Jim Philp

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.

Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction.

In the present study, we proposed methyl tertiary-butyl ether (MTBE) as a solvent for extraction of biosurfactants from Rhodococcus bacterial cultures. After comparison with other well known solvent systems used for biosurfactant extraction, it was found that MTBE was able to extract crude surfactant material with high product recovery (10 g/l), efficiency (critical micelle concentration (CMC), 130-170 mg/l) and good functional surfactant characteristics (surface and interfacial tensions, 29 and 0.9 mN/m, respectively). The isolated surfactant complex contained 10% polar lipids, mostly glycolipids possessing maximal surface activity. Ultrasonic treatment of the extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. Due to certain characteristics of MTBE, such as relatively low toxicity, biodegradability, ease of downstream recovery, low flammability and explosion safety, the use of this solvent as an extraction agent in industrial scale biosurfactant production is feasible.

Alkanotrophic Rhodococcus ruber as a biosurfactant producer

Abstract. 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.

Oil desorption from mineral and organic materials using biosurfactant complexes produced by Rhodococcus species

Rhodococcus strains from the culture collection at the Institute of Ecology and Genetics of Microorganisms, Perm, Russia were examined for biosurfactant production during growth on n-alkanes and the ability to remove oil associated with contaminated sands and oil shale cuttings. Members of the genus, particularly R. ruber, were shown to produce low toxicity surfactants effective in removing oil from surfaces. The extent of desorption was inversely related to the concentration of high molecular weight hydrocarbons, namely asphaltenes and resins. In addition, crude surfactant complexes enhanced the degradation of crude oil, in the short term, when added to contaminated agricultural soil during bioremediation studies utilizing biopiling technology.

Whole cell immobilised biosensors for toxicity assessment of a wastewater treatment plant treating phenolics-containing waste.

Wastewater treatment plants dealing with industrial wastes are often susceptible to overload of toxic influent that can partially or completely destroy treatment for extended periods. An obvious candidate for monitoring toxicity in such wastewater systems is bioluminescent bacteria. However, the natural bioluminescent bacteria can be particularly sensitive to some industrial wastes and therefore their response to normal operational conditions does not reflect the status of the microbial community responsible for treatment. Moreover, the salt dependence of the marine bioluminescent bacteria, and the temperature sensitivity of some strains, further complicate their use. Here we describe the construction of whole cell genetically modified bioluminescent biosensors and their immobilisation for use in monitoring the toxicity of a complex industrial wastewater containing phenolic materials. A hand-held luminometer was designed for laboratory or field use, and the immobilisation system designed with several things in mind: the geometry of the instrument; the need for containment of GM bacteria; the maximisation of the bioavailability of the wastewater to the biosensor. The performance of a candidate GM sensor was compared with Vibrio fischeri in liquid culture and after immobilisation in thin films of poly(vinyl alcohol) (PVA) cryogels. The biosensors were tested against pure phenol and 3-chlorophenol as a reference toxic chemical known to be much more toxic to bacteria than phenol. The biosensors were then tested with the phenolics-containing industrial wastewater. The immobilisation system proved to operate predictably with pure toxicants, and was able to discriminate toxicity of various zones within the wastewater treatment plant.

Ecological and physiological analyses of Pseudomonad species within a phenol remediation system

A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy.

Identification of Rhodococcus equi using the polymerase chain reaction

K.S. BELL, J.C. PHILP, N. CHRISTOFI AND D.W.J. AW. 1996. Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species‐specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.

Identification and environmental detection of Rhodococcus species by 16S rDNA‐targeted PCR

Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. There is a need for improved tests to identify Rhodococcus species. PCR‐based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species‐specific sequences in the 16S rRNA gene, were successfully developed for R. globerulus, R. erythropolis, R. opacus and R. ruber. These tests gave positive results with all or most strains of target species but did not generally cross‐react with other species. Cases of apparent cross‐reaction were shown to be due to prior misclassification of strains of R. opacus as R. erythropolis and of strains of R. ruber as R. rhodochrous. A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R. erythropolis in an environmental sample. Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze‐thaw cycles. Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL‐6B in a spun‐column format. Extracts prepared using a tris‐EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds. A detection limit of 104 cfu g−1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g−1.

Development of bespoke bioluminescent reporters with the potential for in situ deployment within a phenolic-remediating wastewater treatment system

A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system. Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens. These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant. The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l(-1) of phenol. When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri. Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l(-1)) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%). This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment.

Microbial Resources for Global Sustainability

From 2015 onward, sustainability moved high up on the political agenda through several landmark events. The essence of future sustainability is to decrease (and eventually remove) our dependence on fossil resources for fuels, electricity, and materials. The only renewable source of carbon available in huge quantities is biomass. However, building a future production regime based on biomass as a feedstock is fraught with many challenges. The use of biomass in industrial production will clash with the food needs of a population still increasing in the face of drought, soil destruction, and climate change more generally. Here the immense metabolic and genetic diversity of microorganisms can be harnessed as clear routes to fuels and chemicals can already be seen in research. The bigger challenge however is in chemical production at large scale, which has been elusive for most bio-based chemicals, with the most notable exception being ethanol. Many of the issues are as old as microbiology itself—microorganisms were not intended for industrial production. Modern techniques of biotechnology are providing some of the answers. This chapter deals specifically with industrial production of biofuels and bio-based chemicals from microorganisms, and does not deal with the on-going large worldwide efforts in adapting microorganisms to new food challenges. It is this bioeconomy, increasingly utilizing microorganisms, crops, and waste materials that offers the most hope to break out of the myriad of problems created by the over-exploitation of fossil resources.