GB2172898A - Surfactants and their use - Google Patents

Abstract

A product of a microbiological process, which has the characteristic capability of reducing interfacial tension to 10<-2> mN m<-1> or lower against hexadecane. The product may be used in the process of enhanced oil recovery.

Description

SPECIFICATION Surfactants and their use This invention relates to surfactants, and in particular to "biosurfactants" produced by microbiological techniques, and to their use.

The production of surface-active compounds by micro-organisms has been known for well over 50 years.

Although many investigations have been carried out, to characterise biosurfactants and the growth requirements for biosurfactant production, very little quantitative assessment of biosurfactant performance for enhanced oil recovery (EOR) or other applications, e.g. as cleaning and emulsifying agents, has been reported.

For example, Singer petal in a paper presented before the division of Petroleum Chemistry Inc., American Chemical Society, in a symposium at Seattle, 20th to 25th March 1983, disclose that 20 out of 200 bacterial isolates reduced the surface or interfacial tension of spent growth medium. In particular, a mixed culture grown on a hexadecane substrate reduced interfacial tension of spent growth medium to 11.8 mN m'; glycolipids were detected in the medium and a pure culture, identified as the glycolipid producer, was isolated. Maximum glycolipid production was obtained by growing the pure culture on tridecane. The critical micelle concentration (CMC) of a crude glycolipid fraction was 1.5 mg/ml, with a minimum interfacial tension of 0.02 mN m~' against decane, using the drop weight method.The addition of 0.5% pentanol (a co-surfactant), to the glycolipid resulted in a marked decrease in interfacial tension, to a minimum of 6 x 10-5 mN ml against undecane (by comparison with other C616 alkanes).

Chemically-synthesised surfactants are employed by the oil industry for EOR applications. The capillary forces which are responsible for trapping residual oil in the pore structure of reservoir rock, may be overcome by reducing the interfacial tension of the waterflood to 10-3 mN ml or lower. Values of about 10-4 mN m~' are characteristic of the optimised petroleum sulphonate surfactant systems which are widely used in EOR. Petroleum sulphonates are only satisfactory for the intended purpose within a narrow range of sodium chloride concentrations and temperatures, and they are expensive. For best results, blends of surfactants are commonly used.

The present invention is based on the discovery that there are micro-organisms which can be cultivated under suitable conditions to produce biosurfactants which are suitable for use in EOR. The novel biosurfactants are also useful for any purpose in which surface-active characteristics as defined are useful.

A preferred surfactant according to the present invention is the product of a microbiological process and has the characteristic capability of reducing the interfacial tension to 1 of2 mN m1 or less, e.g. 10-3 nM my or less, perhaps to iO4 mN m~' or less, down to 5 x 10d mN m1 or even 10.6 mN m1, or below, against hexadecane or crude oil.

The micro-organisms are aerobic or anaerobic. For use in EOR, aerobes or anaerobes may be cultivated in fermenters, and the desired metabolite purified, above ground, the product then being injected into an oil reservoir in conventional fashion, as a chemically-synthesised surfactant. Surfactant production by an anaerobicprganism would have the advantage of eliminating the cost of aeration during fermentation.

A biosurfactant of the invention may be produced when a suitable isolate, from a chosen source, is grown on a suitable medium. The medium which is used may be a conventional medium, or such a medium after modification, in which substantially only those components essential for growth are present. The most effective media for promoting surfactant synthesis are defined minimal salts media.

The medium should comprise a carbohydrate carbon source, a mineral source and/or trace elements. The carbon source may be an organic compound, e.g. containing up to 6 carbon atoms and atoms other than carbon and hydrogen, e.g. oxygen as, say, hydroxyl or carboxyl groups. Glucose, glycerol and molasses are suitable sources. Preferably all the other components of the medium are inorganic. The most satisfactory carbon source is not necessarily that which is best for growth as such.

A culture may produce more than one surfactant compound. This may be observed as a two-phase reduction in culture surface tension during exponential growth in batch culture. Production of a surfactant in the exponential growth stage suggests that it may be possible to produce it effectively in a continuous process.

