Common Components of Industrial Metal-Working Fluids as

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 51, No. 6 Common Components of Industrial Metal-Working Fluids as Sources of Carbon for Bacterial Growth S. FOXALL-VANAKEN,' J. A. BROWN, JR.,' W. YOUNG,' I. SALMEEN,1* T. McCLURE,2 S. NAPIER, JR.,2 AND R. H. OLSEN3 Research Staff, Ford Motor Company, Dearborn, Michigan 48121'; Franklin Oil Company, Cleveland, Ohio ; and Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan Received 2 January 1986/Accepted 10 March 1986 Water-based metal-working fluids used in large-scale industrial operations consist of many components, but in the most commonly used formulations only three classes of components are present in high enough concentrations that they could, in principle, provide enough carbon to support the high bacterial densities (10' CFU/ml) often observed in contaminated factory fluids. These components are petroleum oil (1 to 5%), petroleum sulfonates (0.1 to 0.5%), and fatty acids (less than 0.1%, mainly linoleic and oleic acids supplied as tall oils). We isolated pure strains of predominating bacteria from contaminated reservoirs of two metalworking systems and randomly selected 12 strains which we tested in liquid culture for growth with each of the metal-working fluid components as the sole source of carbon. Of the 12 strains, 7 reached high density (109 CFU/ml from an initial inoculum of less than 2 x 103) in 24 h, and 1 strain did the same in 48 h with 0.05% oleic or linoleic acid as the carbon source. These same strains also grew on 1% naphthenic petroleum oil but required up to 72 h to reach densities near 108 CFU/ml. One strain grew slightly and the others not at all on the petroleum sulfonates. The four remaining strains did not grow on any of the components, even though they were among the predominating bacteria in the contaminated system. Of the seven strains that grew best on the fatty acids and on the naphthenic petroleum oil, five were tentatively identified as Acinetobacter species and two were identified as Pseudomonas species. Four of the bacteria that did not grow were tentatively identified as species of Pseudomonas, and one could not be identified. Water-based metal-working fluids as they are used in industrial metal working operations often become contaminated by bacteria with counts exceeding 109 CFU/ml (1-3, 5). The detailed composition of these fluids is usually proprietary information of the suppliers, but the fluids used in large industrial systems are generally emulsions consisting of oil, petroleum sulfonates, fatty acids, synthetic detergents, and various additives such as antioxidants, antifoam agents, and other ingredients designed to achieve certain performance objectives (4). The fluids are supplied as a concentrated mixture and used after dilution, typically between 1:15 and 1:40, with water. In large factory operations, the fluid is delivered to the metal-working machines by a system that pumps the fluid from a central reservoir (a capacity typically between 1,000 and 50,000 gallons [3,785 and 189,250 liters, respectively]) and recirculates it back to the reservoir. Many papers over the past 30 years have discussed the identification of the bacteria isolated from contaminated reservoirs, the effects of the bacteria on the emulsion stability, the influences of ph, oil-to-water ratios, and dissolved salts on the growth of the bacteria, and the use of biocides for controlling the growth (1-5). In 1956 Sabina and Pivnick (10) showed that very concentrated suspensions of Pseudomonas strains isolated from used metal-working fluids could oxidize, in a Warburg apparatus, components of metal-working fluids. Other investigators have commented that the bacteria isolated from metal-working fluid systems are capable of degrading the * Corresponding author components of the fluids (6). The capability of oxidizing or degrading these components, however, does not necessarily mean that these bacteria will grow to the high densities observed in contaminated factory systems when these components are the only sources of carbon. To devise means to control the growth in factories, it is useful to know which components serve as the primary carbon sources. The carbon metabolism of the community of bacteria in the contaminated factory reservoirs may be complex, as suggested by the many different strains of bacteria that have been isolated from any one contaminated system. For certain large systems, however, the identification of the main carbon sources may be easier than first appears, because the fluids used in these systems contain only three classes of compounds present in sufficiently high concentrations that they could, in principle, provide enough carbon to support growth to the high densities that are often observed. These classes of compounds are: oil, which constitutes 75 to 85% of the stock fluid; petroleum sulfonates, which constitute up to about 10% of the stock; and fatty acids (mainly oleic and linoleic acids supplied as tall oils) which constitute a few percent of the stock. When these stock solutions are diluted 1:15 to 1:40, even the most abundant carbon sources are not present in very high concentrations. Herein we describe a study of the carbon sources that support bacterial growth in a fluid used in large metalworking operations in the Ford Motor Company. We isolated predominant bacteria from factory metal-working fluid reservoirs and carried out quantitative bacterial growth experiments for 12 strains with each of the main components of the fluid as the sole source of carbon.

