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.     

Alternatives for biosurfactants and bacteriocins extraction from Lactococcus lactis cultures produced under different pH conditions

Aims: Study of the potential of Lactococcus lactis CECT‐4434 as a biosurfactants and nisin (the only bacteriocin allowed to be used in the food industry) producer for industrial applications, exploiting the possibility of recovering separately both metabolites, taking into account that L. lactis is an interesting micro‐organism with several applications in the food industry because it is recognized as GRAS. Methods and Results: The results showed the ability of this strain to produce cell‐bound biosurfactants, under controlled pH, and cell‐bound biosurfactants and bacteriocins, when pH was not controlled. Three extraction procedures were designed to separately recover these substances. Conclusions: The strain L. lactis CECT‐4434 showed to be a cell‐bound biosurfactants and bacterocins producer when fermentations were carried out under uncontrolled pH. Both products can be recovered separately. Significance and Impact of the Study: Development of a convenient tool for the extraction of cell‐bound biosurfactants and bacteriocins from the fermentation broth.     

Potential application of cyclic lipopeptide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations

AIMS: Crude cyclic lipopeptide (CLP) biosurfactants from two Bacillus subtilis strains (DM-03 and DM-04) were studied for their compatibility and stability with some locally available commercial laundry detergents. METHODS AND RESULTS: CLP biosurfactants from both B. subtilis strains were stable over the pH range of 7.0-12.0, and heating them at 80 degrees C for 60 min did not result in any loss of their surface-active property. Crude CLP biosurfactants showed good emulsion formation capability with vegetable oils, and demonstrated excellent compatibility and stability with all the tested laundry detergents. CONCLUSION: CLP biosurfactants from B. subtilis strains act additively with other components of the detergents to further improve the wash quality of detergents. The thermal resistance and extreme alkaline pH stability of B. subtilis CLP biosurfactants favour their inclusion in laundry detergent formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has great significance because it is already known that microbial biosurfactants are considered safer alternative to chemical or synthetic surfactants owing to lower toxicity, ease of biodegradability and low ecological impact. The present study provides further evidence that CLP biosurfactants from B. subtilis strains can be employed in laundry detergents.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

Production of Glycolipid Biosurfactants,Mannosylerythritol Lipids,by a Smut Fungus,Ustilago scitaminea NBRC 32730

A smut fungus Ustilago scitaminea NBRC 32730 on sugar cane (Saccharum) was found to accumulate a large amount of glycolipids in the culture medium. As a result of structural characterization, the main glycolipid was identified as MEL-B, 4-O-β-(2′,3′-di-O-alka(e)noyl-6′-O-acetyl-D-mannopyranosyl)-erythritol. The MEL-B was sufficiently produced from a variety of sugars such as sucrose, glucose, fructose, and mannose. Olive oil and methyl oleate were also available as carbon sources to produce MEL-B. However, these residual oils made product recovery very complicated. Under optimal conditions, a maximum MEL yield of 12.8 g/l was achieved by feeding of sucrose.     

生物表面活性剂修复重金属污染研究进展

重金属在环境中积累会对动植物和人体健康造成危害。生物表面活性剂环境相容性好,在环境污染修复方面的应用日益受到关注。本文介绍了生物表面活性剂及其在重金属污染修复中的应用;生物表面活性剂与重金属络合的机理;影响二者络合的因素(如pH值、表面活性剂浓度、重金属存在形态等);对生物表面活性剂修复重金属污染的前景进行了展望。     

生物表面活性剂生产及应用研究进展

生物表面活性剂主要是由微生物代谢产生的,具有疏水基团和亲水基团的两亲性物质,它们能显著降低表面与界面张力。与化学表面活性剂相比,生物表面活性剂具有毒性低、生物兼容性好、可降解等优点,在众多领域具有良好的应用前景,但生物表面活性剂的高生产成本限制了商业化发展。本文旨在分析微生物表面活性剂的生产,重点是生产过程和代谢途径的优化,以探索产量与成本的关键因素,为生物表面活性剂商业化发展提供解决方案。     

Production of Glycolipid Biosurfactants,Mannosylerythritol Lipids,Using Sucrose by Fungal and Yeast Strains,and Their Interfacial Properties

Glycolipid biosurfactants, mannosylerythritol lipids (MELs), were produced from glucose and sucrose without vegetable oils. Pseudozyma antarctica JCM 10317, Ustilago maydis NBRC 5346, U. scitaminea NBRC 32730, and P. siamensis CBS 9960 produced mainly MEL-A, MEL-A, MEL-B, and MEL-C respectively. The sucrose-derived MELs showed excellent interfacial properties: low critical micelle concentration as well as that of oil-derived MELs.     

