Surface-active compounds and their role in the access to hydrocarbons in Gordonia strains

Three new bacterial strains (M22, BS25 and BS29) belonging to the Gordonia genus were isolated from a site chronically contaminated by diesel. Those Gordonia strains were able to grow using a wide range of straight and branched aliphatic hydrocarbons as carbon and energy sources and to produce at least two classes of surface-active compounds. Emulsifying agents were released in the culture medium when bacteria grew both on hydrocarbons and water-soluble substrates. Cell-bound biosurfactants, which reduce the surface tension, were produced on hydrocarbons; however, their production was significantly lower on water soluble substrates. The relationship of growth phase, surface-active compound production and cell-surface properties was analyzed in kinetic experiments on hydrocarbons. Gordonia sp. BS29 synthesized, and released extracellularly, bioemulsans during the exponential phase with n-hexadecane as carbon and energy source. The production of biosurfactants started in the exponential phase and their concentration increased during the following linear growth. Furthermore, the adhesion of bacterial cells to hydrocarbons decreased during growth. Our results led us to hypothesize a change in the mode by which Gordonia cells access the substrate during growth on hydrocarbons.     

Production and surface-active properties of microbial surfactants

A new source of surface-active compounds produced by microbes was investigated. These biologically derived surfactants (biosurfactants) were found to be extracellular products of two newly isolated Corynebacterium species, which were gorwn on kerosene (Imperial Oil No.9). Batch-growth kinetic studies were carried out in a 14 liter fermentor and the production of biosurfactants was found to be cell growth associated. The surface tension of the whole microbial broth was reduced to a minimum of about 30 dyn/cm, as compared with the surface tension for distilled water of 72 dyn/cm. The crude biosurfactants were recovered from the cell-free broth, freeze-dried, redissolved in water, and their surface-active properties were studied. The biosurfactants reported here were found to be nontoxic and their ability to lower the surface tension of water was found to be comparable to that of sodium dodecylbenzene sulfonate, common commercial synthetic surfactant.     

Production of rhamnolipids by a Pseudomonas alcaligenes strain

Brazil is one of the main producers of palm oil (Ellaus guineeusis). It is a low-cost product that has some interesting industrial qualities, such as its use as the raw material for the production of glycerin and soap as well as its use in the preparation of food. Some renewable sources and agroindustrial wastes have been used extensively in research on the production of biosurfactants of the Pseudomonas strains. However, to our knowledge, no studies have been published on the use of palm oil as a substrate for the synthesis of biosurfactants by Pseudomonas alcaligenes. This paper describes the production and characterization of biosurfactants synthesized by a strain of P. alcaligenes PCL previously isolated from soil that was contaminated with crude-oil. Furthermore, the paper presents the optimization of the production of biological surface-active compounds by applying experimental design tools and their capacity to emulsify hydrocarbons.     

Rhamnolipids as biosurfactants from renewable resources: Concepts for next-generation rhamnolipid production

Several microorganisms are known to produce a wide variety of surface-active substances, which are referred to as biosurfactants. Interesting examples for biosurfactants are rhamnolipids, glycolipids mainly known from Pseudomonas aeruginosa produced during cultivation on different substrates like vegetable oils, sugars, glycerol or hydrocarbons. However, besides costs for downstream processing of rhamnolipids, relatively high raw-material prices and low productivities currently inhibit potential economical production of rhamnolipids on an industrial scale. This review focuses on cost-effective and sustainable production of rhamnolipids by introducing new possibilities and strategies regarding renewable substrates. Additionally, past and recent production strategies using alternative substrates such as agro-industrial byproducts or wastes are summarized. Requirements and concepts for next-generation rhamnolipid producing strains are discussed and potential targets for strain-engineering are presented. The discussion of potential new strategies is supported by an analysis of the metabolism of different Pseudomonas species. According to calculations of theoretical substrate-to-product conversion yields and current world-market price analysis, different renewable substrates are compared and discussed from an economical point of view. A next-generation rhamnolipid producing strain, as proposed within this review, may be engineered towards reduced formation of byproducts, increased metabolic spectrum, broadened substrate spectrum and controlled regulation for the induction of rhamnolipid synthesis.     

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.     

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.     