2 1166 FOXALL-VANAKEN ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Growth of strains on various components of metal-working fluid as sole sources of carbon nlnoa ratio with carbon source Bacte- Metal-working fluidb Oleic acids Fatty Petroleum sulfonate Naphthenic rium acid petroleum oile estersd Al x 105 NTf 30 (100) 30 (30) 7 x 103 (2 x 105) A2 3 x x x i05 3 x 105 NT 4 (5 x 103) 30 (103) 103 (2 x 104) A3 2 x x x x (4) 10 (16) 2 x 104 (2 x 105) A4 2 x x x x x x x 103 (2 x 104) (3 x 104) (2 x 103) A5 2 x x x NT 1 (0) 1 (0) 3 x 104 (2 x 105) P1 8 x x x NT x 103 (104) P x x 105 i05 NT NT NT 3 x 102 (4 x 104) P3 9 x 103 (5 x 105) 5 x 103 (3 x 104) 4 x 103 (106) 4 x 103 (6 x 105) (0) 0 (0) 2 x 102 (5 x 102) P4 40 (102) 30 (3 x 103) 102 (2 x 103) 2 x 102 (7 x 103) NT 1 (1) 1 (1) 5 x 102 (2 x 103) P5 60 (5 x 102) NT 70 (102) NT NT NT NT 3 (102) Ul 3 x 102 (5 x 103) 10 (75) 2 x 102 (2 x 102) 10 (8) NT 10 (102) 10 (5 x 102) 1 (102) U2 20 (1) 20 (1) 6 (2) 3 (2) NT 1 (0) 1 (1) 1 (0) a n is bacterial density after growth pefiod, and no is initial inoculum. The initial inocula were always less than 2 x 103 CFU/ml. When meager growth was observed after 24 h, the growth period was extended to 48 h. Data for 48-h cultures are shown in parentheses. b Data for the complete generic fluid formulated as described in the text. c Growth of every strain on linoleic acid was the same as for oleic acid. d 0. 1%t. e 1%. f NT, Not tested. MATERIALS AND METHODS Contaminated fluids from two different factories were collected as the fluid cascaded off metal-working machines. The same type of fluid was used in the two systems. Pure strains of the predominating organisms were isolated by diluting the contaminated fluid by 104 in a mineral salts solution (12) and then streaking the diluted sample on eosin-methylene blue agar (Difco Laboratories, Detroit, Mich.) or on nutrient agar. Individual colonies were picked from the streaks and restreaked on TNA agar plates (tryptone, 5 g/liter; yeast extract, 2.5 g/liter; dextrose, 1.0 g/liter; NaCl, 8.5 g/liter; Difco Noble agar, 20 g/liter; CaCl2 [0.135 M stock], 10 ml/liter). The eosin-methylene-blue plates typically showed a few hundred colonies. We selected between 25 and 50 colonies, attempting in the process to achieve representative sampling of each morphologically distinguishable type of colony. Single colonies from the latter TNA agar plates were streaked on fresh TNA agar plates and incubated overnight. The bacteria were then scraped from the surface of the agar and suspended in 1.5 ml of phosphate buffer (ph 7.0), and then mixed in a 1:1 ratio with glycerol and stored at -20 C. Preliminary taxonomic data for the pure strains of bacteria were obtained by the rapid NFT test (DMS Laboratories, Flemington, N.J.) and the oxidase test, and by growth on various substrates as the sole sources of carbon. For the latter characterization, bacteria were streaked for isolated colonies on plates containing Noble agar (Difco), mineral salts (12), and the particular substrate (0.2%). A mixed culture from the contaminated fluids was prepared and stored as follows. A 10-ml volume of contaminated fluid was centrifuged, and the bacteria pellet was suspended in a mineral salts solution (12). The resuspended bacteria were streaked on a TNA agar plate, and the plate was incubated (27 C) overnight. The plate was then scraped into the glycerol buffer as described above for the pure strains and stored at -20 C. The growth experiments were carried out on samples of commercially available materials that we believed were representative of those in the fluids used in the factories from which the bacteria were obtained. The detailed composition of the metal-working fluid actually used was not available from the supplier, but our chemical analysis and some limited data from the supplier showed that its general composition was naphthenic petroleum oil, petroleum sulfonates, oleic and linoleic acids, and minor unknown components. Growth experiments were carried out on individual components and on a generic metal-working fluid formulated from commercially available components (Franklin Oil Co., Cleveland, Ohio) and