Complete Genome Sequence of Alcanivorax dieselolei Type Strain B5

Alcanivorax dieselolei B5^T was isolated from oil-contaminated surface water of the Bohai Sea of China and characterized by the efficient degradation of alkane (C₅-C₃₆). Here we report the complete genome of B5^T and genes associated with alkane degradation.     

Isolation and characterization of a biosurfactant‐producing Fusarium sp. BS‐8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m^?1) with a critical micelle concentration of ≥ 1.2 mg mL^?1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L^?1 pure biosurfactant having significant emulsifying index (E₂₄, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1065–1075, 2014     

A cold‐adapted leucine dehydrogenase from marine bacterium Alcanivorax dieselolei: Characterization and l‐tert‐leucine production

l ‐tert‐leucine, an intermediate in the synthesis of several chiral drugs, is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) is the key enzyme. A novel leudh was obtained from the marine bacterium Alcanivorax dieselolei B‐5(T) by PCR. The gene encoded a novel cold‐adapted LeuDH that showed low similarity (less than 50%) to any known proteins; the highest similarity (42.6%) was found for LeuDH from Bacillus cereus. The cold‐adapted LeuDH showed optimal activity at 30℃ and pH 6.5, and was identified to be extremely cold‐adaptive, retaining over 90% activity in the temperature range of 0–37℃. The enzyme exhibited better stability in weak alkali environment (pH 6.0–8.5) than Thermoactinomyces intermedius LeuDH. The best substrate concentration was established, and LeuDH conversion rate in catalyzing trimethylpyruvic acid to l ‐tert‐leucine was 54.6%. The cold activity and its ability to produce l ‐tert‐leucine with excellent performance of enantiomers of choice make it a promising biocatalyst for industrial application under extreme conditions.     

A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1

A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography–mass spectrometry (GC-MS) to be a mixture of l-ornithine lipids, which were composed of l-ornithine and a different couple of iso-3-hydroxyfatty acid (C₁₅–C₁₇) and iso-fatty acid (C₁₅ or C₁₆) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.     

A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation

Biodegradation of petroleum compounds in saline environments seems intricate and needs more attention. In this study, tetracosane was used to enrich alkane-degrading bacteria from oil-contaminated saline soils. Among the isolates, strain Qtet3, with the highest 16s rRNA gene sequence similarity to Alcanivorax dieselolei B-5^T, was able to grow at a wide range of NaCl concentrations and was shown by GC analysis to degrade more than 90% of tetracosane in 10 days. This strain has at least two alkB genes and could grow on crude oil and diesel fuel, and utilize various pure aliphatic hydrocarbon substrates (from C₁₂ to C₃₄). Highly hydrophobic cell surfaces and lack of significant surface tension reduction in the media suggest that the main mechanism of the cells for accessing substrate is to attach directly to hydrocarbon particles. Application of this strain for remediating crude oil-contaminated soils irrigated with defined saline water demonstrated that this halotolerant bacterium could survive and grow in saline soils irrigated with NaCl solutions up to 5% w/v, with the highest hydrocarbon degradation of 26.1% observed at 2.5% NaCl. This strain is promising for future industrial applications especially in bioremediation of saline soils and wastes.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

A novel lipopeptide produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW53

Aims: Our goal was to find a novel, biosurfactant‐producing bacterium from Pacific Ocean deep‐sea sediments. Methods and Results: An oil‐degrading biosurfactant‐producing bacterium TW53 was obtained from deep‐sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34·4 mN m^?1. Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l^?1 HCl. Gas chromatography‐mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C₁₄–C₁₉, and C₁₆H₃₂O₂ was a major component making up 59·18% of the total. However, 3‐hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI‐Q‐TOF‐MS analysis confirmed the composition results and provided their sequence tentatively as Ala‐Ile‐Asp‐Met‐Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30·7 mN m^?1 with a CMC value of 23·7 mg l^?1. These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. Conclusion: The deep‐sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide‐producing bacterium of this genus. The lipopeptides had novel chemical compositions. Significance and Impact of the Study: Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.     

Lipid synthesized by micro‐algae grown in laboratory‐ and industrial‐scale bioreactors

Tetraselmis sp. and Nannochloropsis oculata, cultivated in industrial‐scale bioreactors, produced 2.33 and 2.44% w/w lipid (calculated as the sum of fatty acid methyl esters) in dry biomass, respectively. These lipids contained higher amounts of neutral lipids and glycolipids plus sphingolipids, than phospholipids. Lipids of Tetraselmis sp. were characterized by the presence of eicosapentaenoic acid (that was located mainly in phospholipids), and octadecatetraenoic acid (that was equally distributed among lipid fractions), while these fatty acids were completely absent in N. oculata lipids. Additionally, lipids produced by 16 newly isolated strains from Greek aquatic environments (cultivated in flask reactors) were studied. The highest percentage of lipids was found in Prorocentrum triestinum (3.69% w/w) while the lowest in Prymnesium parvum (0.47% w/w). Several strains produced lipids rich in eicosapentaenoic and docosahexaenoic acids. For instance, docosahexaenoic acid was found in high percentages in lipids of Amphidinium sp. S1, P. parvum, Prorocentrum minimum and P. triestinum, while lipids produced by Asterionella sp. (?) S2 contained eicosapentaenoic acid in high concentration. These lipids, containing ω‐3‐long‐chain polyunsaturated fatty acids, have important applications in the food and pharmaceutical industries and in aquaculture.     