Surface-active lipids in rhodococci

Like other hydrocarbon-oxidising bacteria, rhodococci respond to the presence of alkanes by producing biosurfactant molecules to improve their ability to utilise these hydrophobic compounds as growth substrates. In the rhodococci these surfactants are predominantly glycolipids, the majority of which remain cell-bound during unrestricted growth. Most work has been done on the trehalose mycolates formed by Rhodococcus erythropolis, but nitrogen- limited conditions lead to the production of anionic trehalose tetraesters also.As surfactants, these compounds, whether purified or in crude form, are able to reduce the surface tension of water from 72 mN m-1 to a low of 26, thus making them among the most potent biosurfactants known. They are also able to reduce the interfacial tension between water and a hydrophobic phase (e.g. n- hexadecane) from 43 mN m-1 to values less than one (Table 1). Biosurfactants have about a ten- to 40-fold lower critical micelle concentration than synthetic surfactants. Such properties suggest a range of industrial applications, where a variety of surface-active characteristics are appropriate. Interest in biosurfactants as industrial chemicals results from the toxicity of many petrochemical-derived surfactants. Currently world-wide surfactant production is on a very large scale, and the demand for them is increasing. However, the drive towards less environmentally damaging chemicals makes biosurfactants attractive as they have lower toxicity.The reason they have not achieved a significant market share is the cost of production, which is considerably higher than for synthetic surfactants. This problem is being addressed using several strategies. An approach where there is great scope for improvement with the rhodococci is an understanding of the genetic basis of glycolipid production, which is largely unknown. They may find applications in the near future in the environmental remediation industries, where the requirement for purified molecules is of less importance.This review summarises knowledge of the chemistry, biochemistry and production of Rhodococcus surface-active lipids. Where they have been used, or there is potential for use, in industrial applications is discussed.     

厌氧产表面活性剂微生物提高原油采收率的研究进展

微生物强化采油(microbial enhanced oil recovery,MEOR)是近年来在国内外发展迅速的一项提高原油采收率技术。微生物在油藏中高效生产表面活性剂等驱油物质是微生物采油技术成功实施的关键之一。然而,油藏的缺/厌氧环境严重影响好氧表面活性剂产生菌在油藏原位的生存与代谢活性;油藏注空气会增加开采成本,且注入空气的作用时效和范围难以确定。因此,开发厌氧产表面活性剂菌种资源并强化其驱油效率对于提高原油采收率具有重要意义。本文综述了国内外近年来利用厌氧产表面活性剂微生物提高原油采收率的研究进展,简述了微生物厌氧产表面活性剂的相关驱油机理、菌种资源开发现状以及油藏原位驱油应用进展,并对当前的研究提出了一些思考。     

Biosurfactants: moving towards industrial application.

Chemically synthesized surface-active compounds are widely used in the pharmaceutical, cosmetic, petroleum and food industries. However, with the advantages of biodegradability, and production on renewable-resource substrates, biosurfactants may eventually replace their chemically synthesized counterparts. So far, the use of biosurfactants has been limited to a few specialized applications because biosurfactants have been economically uncompetitive. There is a need to gain a greater understanding of the physiology, genetics and biochemistry of biosurfactant-producing strains, and to improve process technology to reduce production costs.     

Potential commercial applications of microbial surfactants

Surfactants are surface-active compounds capable of reducing surface and interfacial tension at the interfaces between liquids, solids and gases, thereby allowing them to mix or disperse readily as emulsions in water or other liquids. The enormous market demand for surfactants is currently met by numerous synthetic, mainly petroleum-based, chemical surfactants. These compounds are usually toxic to the environment and non-biodegradable. They may bio-accumulate and their production, processes and by-products can be environmentally hazardous. Tightening environmental regulations and increasing awareness for the need to protect the ecosystem have effectively resulted in an increasing interest in biosurfactants as possible alternatives to chemical surfactants. Biosurfactants are amphiphilic compounds of microbial origin with considerable potential in commercial applications within various industries. They have advantages over their chemical counterparts in biodegradability and effectiveness at extreme temperature or pH and in having lower toxicity. Biosurfactants are beginning to acquire a status as potential performance-effective molecules in various fields. At present biosurfactants are mainly used in studies on enhanced oil recovery and hydrocarbon bioremediation. The solubilization and emulsification of toxic chemicals by biosurfactants have also been reported. Biosurfactants also have potential applications in agriculture, cosmetics, pharmaceuticals, detergents, personal care products, food processing, textile manufacturing, laundry supplies, metal treatment and processing, pulp and paper processing and paint industries. Their uses and potential commercial applications in these fields are reviewed. Received: 29 July 1999 / Received revision: 8 November 1999 / Accepted: 9 November 1999     

Designer rhamnolipids by reduction of congener diversity: production and characterization

Background

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.

Results

Here, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.

Conclusions

The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.

