Many microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates. Biosurfactants are more effective, selective, environmentally friendly, and stable than many synthetic surfactants. Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain aliphatic acid, while others, like lipopeptides, lipoproteins, and heteropolysaccharides, are more complex. Rapid and reliable methods for screening and selection of biosurfactant-producing microorganisms and evaluation of their activity have been developed. Genes involved in rhamnolipid synthesis (rhlAB) and regulation (rhlI and rhlR) in Pseudomonas aeruginosa are characterized, and expression of rhlAB in heterologous hosts is discussed. Genes for surfactin production (sfp, srfA, and comA) in Bacillus spp. are also characterized. Fermentative production of biosurfactants depends primarily on the microbial strain, source of carbon and nitrogen, pH, temperature, and concentration of oxygen and metal ions. Addition of water-immiscible substrates to media and nitrogen and iron limitations in the media result in an overproduction of some biosurfactants. Other important advances are the use of water-soluble substrates and agroindustrial wastes for production, development of continuous recovery processes, and production through biotransformation. Commercialization of biosurfactants in the cosmetic, food, health care, pulp- and paper-processing, coal, ceramic, and metal industries has been proposed. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants. Perspectives for future research and applications are also discussed.

Microbial production of surfactants and their commercial potential.

Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S. cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K. marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)

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Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation

Foreign gene expression in yeast: a review.

Two quorum-sensing systems (las and rhl) regulate virulence gene expression in Pseudomonas aeruginosa. The las system consists of a transcriptional activator, LasR, and LasI, which directs the synthesis of the autoinducer N-(3-oxododecanoyl) homoserine lactone (PAI-1). Induction of lasB (encoding elastase) and other virulence genes requires LasR and PAI-1. The rhl system consists of a putative transcriptional activator, RhlR, and RhlI, which directs the synthesis of N-butyryl homoserine lactone (PAI-2). Rhamnolipid production in P. aeruginosa has been reported to require both the rhl system and rhlAB (encoding a rhamnosyltransferase). Here we report the generation of a delta lasI mutant and both delta lasI delta rhlI and delta lasR rhlR::Tn501 double mutants of strain PAO1. Rhamnolipid production and elastolysis were reduced in the delta lasI single mutant and abolished in the double-mutant strains. rhlAB mRNA was not detected in these strains at mid-logarithmic phase but was abundant in the parental strain. Further RNA analysis of the wild-type strain revealed that rhlAB is organized as an operon. The rhlAB transcriptional start was mapped, and putative sigma 54 and sigma 70 promoters were identified upstream. To define components required for rhlAB expression, we developed a bioassay in Escherichia coli and demonstrated that PAI-2 and RhlR are required and sufficient for expression of rhlA. To characterize the putative interaction between PAI-2 and RhlR, we demonstrated that [3H]PAI-2 binds to E. coli cells expressing RhlR and not to those expressing LasR. Finally, the specificity of the las and rhl systems was examined in E. coli bioassays. The las system was capable of mildly activating rhlA, and similarly, the rhl system partly activated lasB. However; these effects were much less than the activation of rhlA by the rhl system and lasB by the las system. The results presented here further characterize the roles of the rhl and las quorum-sensing systems in virulence gene expression.

Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes.

The use and potential commercial application of biosurfactants in the medical field has increased during the past decade. Their antibacterial, antifungal and antiviral activities make them relevant molecules for applications in combating many diseases and as therapeutic agents. In addition, their role as anti-adhesive agents against several pathogens indicates their utility as suitable anti-adhesive coating agents for medical insertional materials leading to a reduction in a large number of hospital infections without the use of synthetic drugs and chemicals. This review looks at medicinal and therapeutic perspectives on biosurfactant applications.

https://repositorium.sdum.uminho.pt/bitstream/1822/5101/1/rodrigues_JAC_2006%5b1%5d.pdf

Biosurfactants: potential applications in medicine

Regulation of glucose metabolism in growing yeast cells

We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up [14C]hexadecane. The addition of small amounts of rhamnolipids restored growth on alkanes and [14C]hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P. aeruginosa.

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

SUMMARY: The respiratory capacity of Saccharomyces cerevisiae growing in continuous culture on glucose and on mixtures of glucose and ethanol was investigated. An oxygen uptake rate of 8 mmo1 g-1 h-1 was found to limit the ability of the organism to degrade a substrate purely oxidatively. On glucose as sole energy and carbon source, this respiration rate was invariably achieved at an identical growth rate and thus at an identical substrate uptake rate when the inlet glucose concentration was varied. The rate of ethanol co-consumption together with glucose was strictly governed by this limiting maximum respirator)' capacity and no repression of respiration was observed at dilution rates where ethanol was excreted by the cells. Hence, a limitation in some step in the oxidative branch of catabolism is likely to be responsible for incomplete oxidation of glucose at high growth rates rather than an undefined action of glucose repression.

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The Role Of Limited Respiration In The Incomplete Oxidation Of Glucose By Saccharomyces Cerevisiae

SUMMARY: When Saccharomyces uvarum was cultivated in continuous culture, it exhibited the typical growth behaviour of a glucose-sensitive yeast. Metabolic changes related to glucose-repressed growth were assessed by an analysis of overall culture parameters (biomass formation, ethanol and acetate production and gas exchange rates) and by measuring the mitochondrial cytochrome content. These functions were mainly affected by the glucose effect; the steady state values of these variables were first established in the chemostat as a function of dilution rate.
The short- and long-term regulation taking place when the cells were submitted to repression was assessed by administering glucose pulses and by shifts in the dilution rate. The primary response of the cells to the initiation of repressed growth was the formation of ethanol and acetate. Since there was no repression of oxygen uptake rate or cytochrome content prior to this response, it was concluded that ethanol and acetate formation was not the consequence of repression of respiratory activity, but resulted from the regulation of pyruvate dehydrogenase and pyruvate decarboxylase activities. Long-term adaptation of the cells occurred within 24 to 48 h of the initiation of repressed growth as manifested by a decrease of mitochondrial cytochrome content to the steady state value corresponding to that of repressed growth.

An Expanded Concept for the Glucose Effect in the Yeast Saccharomyces uvarum: Involvement of Short- and Long-term Regulation

The surface-active compounds of the strain Bacillus licheniformis were isolated and their structure was elucidated. The high surface-active capacity of the crude extract was basically due to traces of long-chain saturated fatty acids, especially of palmitic and stearic acids, to a mixture of small amounts of hydrocarbons with chain lengths of 20 and 22 carbons, and mainly to appreciable amounts of four slightly different lipopeptides. The lipopeptides were found to be a mixture of four closely related compounds. The lipophilic part consisting of i-, n-C14 or i-, ai-C15 β-OH fatty acids was linked to the hydrophilic peptide moiety, which contained seven amino acids (Glu, Asp, Val, three Leu and Ile) by a lactone linkage. Fifteen milligrams per litre of the purified lipopeptide product decreased the surface tension of water from 72 mN m−1 to 27 mN m−1, characterizing the product as a powerful surface-active agent that compares favourably to other (bio)surfactants. Antibiotic activity was demonstrated against bacteria and yeasts.

Othmar Käppeli

Papers

Regulation of glucose metabolism in growing yeast cells

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

The Role Of Limited Respiration In The Incomplete Oxidation Of Glucose By Saccharomyces Cerevisiae

An Expanded Concept for the Glucose Effect in the Yeast Saccharomyces uvarum: Involvement of Short- and Long-term Regulation

Biosurfactants from Bacillus licheniformis: structural analysis and characterization.