. The purified biosurfactants produced were able to cause emulsification of kerosene (19.71-27.14%) as well as vegetable oil (16.91-28.12%) based on the emulsification index. This result suggests that the isolates can be an asset and further work can exploit their optimal potential in industries.

This work is aimed at evaluating the potentiality of adding a rhamnolipid biosurfactant in a
 petroleum-bearing soil. For this purpose, dehydrogenase activity and seed germination
 (Lactuca sativa) testes were performed before the biodegradation assays with different
 concentrations of rhamnolipid (1 to 15mg for 1g of soil). The addition of 1 and 15 mg g-1 of
 rhamnolipid was harmful to the soil environment. The biodegradation assays were carried out
 at room temperature during 45 days in bioreactors containing 450g of a polluted soil with
 different rhamnolipid concentrations varying from 1 to 15 mg g-1. The nutrients were corrected
 through the addition of NH4NO3 and KH2PO4, in a nutritional ratio of C:N:P=100:15:1. The
 humidity was adjusted to 50% of the liquid retention capacity. Besides these assays, a control
 test was conducted without adding rhamnolipid. TPH (Total petroleum hydrocarbon) removal
 and seed germination were evaluated at the end of these experiments. When 4 mg g-1 of
 rhamnolipid were used a TPH removal of about 60% was observed. The biosurfactant
 addition improved all treatments, except for the assays with addition of 1 and 15 mg g-1 in
 which a decrease of the bioremediations rates was observed in the toxicity tests.

Application of nano or biomaterials for enhanced oil recovery (EOR) has been recently much attended by petroleum engineering researchers. However, how would be the displacement mechanisms and how would change the recovery efficiency while nano and biomaterials are used simultaneously is still an open question. To this end, a series of injection tests performed on micromodel containing shale strikes. Three types of biomaterials including biosurfactant, bioemulsifier, and biopolymer beside two types of nanoparticles including SiO2 and TiO2 at different concentrations were used as injection fluids. The microscopic as well as macroscopic efficiency of displacements were observed from analysis of images recorded during the tests. Microscopic observations revealed different mechanisms responsible for oil recovery including: wettability alteration, thinning oil film, interfacial tension (IFT) reduction, and water in oil emulsion formation. Contact angle experiments showed changes in the surface wetness from an oil-wet to neutral-wet/water-wet conditions when a layer of nano-biomaterial covered thin sections of a shaly sandstone. Also the results showed that the presence of shales causes early breakthrough and ultimate oil recovery reduction. Shales act as flow barriers and enhance injection fluid viscous fingering. Displacement efficiency in shaly systems is sharply related to the shale distribution. Oil recovery after breakthrough in shaly systems is progressive and considerable volume of oil in place is recovered after breakthrough. The highest efficiency, 78%, observed while injecting one pore volume of biopolymer and SiO2 nanoparticles. This work illustrates for the first time the mechanisms involved in nano-biomaterial-crude oil displacements.

Aims: This work aimed to demonstrate the feasibility of producing (mono- and di-) rhamnolipids employing a strain of Pseudomonas aeruginosa strain ATCC 9027 employing olive oil as a substrate and some mineral salts. This rhamnolipid is a biosurfactants with multiple applications The CMC of this product under different conditions (filtered, unfiltered, in the presence and absence of Fe and Mg, at different pH values) was assessed. At the end, the UP was assessed in the washing of a TPH contaminated soil.
 Place and Duration of Study: Bioprocess department. Unidad Profesional Interdisciplinaria de Biotecnologia-IPN facilities, during 2016.
 Methodology: Rhamnolipids were produced with P. aeruginosa in olive oil, then by drying the culture broth was generated an unpurified product (UP) that contained 0.19% rhamnolipids. Critical micelle concentration CMC of UP products were evaluated in the presence of Ca2+ or Fe3+ from 0.5 to 2 mM, and pH values from 4 to 10. Finally, this surfactant was assessed in the washing of hydrocarbon-contaminated soils, and compared with other synthetic surfactants.
 Results: It was found that CMCs were similar to those reported in the literature for pure rhamnolipids. The UP products have shown dynamic behavior in the soil washing at concentrations below 176 mg/L because removed 80% of 6,500 mg TPH/Kg from a gravel-sandy soil; the rhamnolipids could be removed TPH through mobilization mechanism.
 Conclusion: It was possible to produce rhamnolipid using olive oil as carbon source and strain of P. aeruginosa ATCC 9027 to levels of 100 mg/L. It was feasible to produce a powder containing 1.19% of rhamnolipids. The UP had better properties as a surfactant than the purified product. The pH affects the CMC of the rhamnolipids in a way that promotes their behavior as ionic surfactant or nonionic surfactant. The ionic strength with Ca 2+ and Fe3+ has an effect on the CMC of rhamnolipids so that the decreases in the range of 35 to 41 mg/L in the presence of 0.5 to 2 mM of metals. The UP rhamnolipids were employed for washing soil contaminated with 6,500 mg/kg increased TPH removal at low concentrations and to be as effective as chemical surfactants. TPH removal observed was about 80% for rhamnolipid with a CMC x 0.074 concentration.