A surfactant may be a secondary metabolite, i.e. a compound which is not essential for exponential growth. This may be observed as a decrease in culture surface tension towards the end of the exponential growth phase.

Reservoir brines may contain salts, e.g. NaCI, in amounts of up to 15% w/v. It is therefore desirable that a surfactant should be effective under these conditions. In general, interfacial tensions of novel biosurfactants were lower in the presence of salt than in its absence.

Biosurfactant-producing bacteria can be and have been obtained from a screening programme of isolates originating from natural samples. Samples of soil, oil-contaminated refinery mud, digested anaerobic sewage sludge, reservoir mudlwater, rodent faeces, estuarine mud and oil-contaminated soil have either been added to enrichment cultures or been streaked directly on to agar plates. Pure isolates can be inoculated into liquid culture; the surface and interfacial tensions of these cultures may be measured using a ring tensiometer (Griffin and George, UK) after growth has occurred. In testing more than 500 isolates, nearly 50 have been found which are capable of significantly reducing culture broth surface and interfacial tensions.

Bacteria gre suitably cultivated aerobically in 250 ml conical flasks containing 50 ml of Medium 5 (see Tablel) with glucose or glycerol as carbon source and incubated on an orbital shaker at 200 rpm. Anaerobic isolates may be cultivated in Bellco culture tubes with butyl rubber bungs under an atmosphere of oxygen-free nitrogen in 10 ml volumes of Medium 1,2,3 or4 (see Table 1); agar plates are incubated in anaerobic jars supplied with hydrogen and carbon dioxide-generating sachets and catalyst.

Table 1 (in its two parts) sets out the compositions of Media 1 to 5; the "trace element solution" given in Table 1A is defined for the relevant Media (1,4 and 5) in Table 1B. Media 1 and 5, for example, are essentially defined minimal salts media; medium 5 is Davis and Mingioli medium (Cruickshank et al, 1975, Medical Microbiology, Volume 2, Churchill Livingstone, Edinburgh, London and New York, p109). Media 2 and 3, for example, contain organic compounds such as yeast extract and are therefore not "defined" media.

TABLE 1A Component Unit per (amount in) Medium litre 1 2 3 4 5 KNO3 g 5.0 2.0 4.5 K2HPO4 g 1.0 1.0 7.0 KH2PO4 g 0.5 0.5 0.5 0.5 3.0 CACI2(1%w/v) ml 1 CACl2.6H20 g 0.1 0.06 MgSO4.7H2O g 0.4 0.06 0.4 0.1 MgCi2.6H2O 9 2.0 NH4CI g 1.0 1.0 0.1 1.0 (NH4)2S04 g 1.0 FeSO4.7H20 g 0.003 0.004 trace element solution ml 1 1 5 ascorbic acid g 0.1 thioglycolicacid g 0.1 yeast extract g 1.0 1.0 glucose g 20.0 citrate g 5.0 Lab M Agar g 15.0 pH,to 7.2 7.4 7.5 7.0 7.2 (with NaOH) TABLE 1B Trace element solution Trace element solution for Media 1 and 4 (g1~1) for Medium 5(1.1) Nitrilotriacetic acid 1.5 FeSO4.7H20 0.5 g FeC12.6H20 0.3 ZnSO4.7H2O 0.5 g MnCi2.4H2O 0.1 MnSO4.3H2O 0.5 g CaCl2.6H20 0.1 H2SO4(0.1 N) 10ml ZnCI2 0.1 CuCl2.2H2O 0.02 H3BO3 0.01 NaMoO4.2H2O 0.01 CoCl2.6H20 0.1 Biosurfactants may be extracted from culture broths with a suitable solvent, preferably 1-butanol or ethyl acetate. For example, equal volumes of culture and solvent are shaken thoroughly, and the aqueous and organic phases are allowed to separate. The organic phase is dried under forced air evaporation, and the residue obtained is redissolved in the appropriate medium at a range of concentrations. This allows the determination of surface and interfacial tension values at the CMC of the biosurfactant.

The following Examples illustrate the invention. The biosurfactant products may yet be characterised as reducing interfacial tension to less than 10.2 or even 10-3 mN m~' against hexadecane under optimised conditions. EOR capability in laboratory models is already indicated.

The biosurfactants which are produced by the procedures described below have been evaluated for oil recovery. The biosurfactants were heated to 100 C and cooled to -20 C, to monitor temperature stability.

Ultralow interfacial tensions of biosurfactants were measured using a spinning drop tensiometer (Kruess, West Germany). The effect of adding 5% (w/v) sodium chloride to biosurfactants, on interfacial tension and dilution of biosurfactants was monitored.

The ability of biosurfactants of the invention to recover residual oil from laboratory sandpacks has been measured. Sandpacks were prepared by pouring fine sand (40 to 100 mesh, 42 to 150 pbm) into a glass column stoppered at both ends by rubber bungs containing inlet and outlet tubes. The packs were evacuated before flooding with water or 1% w/v NaCI. Permeability was calculated by recording the pressure differential across the column at a fixed flow rate. The packs were evacuated once more before saturation with paraffin or crude oil. Water was pumped through the pack by a syringe pump (Sage Instruments, UK) at a flow rate of 60 ml h-' until no further oil was released from the pack.Waterflooding the packs released a proportion of the oil; after flooding with 2 pore volumes of water, less than 1% of the total oil was released by further waterfloods. At this stage, approximately 33% of the initial oil-in-place was retained by the packs as residual oil. Biosurfactant floods were then introduced into sandpacks with a permeability of approximately 25 Darcies from syringe pumps at a flow rate of 60 ml h-1. The volume of any residual oil released by the flood was measured.

Example 1 Isolate 3067 was isolated by streaking samples of anaerobic, digested sewage sludge on to solid Medium 1 containing 2% w/v glucose and incubating plates at 37 C for 2 weeks. It produces a biosurfactant when grown on liquid Medium 1 with 0.4% w/v glycerol as a carbon source at 37 C under anaerobic conditions, which is able to reduce culture broth surface tension to 29.2 mN m1 and interfacial tension to 3.0 mN m~1. Slightly higher surface tension values were observed when glucose, trisodium citrate or succinate was used instead of glycerol.

The isolate also grows aerobically on nutrient agar, producing a green pigment and a fluorescent yellow pigment; this suggests that the organism is Pseudomonasaeruginosa. Surface tension decreases towards the end of the exponential growth phase in batch culture, indicating that the surfactant is a secondary metabolite. The pH of the culture increases from 6.5 to 8.0 during growth and surfactant production.

Concentration of the 3067 biosurfactant to the CMC, after extraction with ethyl acetate, reduces surface tension to 28.5 mN m1 and interfacial tension to 1.5 mN m-l. The surface and interfacial tension values of the culture broth are a little above the CMC of the biosurfactant.

In three directly comparative tests, isolate 3067 reduced surface tension to values of 34.5, 52.5 and 55.0 mN m~1 when grown on Media 1, 2 and 3, respectively. This indicates that more surfactant was produced on the defined minimal salts Medium 1 than on the other media which contain complex organic compounds.

Example 2 Isolate 3090 was isolated from digested, anaerobic sewage sludge on solid Medium 3 containing 2% w/v molasses at 37 C under anaerobic conditions. It produces a biosurfactant which reduces culture broth surface tension to 35.5 mN m~ when grown at 37 C on liquid Medium 3 with 3% molasses as carbon source, under anaerobic conditions. Surface tension decreases throughout the exponential growth phase. The surfactant is associated with the cells since centrifugation of the culture increases surface tension to 55.0 mN ml. Concentration of the biosurfactant, after extraction with 1-butanol, reduces surface tension to 24.6 mN -1 interfacial to m and interfacial tension to less than 0.1 mN m1 at the CMC; the surfactant requires concentration by a factor of 12.5 to reach its CMC.The interfacial tension of the surfactant decreases on the addition of 5% w/v sodium chloride.