containing naphthenic petroleum oil (84.5%), tall oil (2%), esterified animal fatty acids (0.5%), petroleum sulfonate with an average molecular weight of 450 (10%), triethanolamine (1.5%), and ethoxylated octylphenol nonionic surfactant (1%). This stock fluid was diluted as described below to specified concentrations (see below). Tall oil is composed of roughly equal concentrations of oleic and linoleic acids which together constitute about 98% of the tall oil. Growth experiments were carried out on linoleic and oleic acids (clear oleic acid [A-195] and purified linoleic acid [A-165], Fisher Scientific Co., Pittsburgh, Pa.) rather than on the less well defined tall oils. A total of 70 isolates were characterized, and 12 strains which were metabolically distinguishable from each other were selected from among them for growth experiments. Growth experiments were carried out as follows. A 100-,lI amount of the cultures that were stored in glycerol at -20 C was used to inoculate 20 ml of a nutrient broth (tryptone, 5 g/liter; yeast extract, 2.5 g/liter; dextrose, 1.0 g/liter, NaCl, 5 g/liter, KNO3, 4 g/liter) which was then incubated at 27 C for 14 to 18 h in a shaker incubator. These cultures were centrifuged at 10,000 rpm) (Sorvall SS34 head; Ivan Sorvall, Inc., Norwalk, Conn.) for 15 min at 27 C and suspended in 20 ml of the mineral salts solution. This washed culture was diluted by 104, and 200,ul of it was used to inoculate 20 ml of mineral salts to which was added either metal-working fluid or one of the components as the sole carbon source. Immediately after the inoculation of the latter, an aliquot was removed and plated (undiluted and diluted 10-fold) on nutri-

3 VOL. 51, 1986 BACTERIAL GROWTH IN METAL-WORKING FLUIDS 1167 TABLE 2. Growth on metal-working fluid without fatty acids nlnoa ratio with carbon source (%): Bacte- Metal-working fluid Fatty acid-free fluid rium Al 10S 7 x (103) 130 (2 x 103) 30 (4 X 102) A5 2 x x x x P3 9 X x x 103 <1 (50) 4 (3 x 102) 20 (103) P (100) 30 (500) 20 (80) Mixedb l X X X (4 X 103) 50 (7 X 104) 20 (5 X 104) a See Table 1, footnote a. b A mixed population was obtained directly from a factory sample as described in the text. ent agar to determine the initial viable inoculum. The liquid cultures were then incubated at 27 C as above and aliquots were removed to determine counts after specified periods (see below). RESULTS AND DISCUSSION The main results of the experiments in which the isolated strains of bacteria were grown on the components of the metal-working fluids as the sole source of carbon were recorded (Table 1) as ratios (n/no, where no was the initial inoculum and n was the count after the growth period). The initial inocula were always less than 2 x 103 CFU/ml. Bacterial densities were determined after 24 and 48 h. Growth of every strain on linoleic acid was the same as that on oleic acid. The bacteria were not tested for growth in liquid culture with triethanolamine or with the synthetic TABLE 3. Classification of bacteria in growth experiment Bacte- Oxidase Nodea Identification (%)b Al Acinetobacter calcoaceticus subsp. anitratus (99.9) A Acinetobacter haemolyticus (99.5) A Acinetobacter haemolyticus A Acinetobacter calcoaceticus subsp. iwoffi (99) AS Acinetobacter haemolyticus (99.5) P Pseudomonas putdia (88.3) P Pseudomonas stutzeri (97.8) P Pseudomonas alcaligenes (62.8) Alcaligenes denitrificans (17.8) P Pseudomonas putrefaciens (99.8) PS Pseudomonas cepacia (99.9) Ul Alcaligenes dentrificans (93.6) U Pseudomonas sp. (59) a Numerical classification code from rapid NFT test. b Percent identification according to NFT code. c Lack of growth on galactose distinguishes it from A. calcoaceticus subsp. anitratus. surfactant as the sole source of carbon. In plating experiments, however, none of the bacteria showed growth with these compounds as the carbon source. The data (Table 1) show that 7 out of 12 strains (5 Acinetobacter strains [Al to AS] and 2 Pseudomonas strains [P1 and P2]) reached high densities (about 109 CFU/ml) in 24 h, and one Pseudomonas strain (P3) reached about 109 CFU/ml in 48 h on oleic acid as the sole source of