Production and structural elucidation of trehalose tetraesters (biosurfactants) from a novel alkanothrophic Rhodococcus wratislaviensis strain

Aims:  To isolate a biosurfactant-producing bacterial strain and to identify and characterize the chemical structure and properties of its biosurfactants.
Methods and Results:  The bacterium Rhodococcus wratislaviensis BN38, isolated from soil, was found to produce glycolipid biosurfactants when grown on 2% n -hexadecane. The glycolipids were isolated by chromatography on silica gel columns and their structures elucidated using a combination of multidimensional NMR and ESI-MS/MS techniques. The main product was identified as 2,3,4,2'-trehalose tetraester with molecular mass of 876 g mol⁻¹. It was also noted that the biosurfactant was produced under nitrogen-limiting conditions and could not be synthesized from water-soluble substrates. The purified product showed extremely high surface-active properties.
Conclusions:  The glycolipid biosurfactant produced by the alkanothrophic strain R. wratislaviensis BN38 was characterized to be 2,3,4,2'-trehalose tetraester which exhibited high surfactant activities.
Significance and Impact of the Study:  Strain BN38 of R. wratislaviensis is a potential candidate for use in bioremediation applications or in biosurfactant exploration.     

Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.     

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P相似文献   

An oil-degrading bacterium: Rhodococcus erythropolis strain 3C-9 and its biosurfactants

AIMS: To isolate a biosurfactant-producing bacterium and find new products within its culture. METHODS AND RESULTS: A biosurfactant-producing bacterium identified as Rhodococcus erythropolis (3C-9 strain) was isolated from seaside soil. When n-hexadecane was supplied as the sole carbon source, two types of biosurfactants (free fatty acids and glycolipids) were detected in the supernatant of the bacterial culture by use of thin layer chromatography (TLC). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the former consisted of at least 12 free fatty acids of chain lengths from C(9) to C(22); and the latter contained 2 kinds of glycolipids (a glucolipid and a trehalose lipid), which were detected by use of TLC, as well as GC-MS. The hydrophobic moieties of both glycolipids consisted of seven types of straight-chain fatty acids of varying compositions, with chain lengths ranging from C(10) to C(18). It was also noted that biosurfactants of strain 3C-9 were produced in a manner that was growth-related and cannot be synthesized from water-soluble substrates. The effects to enhance the solubility of polycyclic aromatic hydrocarbons and the degradation rate of hexadecane were also tested. CONCLUSIONS: The biosurfactants produced by strain 3C-9 of R. erythropolis included two kinds of glycolipids, as well as free fatty acids. These biosurfactants were notably different from those of previously reported Rhodococcus species, both in terms of their structure and chemical composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain 3C-9 of R. erythropolis is a competitive candidate for use in oil spill cleanup operations, or in new biosurfactant exploration. The findings in this report show that Rhodococcus is a natural reservoir of new biosurfactants.     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha C(10)-C(12), respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha-C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha-C10-C12, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

耐热木霉菌株筛选及其对热作区香蕉促生效应的研究

[目的]为筛选安全、高效的耐热木霉功能菌株,有效促进热作区香蕉的生产,本研究从热作区土壤分离筛选适温范围较宽的木霉菌株,研制木霉生物有机肥,并研究其对香蕉枯萎病发病率、果实产量及品质的影响.[方法]通过稀释涂布法筛选出木霉菌株,根据菌株在不同温度下的生长情况、拮抗尖孢菌能力及酶活强弱进行菌株复筛;将复筛所得菌株试制成生...     

A Novel Biosurfactant Produced by Aureobasidium pullulans L3-GPY from a Tiger Lily Wild Flower,Lilium lancifolium Thunb.

Yeast biosurfactants are important biotechnological products in the food industry, and they have medical and cosmeceutical applications owing to their specific modes of action, low toxicity, and applicability. Thus, we have isolated and examined biosurfactant-producing yeast for various industrial and medical applications. A rapid and simple method was developed to screen biosurfactant-producing yeasts for high production of eco-friendly biosurfactants. Using this method, several potential niches of biosurfactant-producing yeasts, such as wild flowers, were investigated. We successfully selected a yeast strain, L3-GPY, with potent surfactant activity from a tiger lily, Lilium lancifolium Thunb. Here, we report the first identification of strain L3-GPY as the black yeast Aureobasidium pullulans. In addition, we isolated a new low-surface-tension chemical, designated glycerol-liamocin, from the culture supernatant of strain L3-GPY through consecutive chromatography steps, involving an ODS column, solvent partition, silica gel, Sephadex LH-20, and an ODS Sep-Pak cartridge column. The chemical structure of glycerol-liamocin, determined by mass spectrometry and nuclear magnetic resonance spectroscopy, indicates that it is a novel compound with the molecular formula C₃₃H₆₂O₁₂. Furthermore, glycerol-liamocin exhibited potent biosurfactant activity (31 mN/m). These results suggest that glycerol-liamocin is a potential novel biosurfactantfor use in various industrial applications.     

Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28

AIMS: The influence of biosurfactant compounds produced by a strain of Pseudomonas fluorescens on the adhesion of Listeria monocytogenes LO28 to polytetrafluoroethylene (PTFE) and AISI 304 stainless steel surfaces was investigated. METHODS AND RESULTS: The biosurfactant was produced according to a simple, novel technique based on cultivation on nutrient agar. Adhesion studies were performed using L. monocytogenes cells cultured at 20 or 37 degrees C. CONCLUSIONS: A substrate-dependent behaviour of the LO28 strain (larger number of cells adhering to stainless steel than to PTFE), and a significant reduction (< 90%) in microbial adhesion levels through the prior adsorption of biosurfactants on stainless steel surfaces, which can be related to a change in the electron-donor characteristics of this substratum, was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The prior adsorption of biosurfactants on solid surfaces may constitute a new and effective means of combating the implantation of pathogenic micro-organisms in food processing plants.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

Isolation and characterization of hydrocarbon-degrading and biosurfactant-producing yeast strains obtained from a polluted lagoon water

Microbial surfactants are environmentally friendly products with amazing properties and spectrum of applications. It is therefore, not surprising that research has increased in recent time with the objectives of sourcing for novel surface-active compounds with dual functions in oil and pharmaceutical industries. Evaluation of hydrocarbon degrading potentials and emulsifying activities indicated that biosurfactants were produced by two newly isolated and promising yeast strains, Saccharomyces cerevisiae and Candida albicans, obtained from a polluted lagoon water. Both strains were able to grow effectively on crude oil and diesel as sole sources of carbon and energy. Growth curves on diesel were obtained to establish the relation between cell growth and biosurfactant production. The growth peak was on the 8th day while the specific growth rate ranged insignificantly (P < 0.05) between 0.46 and 0.48 day⁻¹. Interestingly, biosurfactant was detected on the 2nd day when growth was almost inexistent, with maximal production obtained at stationary/death phase of growth. The partially-purified biosurfactants exhibited antimicrobial activities by completely inhibiting the growth of clinical strains of Escherichia coli and Staphylococcus aureus at all concentrations tested. Although C. albicans appeared to be a better diesel-utilizer and biosurfactant-producer (E₂₄ = 64.2%), the potency of its surfactant was smaller than that of S. cerevisiae. These strains represent a new class of biosurfactant producers that have potential for use in a variety of biotechnological and industrial processes particularly in the pharmaceutical industry.     

Isolation and characterization of thermotolerant yeasts for the production of second-generation bioethanol

The purpose of this study was to isolate, identify, and characterize the thermotolerant yeasts for use in high-temperature ethanol fermentation. Thermotolerant yeasts were isolated and screened from soil samples collected from the Mekong Delta, Vietnam, using the enrichment method. Classification and identification of the selected thermotolerant yeasts were performed using matrix-assisted laser desorption ionization/time-of-fight mass spectrometry (MALDI-TOF/MS) and nucleotide sequencing of the D1/D2 domain of the 26S rDNA and the internal transcribed spacer (ITS) 1 and 2 regions. The ethanol production by the selected thermotolerant yeast was carried out using pineapple waste hydrolysate (PWH) as feedstock. A total of 174 yeast isolates were obtained from 80 soil samples collected from 13 provinces in the Mekong Delta, Vietnam. Using MALDI-TOF/MS and nucleotide sequencing of the D1/D2 domain and the ITS 1 and 2 regions, six different yeast species were identified, including Meyerozyma caribbica, Saccharomyces cerevisiae, Candida tropicalis, Torulaspora globosa, Pichia manshurica, and Pichia kudriavzevii. Among the isolated thermotolerant yeasts, P. kudriavzevii CM4.2 displayed great potential for high-temperature ethanol fermentation. The maximum ethanol concentration (36.91 g/L) and volumetric ethanol productivity (4.10 g/L h) produced at 45 °C by P. kudriavzevii CM4.2 were achieved using PWH containing 103.08 g/L of total sugars as a feedstock. These findings clearly demonstrate that the newly isolated thermotolerant yeast P. kudriavzevii CM4.2 has a high potential for second-generation bioethanol production at high temperature.     

Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

Aims: Plant growth‐promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping‐off of cucumber evaluated. Methods and Results: The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping‐off of cucumber, since both wild type strain 267 and its biosurfactant‐deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC‐MS and MS‐MS analyses. Conclusions: The biosurfactants produced by Ps. putida 267 were identified as putisolvin‐like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping‐off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study: Pseudomonas putida 267 suppresses Phy. capsici damping‐off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin‐like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.     

Importance of 3-hydroxy fatty acid composition of lipopeptides for biosurfactant activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r2= 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r2= 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Antimicrobial biosurfactants from marine Bacillus circulans: extracellular synthesis and purification

Aims:  To purify the biosurfactant produced by a marine Bacillus circulans strain and evaluate the improvement in surface and antimicrobial activities.
Methods and Results:  The study of biosurfactant production by B. circulans was carried out in glucose mineral salts (GMS) medium using high performance thin layer chromatography (HPTLC) for quantitative estimation. The biosurfactant production by this strain was found to be growth-associated showing maximum biosurfactant accumulation at 26 h of fermentation. The crude biosurfactants were purified using gel filtration chromatography with Sephadex^® G-50 matrix. The purification attained by employing this technique was evident from UV–visible spectroscopy and TLC analysis of crude and purified biosurfactants. The purified biosurfactants showed an increase in surface activity and a decrease in critical micelle concentration values. The antimicrobial action of the biosurfactants was also enhanced after purification.
Conclusions:  The marine B. circulans used in this study produced biosurfactants in a growth-associated manner. High degree of purification could be obtained by using gel filtration chromatography. The purified biosurfactants showed enhanced surface and antimicrobial activities.
Significance and Impact of the Study:  The antimicrobial biosurfactant produced by B. circulans could be effectively purified using gel filtration and can serve as new potential drugs in antimicrobial chemotherapy.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

Characterization and properties of biosurfactants produced by a newly isolated strain <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> DCS1 and their applications in enhancing solubility of hydrocarbon

Six biosurfactant-producing bacteria were isolated from hydrocarbon contaminated soils in Sfax, Tunisia. Isolates were screened for biosurfactant production by different conventional methods including hemolytic activity, surface tension reduction, drop-collapsing and oil displacement tests. All these screening tests show that all the isolates behave differently. Among the isolated bacteria, DCS1 strain was selected for further studies based on its highest activities and it was identified as Bacillus methylotrophicus DCS1. This strain was found to be a potent producer of biosurfactant when cultivated in mineral-salts medium supplemented with diesel oil (2 %, v/v) as a sole carbon source. Physicochemical properties and stability of biosurfactants synthesized by B. methylotrophicus DCS1 were investigated. The produced biosurfactants DCS1, from Landy medium, possess high surface activity that could lower the surface tension of water to a value of 31 from 72 mN m^?1 and have a critical micelle concentration (CMC) of 100 mg L^?1. Compared with SDS and Tween 80, biosurfactants showed excellent emulsification activities against different hydrocarbon substrates and high solubilization efficiency towards diesel oil. Biosurfactants DCS1 showed good stability in a wide range of temperature, pH and salinity. These results suggested that biosurfactants produced by B. methylotrophicus DCS1 could be an alternative to chemically synthesized surfactants for use in bioremediation processes to enhance the solubility of hydrophobic compounds.     

Production of water-soluble surface-active exolipids by Torulopsis apicola

Summary Several Torulopsis yeasts were screened for production of extracellular surface-active compounds. One strain, Torulopsis apicola IMET 43747, was studied in greater detail. Both on nalkanes and on carbohydrates it produced a mixture of water-soluble biosurfactants with remarkable interfacial activities and surface-tension values around 30 mN m^-1 and interfacial tension below 1 mN m^-1. Most of the biosurfactants are produced in the late exponential and in the early stationary growth phase. Production was increased by using hydrophobic compounds as the carbon source. The yields on n-alkanes were influenced by the concentrations of both the carbon source and the yeast extract. The effects of one purified biosurfactant on microbial growth on nalkanes and its antibacterial and antiphagal activities reveal new physiological aspects of biosurfactant generation by T. apicola.