BioLumix微生物荧光光电检测系统在化妆品中的应用评估

本研究将用于化妆品中Bio Lumix微生物荧光光电检测系统与《化妆品安全技术规范2015》微生物检测法进行了比较。结果显示两种方法结果基本一致,对于《化妆品安全技术规范2015》微生物检测法检测化妆品,出具可靠的检测报告需要5 d,而Bio Lumix系统快速检测化妆品细菌项目和霉菌、酵母项目可在16 h和35 h内分别发出预警,并可控制在48 h内完整地输出检测报告。Bio Lumix微生物荧光光电检测系统具有检测时间短、检测效率高、操作简单等优点。     

Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum

Biosurfactants are microbial secondary metabolites. The most studied are rhamnolipids, which decrease the surface tension and have emulsifying capacity. In this study, the production of biosurfactants, with emphasis on rhamnolipids, and diesel oil degradation by 18 strains of bacteria isolated from waste landfill soil contaminated by petroleum was analyzed. Among the studied bacteria, gram‐positive endospore forming rods (39%), gram positive rods without endospores (17%), and gram‐negative rods (44%) were found. The following methods were used to test for biosurfactant production: oil spreading, emulsification, and hemolytic activity. All strains showed the ability to disperse the diesel oil, while 77% and 44% of the strains showed hemolysis and emulsification of diesel oil, respectively. Rhamnolipids production was observed in four strains that were classified on the basis of the 16S rRNA sequences as Pseudomonas aeruginosa. Only those strains showed the rhlAB gene involved in rhamnolipids synthesis, and antibacterial activity against Escherichia coli, P. aeruginosa, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora, and Ralstonia solanacearum. The highest production of rhamnolipids was 565.7 mg/L observed in mineral medium containing olive oil (pH 8). With regard to the capacity to degrade diesel oil, it was observed that 7 strains were positive in reduction of the dye 2,6‐dichlorophenolindophenol (2,6‐DCPIP) while 16 had the gene alkane mono‐oxygenase (alkB), and the producers of rhamnolipids were positive in both tests. Several bacterial strains have shown high potential to be explored further for bioremediation purposes due to their simultaneous ability to emulsify, disperse, and degrade diesel oil. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:262–270, 2016     

生物表面活性剂产生菌的筛选及表面活性剂稳定性研究

大庆油田油泥样品经富集培养,平板分离,获得52株菌。排油性实验和表面张力测定表明,菌株B22、B24、B2s产生的表面活性剂表面活性稳定,表面张力较低。温度、pH和NaCl浓度实验证实,细菌B22,产生的生物表面活性剂可耐受120℃高温,另2种生物表面活性剂可耐受80℃;3种细菌生物表面活性剂对pH有广泛适应性,1322pH适应范围为4．0～13．0,B24、B25的pH适应范围为2．0～13．0;NaCl浓度对表面活性剂的生物活性影响不大。将3株菌的生物表面活性剂用于室内油泥处理实验,72h石油去除率达70％以上。     

Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms

Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. A search for optimal growth conditions for soil bacteria is of a great importance in order to isolate various xenobiotic degraders. Bacteria consortium A43 was isolated from soils contaminated with explosives. In the presence of carbohydrate and plant extract, an addition of TNT to the solidified minimal medium stimulated the growth of the tested bacteria, as compared to other bacteria consortium isolated from the same soils. Reducing sugars as carbohydrates, and cabbage leaf extract as a plant extract were used in these experiments. Cultivation of the A43 in liquid medium of the same content showed that addition of cabbage leaf extract alone to medium is much more efficient for TNT degradation by growing biomass as compared to addition of carbohydrate alone.     

好氧氨氧化微生物系统发育及生理生态学差异

作为好氧氨氧化的驱动者,氨氧化古菌(Ammonia-oxidizing archaea,AOA)和细菌(Ammonia-oxidizing bacteria,AOB)一直是氮的生物地球化学循环的研究热点之一。由于它们的相对丰度、群落结构和活性因环境而异,目前二者对全球氮循环的相对贡献仍存在争议。对培养物和环境样品的动力学、基因组学等研究结果表明,这种差异主要是由AOA和AOB的生理生态学差异导致的。氨浓度、pH、溶氧、温度等环境因素以及代谢途径等生理因素导致AOA和AOB的生态位分化。通过比较AOA和AOB在系统发育、对环境因子的响应以及代谢途径等方面的差异,对好氧氨氧化微生物相关研究成果进行概括和总结,以便深入了解它们在不同环境中对氮循环的相对贡献;同时对好氧氨氧化微生物今后的研究重点进行了展望。     