Characteristics of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA supramolecular complexes at various developmental stages

Differences in viscoelasticity (η) and molecular mass (M) values, as well as in the fatty acid profile of lipids in DNA supramolecular complexes (SC), isolated from Pseudomonas aurantiaca cultures at the exponential and stationary growth phases, were established for the first time. Typical characteristics of DNA SC from actively growing cells were the following: η = 315 ± 15 dl/g, M_DNA = 39 × 10⁶ Da, C_16:0 > C_18:0 > C_18:1 present as basic fatty acids (FA) in a pool of loosely DNA-bound lipids; the tightly DNA-bound lipid fraction consisted of only two acids C_18:0 > C_16:0. Significantly higher values of viscoelasticity η = 779 ± 8 dl/g and M_DNA = 198 × 10⁶ Da were observed for DNA SC of the stationary phase cells; one more FA, C_14:0, was detected in the loosely bound lipid fraction, while lipids tightly bound to DNA contained mainly C_16:0 > C_18:1 > C_18:0 > C_14:0 FA. The content of saturated FA in the DNA-bound lipids in the stationary phase cells was twice as high than in the exponential phase cells. The fraction of tightly bound lipids from the stationary phase cells contained nine times more unsaturated fatty acids than the fraction from proliferating cells. These differences in FA composition of DNA-bound lipids demonstrate the importance of lipids for the structural organization and functioning of genomic DNA during bacterial culture development.     

Purification,crystal structure and antimicrobial activity of phenazine‐1‐carboxamide produced by a growth‐promoting biocontrol bacterium,Pseudomonas aeruginosa MML2212

Aim: To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Methods and Results: Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide‐spectrum antimicrobial activity was cultured in Kings’B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two‐step silica‐gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220–222°C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223·3 by mass spectral analysis. Further, it was characterized by IR, ¹H and ¹³C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X‐ray diffraction study and deposited in the Cambridge Crystallographic Data Centre ( http://www.ccde.com.ac.uk ) with the accession no. CCDC 617344 . Conclusion: The crystal compound was undoubtedly identified as phenazine‐1‐carboxamide (PCN) with the empirical formula of C₁₃H₉N₃O. Significance and Impact of the Study: As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.     

Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry

The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The crude surfactant was purified by silica gel column chromatography with a yield of 230 mg of the purified biosurfactant per liter of foam. TLC, IR spectroscopy, ¹H and ¹³C NMR and Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the C₁₄/Leu₇ surfactin was 70 μM, with emulsification efficiency after 24 h (E₂₄) of 67.6% against crude oil. Raw glycerol represents an abundant and renewable carbon source and provides an opportunity for reducing the cost of biosurfactant production and may add value to biodiesel production by creating new commercial applications for this by-product.     

Structure characterization of a methylated ester biosurfactant produced by a newly isolated <Emphasis Type="Italic">Dietzia cinnamea</Emphasis> KA1

Biosurfactans are amphiphilic compounds synthesized by a wide group of microorganisms and tend to interact with surfaces of different polarities. In the present study we purified and characterized a biosurfactant produced by Dietzia cinnamea KA-1 when cultured by n-hexadecane as sole carbon source. The crude biosurfactant was extracted with ethyl acetate and purified by freezing at–20°C and then silica Gel column chromatography. The purified biosurfactant applied for more characterization using Elemental analysis (CHNS), Fourier Transform Infrared Spectroscopy (FTIR), Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (¹H and ¹³C-NMR) analysis. CHNS analysis showed the presence of C (74.92%) and H (11.63%) but not N or S. Functional groups of OH, CH₂, CH₃, C=O and aliphatic C?O revealed by FTIR analysis. The presence and position of these groups were confirmed by NMR analysis, and molecular mass of biosurfactant calculated using MS analysis. Finally, the product characterized as a methylated ester compound with molecular formula of C₂₁H₄₂O₄. This is the first report of biosurfactant of species D. cinnamea identified as ester, furthermore the ester was found to be in the methylated form.     