Isolate 3090, when grown on Medium 4, reduces culture broth surface tension to a lower value than when grown on a rich medium containing yeast extract, ascorbic acid and thioglycolic acid in addition to mineral salts.

Example 3 Isolate 1101 was isolated from an aerobic enrichment culture containing oil-contaminated refinery mud by streaking on to nutrient agar at 28 C. It produces a biosurfactant and a green pigment when grown in Medium 5 supplemented with 0.4% w/v glycerol at 37 C. It has been identified by biochemical tests as Pseudomonas aeruginosa. Biosurfactant production reduces surface tension to 31.3 mN m~ and interfacial tension to 2.5 mN m~1. Concentration of the biosurfactant by a factor of 5 is required to reach the CMC, after extraction with chloroform. Surface tension is reduced to 28.5 mN m1 and interfacial tension to less than 0.1 mN m1 at the CMC. The efficiency of the biosurfactant is increased by 8 to 10 fold on the addition of 5% w/v sodium chloride.

Example 4 Isolate 1165 was isolated directly from soil on nutrient agar at 37 C under aerobic conditions. It is a Gram-negative, spore-forming rod and has been identified as Bacillus sp. The biosurfactant produced by this isolate reduces culture broth surface tension to 27.6 mN m~' and interfacial tension to less than 0.1 mN m~' when grown at 37 C on liquid Medium 5 supplemented with 0.4% w/v glucose, on an orbital shaker. Surface tension is reduced in two phases during exponential growth in batch culture, suggesting that more than one surfactant compound is produced. Four components are separated from a l-butanol extract of the biosurfactant by thin layer chromatography in a chloroform/methanol/ammonium hydroxide (65:30:5 v/v/v) solvent system.Only one of these components has surface active properties. Amino-acids are released from the biosurfactant compound(s) on acid hydrolysis, suggesting the incorporation of a cyclic or linear peptide.

The biosurfactant retains surface activity for at least one hour at 100 C and for at least two weeks at -20 C.

The interfacial tension of the 1165 culture supernatant, after centrifugation at 10,000 g for 5 minutes to remove cells, is 0.032 mN m~1 against hexadecane, as measured by the spinning drop tensiometer.

Extraction of the biosurfactantwith 1-butanol increases interfacial tension against hexadecane to 0.093 mN m 1; this value is decreased, however, by the addition of 2.5% w/v sodium chloride.

Isolate 1165 supernatant recovers between 37.7 and 45.0% of the residual oil from a laboratory sandpack in the fourth pore volume of the surfactant flood; biosurfactant extracted with l-butanol and redissolved in Medium 5 recovers between 55.0 and 87.7% of the residual oil in the second pore volume.

Isolate 1165 has been deposited atthe National Collection of Industrial Bacteria. The deposition date is 29th March 1985. The accession number is 12059.

Claims (9)

1. A product of a microbiological process, which has the characteristic capability of reducing interfacial tension to 10.2 mN m1 or lower against hexadecane.

2. A product according to claim 1, which has the characteristic capability of reducing interfacial tension to l02to 10-5 mN rn1 against hexadecane.

3. A process which comprises growing a bacterium on a nutrient medium, and recovering a product having the characteristic capability of reducing interfacial tension to 10.2 mN m1 or lower against hexadecane.

4. A process according to claim 3, in which the medium is a defined minimal salts medium comprising substantially only essential nutrients for growth.

5. A process which comprises growing a bacterium on a minimal salts medium comprising substantially only essential nutrients for growth, and recovering a product having the characteristic of reducing interfacial tension.

6. A process according to any of claims 3 to 5, in which the medium contains a carbon source and no other organic materials.

7. A process according to any of claims 3 to 6, in which the medium contains, as the carbon source, an organic compound containing hydroxyl and/or carboxyl groups.

8. A method for enhanced oil recovery by using a surfactant, in which the surfactant is the product of a microbiological process.

9. A method according to claim 8, in which the surfactant is a product according to claim 1 or claim 2 or the product of a process according to any of claims 3 to 7.