Biodegradation of xanthan by salt-tolerant aerobic microorganisms

Summary Three salt-tolerant bacteria which degraded xanthan were isolated from various water and soil samples collected from New Jersey, Illinois, and Louisiana. The mixed culture, HD1, contained aBacillus sp. which produced an inducible enzyme(s) having the highest extracellular xanthan-degrading activity found. Xanthan alone induced the observed xanthan-degrading activity. The optimum pH and temperature for cell growth were 5–7 and 30–35°C, respectively. The optimum temperature for activity of the xanthan-degrading enzyme(s) was 35–45°C, slightly higher than the optimum growth temperature. With a cell-free enzyme preparation, the optimum pH for the reduction of solution viscosity and for the release of reducing sugar groups were different (5 and 6, respectively), suggesting the involvement of more than one enzyme for these two reactions. Products of enzymatic xanthan degradation were identified as glucose, glucuronic acid, mannose, pyruvated mannose, acetylated mannose and unidentified oligo- and polysaccharides. The weight average molecular weight of xanthan samples shifted from 6.5·10⁶ down to 6.0·10⁴ during 18 h of incubation with the cell-free crude enzymes. The activity of the xanthan-degrading enzyme(s) was not influenced by the presence or absence of air or by the presence of Na₂S₂O₄ and low levels of biocides such as formaldehyde (25 ppm) and 2,2-dibromo-3-nitrilopropionamide (10 ppm). Formaldehyde at 50 ppm effectively inhibited growth of the xanthan degraders.     

Microbial biosurfactants: A broad analysis of properties,applications, biosynthesis,and techno-economical assessment of rhamnolipid production

Biosurfactants are surface-active molecules originated from renewable resources, which are produced by microbial fermentation or chemical/enzymatic catalysis. These molecules present important advantages as compared to petrochemical surfactants, given their resistance to extreme conditions, biodegradability, specificity, and environmental compatibility. Besides that, the high production costs hinder its commercialization. In this way, this article aimed to analyze microbial biosurfactants production, focusing on the optimization of metabolic pathways and production processes, to identify key aspects and provide alternatives to allow a cost-effective production at industrial scale. This was achieved by a broad analysis of biosurfactants properties, applications, and biosynthetic pathways (in terms of yield, cofactors, and energy), in addition to an assessment of production-associated costs. As a result of the present extensive data survey and analysis, key production aspects are disclosed. The metabolic pathway yield analysis demonstrated that production of biosurfactants can be significantly improved (highest theoretical yield was 0.47 g_{biosurfactant}/g_substrate) by the use of biomolecular engineering techniques to generate optimized synthetic pathways. With an alternative proposed pathway for surfactin, yield was improved and imbalance in cofactors and ATP was reduced. Analysis of productive costs indicated that to make rhamnolipids commercial production feasible, the main efforts should focus on lowering substrate costs as well as the identification of energy-efficient unit operations to lower electricity cost, since these parameters accounted for 19.36 and 78.22%, respectively, of the production costs. The data generated by this analysis highlight the need for multidisciplinary collaboration to make rhamnolipids economically feasible, including biomolecular engineering and process intensification.     

球形红细菌污泥颗粒化及降解氯苯

投加絮凝剂是促使微生物快速形成污泥颗粒的一种有效手段,通过研究在不同絮凝剂下生成的生物絮体的形态和沉降性能,推荐选用聚合氯化铝(PAC)作为促进光合细菌球形红细菌形成污泥颗粒的絮凝剂。PAC的最佳投加量范围为140-160mg/L,其中,PAC投加量150mg/L时,促进污泥颗粒化的效果最好。考察球形红细菌污泥颗粒降解氯苯的环境条件,结果表明球形红细菌污泥颗粒降解氯苯的最佳条件为好氧、pH7.0、30°C。     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

The algicidal activity of the rhamnolipid biosurfactants (the mixture of Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀) produced by Pseudomonas aeruginosa was investigated in the present paper. The results indicated that the biosurfactants had potential algicidal effects on the harmful algal bloom (HAB) species, Heterosigma akashiwo. The growth of H. akashiwo was strongly inhibited in medium containing rhamnolipids (0.4–3.0 mg L⁻¹); moreover, the rhamnolipids showed strong lytic activity toward H. akashiwo at higher concentrations (≥4.0 mg L⁻¹). In addition, the effects of the rhamnolipids on the growth of Gymnodinium sp. and Prorocentrum dentatum, another two kinds of HAB species, were also studied. Compared with the dramatic algicidal effect on H. akashiwo, the cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg L⁻¹), while the cells of Gymnodinium sp. were not suppressed with the same treatment, indicating the rhamnolipids had the potential for the selective control of HABs.Morphometric analysis at ultrastructural level by transmission electron micrographs indicated that the extent of ultrastructural damage of the alga was severe at high concentrations of rhamnolipids and during extended periods of contact. The first response occurred in the plasma membrane which partly disintegrated. The lack of membrane facilitated the rhamnolipid biosurfactants into the cells and allowed damage to other organelles, which resulted in the injury of chloroplast, vacuolization of mitochondria and deformation of the cristae, disruption of nuclear membrane and condensation of chromatin in nucleus, suggesting that the lytic activity of rhamnolipids was mainly due to their powerful surfactivity and their tendency to cohere on the surface of phospholipids bimolecular layer of the cells and further destroyed the layers, and then the structure of quasi-membrane configurations inside the cells was disintegrated, following by the irreversible damage to the ultrastructure and the loss of the function of organelles, consequently leading the cells to lyse.     

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na⁺, 2Na⁺ and K⁺), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.     

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function-based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic-based screens.