LIPID COMPOSITION OF CHLORARACHNIOPHYTES (CHLORARACHNIOPHYCEAE) FROM THE GENERA BIGELOWIELLA,GYMNOCHLORA, AND LOTHARELLA1

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast‐associated glycolipids, storage triglycerides, and cytoplasmic membrane‐associated polar lipids were characterized. Glycolipid‐associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0). Triglyceride‐associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n‐3)] dominant along with a number of minor C₁₈ and C₂₀ fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans Moestrup. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences were transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.     

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum‐contaminated soil

Aims: To isolate and characterize the biosurfactant‐producing micro‐organism from petroleum‐contaminated soil as well as to determine the biochemical properties of the biosurfactant. Methods and Results: A novel rhamnolipid‐producing Pseudomonas aeruginosa (GenBank accession number GQ241355 ) strain was isolated from a petroleum‐contaminated soil. Surface active compound was separated by solvent extraction of the acidified culture supernatant. The extract was able to reduce the surface tension of water from 72 to 44 mN m^?1 at a critical micelle concentration of 11·27 ± 1·85 mg l^?1. It showed better activity (based on microdilution method) against Gram‐positive (≤ 31 mg ml^?1) bacteria and filamentous fungi (≤ 50 mg ml^?1) than Gram‐negative bacteria (≥ 125 mg ml^?1) with mild toxicity (HC₅₀– 38 ± 8·22 μg ml^?1) to red blood cells. Fourier transform infrared spectroscopy revealed the presence of aliphatic chain, hydroxyl groups, ester and glycosidic bonds. Presence of nineteen rhamnolipid homologues with variation in chain length and saturation was revealed from liquid chromatography coupled to mass spectrometry with electrospray ionization. Conclusion: The results indicate that the isolated biosurfactant has a novel combination of rhamnolipid congeners with unique properties. Significance and Impact of the Study: This study provides a biosurfactant, which can be used as a biocontrol agent against phytopathogens (Fusarium proliferatum NCIM 1105 and Aspergillus niger NCIM 596) and exploited for biomedical applications.     

Characterisation of a biosurfactant produced by a Bacillus cereus strain tolerant to cadmium and isolated from green coffee grain

In this work, a Gram-positive bacterium with bacillus-type morphology was isolated from low-quality coffee beans in a nutritive medium supplemented with 178 μM of Cd [Cd(NO₃)₂ 4H₂O]. PCR showed 99% similarity of the isolated bacteria with Bacillus cereus QD232. This bacterium produced a biosurfactant after 120 h of growth with an average production of 480 mg l⁻¹ and was able to emulsify various hydrocarbons such as diesel (60%), cyclohexane (48%), benzene (48%), isooctane (47%) and toluene (40%). The molecular weight of the biosurfactant, as determined by high-performance liquid chromatography, was 34,194 Da. The cell-free media had a surface tension of 45 mN m⁻¹ and a critical micellar concentration in the range of 0.2–2.5% (v/v), as evaluated by surface tension and conductance, respectively. The emulsifying agent maintained its properties over a pH range from 6 to 10. The composition of the biosurfactant was 53% proteins, 44.4% lipids and 2.6% carbohydrates. Only a few reports have described the production of biosurfactant from Bacillus cereus strains, and the results from this study show that the biosurfactant properties of Bacillus cereus may have potential environmental applications.     

Stereoselective Determination of Metoprolol and its Metabolite α‐Hydroxymetoprolol in Plasma by LC‐MS/MS: Application to Pharmacokinetics during Pregnancy

Metoprolol is available for clinical use as a racemic mixture. The S‐(?)‐metoprolol enantiomer is the one expressing higher activity in the blockade of the β₁‐adrenergic receptor. The α‐hydroxymetoprolol metabolite also has activity in the blockade of the β₁‐adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α‐hydroxymetoprolol in plasma using high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS). 1‐ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α‐hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC‐MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α‐hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100‐mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)^0?∞ values for S‐(?)‐metoprolol (AUC_S‐(?)/AUC_R‐(+) = 1.81) and the favoring of the formation of the new chiral center 1′R of α‐hydroxymetoprolol (AUC^0?∞_1′R/1′S = 2.78). Chirality, 25:1–7, 2013. © 2012 Wiley Periodicals, Inc.     