carbon. The oleic acid concentrations that were tested spanned the range of those in typical commercial metal-working fluids. Thus, the concentrations of fatty acids in these metalworking fluids were sufficient to support the high bacterial densities observed in contaminated factory reservoirs. Strains A3 and A4 grew almost as well on the esterified animal fatty acids as they did on oleic acid. If bacterial esterases converted the esters to fatty acids, then the metabolic pathway of the fatty acid esters may have been the same as that of oleic acid. The strains grew on Tween 80 (data not shown), which implies that they have the requisite esterase activity. Strains Al to A5, P1, and P2 also grew on the naphthenic petroleum oil, but the growth rates were less than those with oleic acid as the carbon source. For two of the strains (A5 and P2) we carried the growth experiment to 96 h. For strain A5, n/no was 8 x 105 and 2 x 106 at 72 and 96 h, respectively. For strain P2, n/no was 5 x 105 and 9 x 105 at 72 and 96 h, respectively. Thus for at least some of the strains, the naphthenic petroleum oil supported bacterial densities as high as those observed for oleic acid, although the growth rates with the naphthenic petroleum oil were much less than those with oleic acid. Four of the strains grew only slightly on the intact fluid and on the individual components. These four strains, however, were among the predominating strains in the contaminated systems, because they were obtained from 104-fold dilutions of the samples obtained from the factories. It appears, therefore, that these latter strains may reach high densities in the factory reservoirs because of cross feeding among the bacteria in the mixed populations found in the factories. We did not carry out experiments with mixtures of the bacteria. The foregoing data suggest that the growth rate of bacteria in factory systems may be controllable, in part, by avoiding the use of fatty acids in the fluids. The same conclusion was reached by Sabina and Pivnick (10) on the basis of oxygen uptake measured in a Warburg apparatus. We tested this possibility by measuring the growth of selected pure strains

4 1168 FOXALL-VANAKEN ET AL. APPL. ENVIRON. MICROBIOL. TABLE 4. Differentiation of Acinetobacter spp. ~~~~~~~~~~~~~~Paracou- Bacte- ~~~~~~~~~ Resistance to: Baucte- Identification Lactate Starch Adipic Parahydroxy- Benzoic- Tryosine maric Renistaepto: acd cillen mycin cyline AS A. haemolyticus (Brown colonies) A2 A. haemolyticus A3 A. haemolyticus /- +/- - Al A. calcoaceticus subsp anitratus A4 A. calcoaceticus subsp iwoffi and of the mixed culture on a mixture containing all of the components except the tall oil and the fatty acid esters. When these were omitted, the mixture still formed an emulsion, with emulsion particles as viewed in a light microscope about the same size as those observed for the complete mixture (Table 2). Strains Al and A5 and the mixed culture reached high densities in 24 h on the intact metal-working fluid but increased in density only 10-fold in 24 h when grown on the fluid without fatty acids and esters. After 48 h however, the fluid without fatty acids and esters showed some slight growth of strains Al and A5 which was comparable to that observed on the naphthenic petroleum component (Table 1). Thus in the model fluid, the fatty acids were the only carbon source that supported rapid growth of the bacteria. The classifications of the bacteria were determined by the NFT battery and by data for growth on various substrates (Tables 3 and 4). The NFT test battery did not distinguish among strains A2, A3, and A5. A3 was distinguishable from A2 and A5 by its growth on various carbon sources (Table 4). A5 showed a brown pigment and A2 did not when tyrosine was the carbon source on agar plates. Antibiotic resistance did not unambiguously distinguish A2 from A5 (Table 4). All the bacteria that we assigned to the genus Acinetobacter grew in liquid culture on oleic acid, hexadecane (data not shown), and naphthenic petroleum oils. Growth on these classes of compounds was consistent with the general hydrocarbon metabolic traits described (7, 9, 