     

Biological activity of glycolipids produced by microorganisms: New trends and possible therapeutic alternatives

Several biological processes in prokaryotic and eukaryotic organisms require the presence of glycolipids (biosurfactants), compounds with both hydrophilic and hydrophobic groups in their structure. They constitute the backbone of different metabolic functions and biological structures such as cell membranes. Besides being structural components, glycolipids show surface activity in the interfaces and are mainly produced by microorganisms. Interest in biosurfactants has increased considerably in recent times due to their applications in the environmental, oil, food, and pharmaceutical industries, since they have unique properties such as low toxicity, high biodegradability, environmentally friendly, foaming capacity, high selectivity and specificity at extreme temperatures, pH and salinity, as well as biological activity. All of these properties are considered advantages over other chemical surfactants, and therefore glycolipids are considered a good alternative, given the current interest on sustainable development. The present work shows a general view of bio-surfactants of microbial origin, particularly of glycolipids, referring to several studies on their biological activity that have revealed their great potential in the medical–biological field, discovering interesting possibilities for their therapeutic application in the near future.     

A molecular mechanism that stabilizes cooperative secretions in Pseudomonas aeruginosa

Bacterial populations frequently act as a collective by secreting a wide range of compounds necessary for cell-cell communication, host colonization and virulence. How such behaviours avoid exploitation by spontaneous 'cheater' mutants that use but do not contribute to secretions remains unclear. We investigate this question using Pseudomonas aeruginosa swarming, a collective surface motility requiring massive secretions of rhamnolipid biosurfactants. We first show that swarming is immune to the evolution of rhlA(-) 'cheaters'. We then demonstrate that P. aeruginosa resists cheating through metabolic prudence: wild-type cells secrete biosurfactants only when the cost of their production and impact on individual fitness is low, therefore preventing non-secreting strains from gaining an evolutionary advantage. Metabolic prudence works because the carbon-rich biosurfactants are only produced when growth is limited by another growth limiting nutrient, the nitrogen source. By genetically manipulating a strain to produce the biosurfactants constitutively we show that swarming becomes cheatable: a non-producing strain rapidly outcompetes and replaces this obligate cooperator. We argue that metabolic prudence, which may first evolve as a direct response to cheating or simply to optimize growth, can explain the maintenance of massive secretions in many bacteria. More generally, prudent regulation is a mechanism to stabilize cooperation.     

Bio-surfactants

Interest in microbially produced biosurfactants has increased recently, due mainly to their potential as agents in enhanced oil recovery. A variety of microbes and their products have been assessed for their surface-active properties, and it has been suggested that biosurfactants may prove useful in a broad spectrum of potential applications which presently utilise synthetic surfactants. The most commonly produced biosurfactants tend to be glycolipids, usually a mono- or di-saccharide attached to a fatty acid, but more complex molecules such as lipopeptides, lipoproteins, and lipo-heteropoly-saccharides have been isolated and studied. Biosurfactant production by microbes is often but not invariably enhanced by the addition of hydrocarbon to the growth medium, and needs to be optimised by controlling such factors as carbon source, nitrogen source and concentrations, aeration and metal ions. Biosurfactants have been shown to be as effective, if not more so, than many conventional synthetic surfactants and their future utilisation may depend utilimately upon the prevailing economics for their production.     

Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.     

Microbial biosurfactants production, applications and future potential

Microorganisms synthesise a wide range of surface-active compounds (SAC), generally called biosurfactants. These compounds are mainly classified according to their molecular weight, physico-chemical properties and mode of action. The low-molecular-weight SACs or biosurfactants reduce the surface tension at the air/water interfaces and the interfacial tension at oil/water interfaces, whereas the high-molecular-weight SACs, also called bioemulsifiers, are more effective in stabilising oil-in-water emulsions. Biosurfactants are attracting much interest due to their potential advantages over their synthetic counterparts in many fields spanning environmental, food, biomedical, and other industrial applications. Their large-scale application and production, however, are currently limited by the high cost of production and by limited understanding of their interactions with cells and with the abiotic environment. In this paper, we review the current knowledge and the latest advances in biosurfactant applications and the biotechnological strategies being developed for improving production processes and future potential.     

Alkanotrophic Rhodococcus ruber as a biosurfactant producer

In this report we examined the structure and properties of surface-active lipids of Rhodococcus ruber. Most historical interest has been in the glycolipids of Rhodococcus erythropolis, which have been extensively characterised. R. erythropolis has been of interest due to its great metabolic diversity. Only recently has the metabolic potential of R. ruber begun to be explored. One major difference in the two species is that most R. ruber strains are able to oxidise the gaseous alkanes propane and butane. In preparation for investigation of the effects of gas metabolism on biosurfactant production, we set out to characterise the biosurfactants produced during growth on liquid n-alkanes and to compare these with R. erythropolis glycolipids.     

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%.