A stearoyl‐acyl carrier protein desaturase,NbSACPD‐C,is critical for ovule development in Nicotiana benthamiana

Stearoyl‐acyl carrier protein desaturase (SACPD) activity is essential for production of the major unsaturated fatty acids (UFAs) in plant lipids. We report here the characterization of three SACPD genes from Nicotiana benthamiana, NbSACPD‐A, ‐B, and ‐C. All three genes share high similarity to AtSSI2/FAB2 (Suppressor of Salicylic acid‐Insensitivity2/Fatty Acid Biosynthesis2), the primary SACPD isoform in Arabidopsis. Knocking down the expression of individual or combinations of NbSACPDs by an artificial microRNA approach resulted in significantly reduced accumulation of 18C UFAs and elevated levels of 18:0‐FA (Fatty acids) in leaves, indicating that all three genes participated in fatty acid desaturation. The triple knockdown (KD) plants displayed severe growth phenotypes, including spontaneous cell death and dwarfing. While no vegetative morphologic abnormality was observed in NbSACPD‐A, ‐B, or ‐C KD plants, strikingly, NbSACPD‐C KD plants produced small fruits with aborted ovules. Reciprocal crosses with wild‐type and NbSACPD‐C KD plants revealed that knocking down NbSACPD‐C expression caused female, but not male, sterility. Furthermore, arrested ovule development and significantly altered lipid composition in ovaries were observed in NbSACPD‐C KD plants, consistent with the predominant NbSACPD‐C expression in ovules. The ovule development defect was fully complemented by coexpressing an amiRNA‐resistant NbSACPD‐C variant in the NbSACPD‐C KD background, further supporting a specific requirement for NbSACPD‐C in female fertility. Our results thus indicated that NbSACPD‐C plays a critical role maintaining membrane lipid composition in ovule development for female fertility in N. benthamiana, complementing and extending prior understanding on the well‐demonstrated roles of SACPDs in biotic and abiotic stresses.     

Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil

Aims: To screen and identify biosurfactant producers from petroleum‐contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. Methods and Results: We successfully isolated three biosurfactant producers from petroleum‐contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57·5% increase of the growth‐associated biosurfactant production by Acinetobacter sp. YC‐X 2 with an optimized medium: beef extract 3·12 g l^?1; peptone 20·87 g l^?1; NaCl 1·04 g l^?1; and n‐hexadecane 1·86 g l^?1. Biosurfactant produced by Acinetobacter sp. YC‐X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121°C) and high salinities [up to 18% (w/v) Na⁺ and Ca²⁺], which was more sensitive to Ca²⁺ than Na⁺. Conclusions: Two novel biosurfactant producers were isolated from petroleum‐contaminated soil. Biosurfactant from Acinetobacter sp. YC‐X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. Significance and Impact of the Study: The fact, an increasing demand of high‐quality surfactants and the lack of cost‐competitive bioprocesses of biosurfactants for commercial utilization, motivates researchers to develop cost‐effective strategies for biosurfactant production through isolating new biosurfactant producers with special surface‐active properties and optimizing their cultural conditions. Two novel biosurfactant producers in this study will widen our knowledge about this kind of micro‐organism. This work is the first application of RSM designs for cultural optimization of biosurfactant produced by Acinetobacter genus and the first report that biosurfactant may be more sensitive to Ca²⁺ than Na⁺.     

Biosignatures present in a hydrothermal massive sulfide from the Mid-Atlantic Ridge

Mid‐ocean spreading and accompanying hydrothermal activities result in huge areas with exposure of minerals rich in reduced chemicals – basaltic and peridotitic rocks as well as metal sulfide precipitates – to the oxygenated seawater. Oxidation of Fe and S present in these rocks provides an extensive long‐term source of energy to lithotrophs. Investigation of lipid biomarkers and their carbon isotope ratios from a massive iron sulfide of an inactive sulfide mound or inactive chimney sampled at the western flank of the Turtle‐Pits hydrothermal field (Mid‐Atlantic Ridge, 5°S) revealed a unique lipid distribution. The bacterial fauna appears to be dominated by chemolithotrophs with a distinct lipid composition mainly comprising of iso‐branched fatty acids and nonisoprenoidal dialkyl glycerol diethers partially including the very rare macrocyclic cores with 30–35 carbon atoms (including 13,16‐dimethyloctacosane and 5,13,16‐trimethyloctacosane). The Bacteria are accompanied by most likely hydrogen/CO₂‐dependent methanogenic Archaea (e.g. Methanococcus) as well as other Archaea with a different life style (e.g. Ferroplasma). Alike some of the bacterial lipids the archaeal lipids predominantly consist of macrocyclic diethers including one C₄₀ and one C₄₁ isoprenoid. Structural homologues of the latter are so far only reported from a methanogenic archaeum and a Pleistocene sulfur deposit. Compound‐specific analyses of the stable isotope ratios revealed δ¹³C values for the majority of bacterial and archaeal lipid components of about 0‰ (vs. VPDB), indicative for chemolithoautotrophically fixed carbon which is, for distinct pathways, accompanied by only negligible fractionations. However, the presence of methanogenic Archaea is indicated by ¹³C‐depleted isoprenoidal lipids (δ¹³C ~ –50‰) characteristic for certain CO₂‐reducing methanogens synthesizing lipids via acetyl CoA.     

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

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