11) for Acinetobacter spp. Juni (7) has suggested that species of Acinetobacter are important natural degraders of hydrocarbons in the soil. Of all the species of Pseudomonas described in Bergey's Manual of Systematic Bacteriology (8) and in reports on hydrocarbon metabolism (9, 11), the only one that grows well on hydrocarbons or fatty acids with more than 10 carbon atoms is Pseudomonas aeruginosa. The metabolic capabilities of P1 and P2 (Tables 1 and 3), tentatively identified as species of Pseudomonas, suggest that the assimilation of longer-chain hydrocarbons by species of this genus may not be restricted to P. aeruginosa. The validity of this suggestion remains to be seen. In summary, the results and conclusions from these experiments are the following. We randomly selected 12 strains of the predominant bacteria from a contaminated metalworking fluid system and sought to determine which components of the fluid as carbon sources support growth of these bacteria. We found that 7 out of the 12 strains reached high densities in 24 h with fatty acids as the sole carbon source. Five of the strains grew on the naphthenic petroleum oil component as the sole carbon source but at a rate that was much less than that observed when fatty acids were the sole carbon source. Thus in a sense, fatty acids appeared to be the primary carbon source in supporting initial bacterial growth in the metal-working fluid reservoirs. It will be difficult to avoid bacterial contamination of any metalworking fluid that is formulated with fatty acids. Even at very low concentrations (0.005%) (data not shown), the fatty acids supported growth of some strains of bacteria to a density of 109 CFU/ml. There were bacteria among the predominating ones in the contaminated system that did not grow in liquid culture on any of the components. Presumably these grew in the factory reservoirs because of some type of cometabolism. This suggests that if means can be found to control the growth of the bacteria that use the fatty acids, then the growth of all the bacteria may be controllable. Even if metal-working fluids can be formulated without fatty acids, however, it still may be difficult to avoid low concentrations of fatty acids in factory reservoirs, because certain commercial flood cleaners contain fatty acids and in many factory situations it will not be easy to prevent these from being washed into the metal-working fluid reservoirs. We have seen that very low concentrations of fatty acids will still support growth of some strains of bacteria. LITERATURE CITED 1. Bennett, E The biology of metal-working fluids. Lubr. Eng. 28: Bennett, E The deterioration of metal cutting fluids. Prog. Ind. Microbiol. 13: Chater, K. W. A., and J. L. Shennan The philosophy and compatibility of biocide additions, p In K. W. A. Chater and E. C. Hill (ed.), Monitoring and maintenance of aqueous metal-working fluids. John Wiley & Sons, Inc., New York. 4. Cookson, J An introduction to cutting fluids. Tribiol. Int. 10: Hill, E. C Microbial infection of cutting fluids. Tribiol. Int. 10: Hill, E. C Microorganisms: number, types, significance, detection. p In K. W. A. Chater and E. C. Hill (ed.), Monitoring and maintenance of aqueous metal-working fluids. John Wiley & Sons, Inc., New York. 7. Juni, E Genetics and physiology of Acinetobacter. Annu. Rev. Microbiol. 32: Palleroni, N. J Pseudomonas, p In N. R. Krieg and J. G. Holt (ed.) Bergey's manual of systematic bacteriology, vol. I. The Williams & Wilkins Co., Baltimore. 9. Perry, J. J Microbial metabolism of cyclic alkanes, p.

5 VOL. 51, 1986 BACTERIAL GROWTH IN METAL-WORKING FLUIDS In R. E. Atlas (ed.), Petroleum microbiology. Macmillan Publishing Co., Inc. New York. 10. Sabina, L. R., and H. Pivnick Oxidation of soluble oil emulsions and emulsifiers by Pseudomonas oleovorans and Pseudomonas formicans. Appl. Microbiol. 4: Singer, M. E., and W. R. Finnerty Microbial metabolism of straight-chain and branched alkanes, p In R. E. Atlas (ed.), Petroleum microbiology. Macmillan Publishing Co., New York. 12. Stainer, R. Y., N. J. Palleroni, and M. Doudoroff The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43: