Received for publication July 28, The ability of psychrophiles to develop anaerobically. matter in nature and spoilage of foods.

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1 ANAEROBIC GROWTH OF PSYCHROPHILIC BACTERIA J. UPADHYAY AND J. L. STOKES Department of Bacteriology and Public Health, lvashington State University, Pullman, Washington Received for publication July 28, 1961 ABSTRACT UPADHYAY, J. (Washington State University, Pullman) AND J. L. STOKES. growth of psychrophilic bacteria. J. Bacteriol. 83: Facultatively anaerobic psychrophilic bacteria are widely distributed in nature and can be readily isolated from a variety of foods, soil, sewage, and other habitats. Strictly anaerobic psychrophiles, however, could not be isolated. Temperature and oxygen have striking effects on the growth and metabolism of the psychrophiles. Sugars which are fermented at 2 C may not be fermented at C or gas may not be produced. Under both aerobic and anaerobic conditions, decreases in incubation temperatures increase the duration of the lag, exponential, stationary, and death phases. Elimination of 2 increases the lag period, allows the cells to survive much longer at low temperatures (but accelerates their death at 2 C and higher), and, finally, reduces the extent of growth. M1aximal cell populations are obtained aerobically at 5 C and anaerobically at 25 C. Growth is most rapid in the range of 2 to 35 C, and maximal growth temperatures range from 25 to 45 C. Neither of these cardinal temperatures is appreciably affected by the presence or absence of 2- Pseudomonas, Achromobacter, and Flavobacterium. It is reasonable to expect that some species of these genera can grow anaerobically, as well as with oxygen, especially since some of the strains can ferment carbohydrates. But, in general, anaerobic growth has not been studied. The ability of psychrophiles to develop anaerobically would be of importance with reference to their role in transformation of organic matter in nature and spoilage of foods. The present investigations were undertaken to determine to what extent strict and facultative anaerobic psychrophilic bacteria occur in nature. Strict anaerobes were not found but a large number of facultative anaerobes were readily isolated from soil, sewage, and various foods. Some of the properties of these bacteria and their kinetics of growth, with and without 2, are described in the present paper. MATERIALS AND METHODS A selective enrichment culture technique was used to isolate anaerobic psychrophilic bacteria. This involved anaerobic cultivation at C of the mixed, natural, microbial populations of soil, sewage, river mud, raw and pasteurized milk, ice cream, cheddar cheese, and a variety of meats and fish in a modified brain heart infusion (BHI) broth. The commercial medium was enriched with.5%7 yeast extract, the phosphate content was raised to.5%, the glucose level to 1%, and the medium was adjusted to ph 7.. Occasionally, ph 6 or 8 was used. Sterile glass-stoppered bottles of 6-ml capacity were partially filled with freshly heated and Psychrophilic bacteria are those which grow well at C. Their ecology and properties have been reviewed recently (Ingraham and Stokes, 1959). Virtually all investigations on psychrophilic bacteria have been carried out under aerobic conditions. Muller (193) appears to be cooled sterile medium, inoculated with microbial the only investigator to have reported anaerobic source material, and then completely filled with growth. His Bacterium A and Bacterium C, medium and incubated at C. The inoculated gram-negative, nonspore-forming rods, grew both clear medium usually became slightly turbid aerobically and anaerobically. In contrast, within 1 week (due to microbial growth) and Straka and Stokes (196) found that none of 3 markedly turbid within 2 weeks. This occurred strains of psychrophilic bacteria from Antarctica could grow anaerobically. Most strains of tures, plates were prepared from the modified with all of the enrichments. To isolate pure cul- psychrophiles so far isolated belong to the genera BHI broth solidified with 1.5% agar, and these 27 Downloaded from on January 16, 219 by guest

2 19621 ANAEROBIC GROWTH OF PSYCHROPHILES 271 were streaked with material from the enrichment cultures. The plates were incubated anaerobically in sealed jars under hydrogen at C for 2 weeks. Single colonies on the plates were picked, restreaked, and the plates incubated as described. This process was repeated to obtain pure cultures. Portions of single colonies from the second plates were inoculated in duplicate onto slants of the modified BHI agar, and one slant was incubated anaerobically under a pyrogallic acid seal and the other aerobically at C. All cultures thus isolated grew both anaerobically and aerobically and, therefore, are facultative. Subsequently, the stock cultures were maintained aerobically. Also, for most subsequent experiments, cultures were incubated at 2 C rather than C to hasten growth. In a number of experiments, however, comparative determinations were made at both temperatures. The cultures were examined for morphology, gram stain reaction, production of indole, and fermentation of glucose, sucrose, and lactose by the usual methods. Fermentation tests at C were incubated for as long as 12 days; those at 2 C were observed for 15 days. A detailed comparison was made of the kinetics of growth of strain 82 (a gram-negative rod), under aerobic and anaerobic conditions in the range of to 35 C at 5-degree intervals. Aerobiosis was obtained and maintained by use of shallow layers of medium, i.e., 12 ml of medium in a 1-liter Erlenmeyer flask, and by frequent shaking of the cultures during incubation. For anaerobic cultivation, the bacteria were grown in 3-ml glass-stoppered bottles completely filled with medium; one bottle culture was used for each growth determination. Growth was measured by means of plate counts. Samples of the cultures were diluted in.1% peptone to avoid possible destruction of the cells (Straka and Stokes, 1957), and were plated onto modified BHI agar. RESULTS A total of 83 pure cultures of bacteria was isolated from various foods, soils, sewage, and other substrates by selective enrichment under anaerobic conditions at C. All of the isolates were psychrophiles, since they grew well at C within 2 weeks, and were facultative anaerobes, since they developed both with and without 2; 47 % of the strains were gram-negative rods, 41 % were gram-positive rods, and the remaining 12% were gram-negative cocci. Fermentation characteristics. Thirty-two strains were examined for the production of indole and the fermentation of glucose, sucrose, and lactose at and 2 C. Only one strain formed indole, and this occurred at 2 C but not at C. Five strains failed to ferment any of the three sugars at both temperatures; the remaining 28 strains fermented one or more of the sugars, usually with the production of only acid. Several strains formed both acid and gas. Fermentations were much slower at C than at 2 C. Final results were usually available in 2 days at 2 C, whereas at least 7 days and frequently as long as 17 to 29 days were required at C for visible fermentation. There were some interesting differences in fermentation at the two temperatures. Three strains which fermented glucose at 2 C failed to ferment glucose at C, and seven strains which fermented lactose at 2 C failed to ferment at C despite greatly extended incubation periods. Also, two strains which formed both acid and gas from glucose at 2 C produced only acid at C. Data for five representative strains are given in Table 1. Metabolic activity at C may be less, therefore, than at higher temperatures, due TABLE 1. Effect of temperature on the fermentation of sugars by psychrophilic bacteria* Strain Gram stain OC 2C Glucose Sucrose Lactose Glucose Sucrose Lactose Downloaded from on January 16, 219 by guest Rod (25) _ AG AG AG AG AG AG Rod (36) + A A A A A A Rod (49) _ A - - Rod (63) _ A - AG AG Coccus (65) + A A A A A * Symbols used: A, acid produced; G, gas formed; -, neither acid nor gas formed.

3 272 UPADHYAY AND STOKES [VOL. 83 probably to inhibition of enzyme formation or enzyme activity by low temperatures. Cardinal temperatures. The optimal and maximal growth temperatures were determined with 1 strains, randomly chosen. The cultures were inoculated onto slants in replicate. One set was incubated aerobically and the other anaerobically under pyrogallic acid seals at temperatures ranging from to 5 C. The rate and extent of TABLE 2. Optimal and maximal growth temperatures, aerobically and anaerobically, of psychrophilic bacteria Temperature, C Strain Gram stain Opti- Maxi- Opti- Maximum mum mum mum Rod (12) Rod (25) Coccus (56) Rod (76) Rod (82) growth were observed visually. Data for some of the strains are given in Table 2. The optimal temperatures ranged from 2 to 3 C, and the maximal temperatures from 25 to 4 C, although a few strains, not listed in the table, had a maximal temperature of 45 C. In general, the optimal and maximal temperatures were the same aerobically and anaerobically. Occasionally, a strain such as strain 76 exhibited a lower maximal temperature under anaerobic conditions. Kinetics of anaerobic and aerobic growth. A detailed investigation was made of the kinetics of growth of one of the gram-negative rods, strain 82, with and without oxygen, at, 5, 15, 2, 25, 3, and 35 C. The modified BHI broth was used. As previously described, aerobic conditions were maintained by use of shallow layers of broth, and anaerobiosis by use of glass-stoppered bottles. A 1-day-old culture grown at 3 C served as inoculum. The course of growth was followed by means of p)late counts. To conserve space, data on cell multiplication at only four temperatures,, 5, 2, and 35 C, are given in Table 3, and data on the duration of the TABLE 3. Multiplication of strain 82 in modified BHI broth, aerobically and anaerobically, at various temperatures Days of incubation Viable bacteria per ml C 5C 2C 35C 1.4 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 1, 1.4 X X X 17 Downloaded from on January 16, 219 by guest

4 1962] ANAEROBIC GROWTH OF PSYCHROPHILES 273 TABLE 4. Growth phases of strain 82 in modified BHI broth, aerobically and anaerobically, at various temperatures Temperature Growth C 5 C 2 C 35 C Duration, hr Lag phase Exponential phase Stationary phase Decline phase Generation time, min Maximum cell count per ml X 1-' Days to reach maximum various growth phases, generation times, and maximal cell yields at these same temperatures are given in Table 4. The lag phase lengthened greatly as the incubation temperature was lowered. ally, it lasted for 96 hr at C, but only 3 hr at 35 C. It was longer under anaerobic conditions, continuing for 235 hr at C compared to only 96 hr aerobically. Also, the duration of the exponential phase increased as the incubation temperature decreased. It continued, aerobically, for 288 hr at C, but only 18 hr at 35 C. Its duration was about the same anaerobically as aerobically at C, but shorter anaerobically at 35 C. The stationary phase was also greatly extended at C, but was not very much affected by the presence or absence of 2. The phase of decline or death continued for a much longer period at the lower temperatures and lasted for 638 hr at C, compared to 26 hr at 2 C and 57 hr at 35 C, aerobically. Oxygen had a striking effect on this phase of the growth cycle, in that death of the cells was much more rapid at low temperatures in the presence of 2 than in the absence of 2- This was especially evident at C, where the death phase lasted for 638 hr aerobically and approximately 1 times as long (6144 hr) anaerobically. At 2 C and above, the reverse was true, and cells died more rapidly under anaerobic conditions. Generation times were greatly lengthened at the lower temperatures. The generation time aerobically at C was 1274 min, compared to only 6 min at 35 C. They were influenced somewhat by 2, being longer anaerobically at C and shorter anaerobically at 35 C. The maximal yield of cells, under aerobic conditions, was about twice as large at C compared to that at 35 C, and, in general, was greater at the lower incubation temperatures. In contrast, under anaerobic conditions, the maximal yield of 96 million cells per ml was obtained at 25 C, and the yield decreased as the temperature was either lowered or raised. Also, cell yields were greatly reduced under anaerobic conditions. For example, at C 12 million cells per ml of culture were obtained aerobically but only 23 million cells per ml anaerobically (approximately one-fifth as many). This is consistent with the fact that much more energy is released by aerobic metabolism. However, because of the slowness of growth at the lower temperatures, a much longer time was required for the cultures to reach maximal populations; 2 days were needed for maximal growth at C, but only 1 day at 35 C. Although the detailed data for all of the temperatures investigated between and 35 C are not given, the results obtained with the intermediate temperatures are similar to those presented for the four temperatures. However, the temperatures at which transitions occurred were established by the more complete data. Thus, generation times were longer anaerobically than aerobically at the temperatures up to 15 C, and were shorter at 2 C and higher temperatures. Likewise, the transition to a markedly shorter stationary phase, anaerobically, began at 3 C. The change from extended death times anaerobically at lower temperatures to more rapid death at higher temperatures occurred at 2 C. Also Downloaded from on January 16, 219 by guest

5 274 UPADHYAY AND STOKES [VOL. 83,, 14 w D 12- z 2 1 w - 8- z w zw 2. D AEROB IC ANAEROB IC TEMPERATURE(C) 1 2 FIG. 1. Generation times of strain 82, aerobically DAYS - X1 11L^ 7..4A-^ >@n 4M>4 ana anaero crtcaacy, at varwzous temperatures itn FIG. 3. Growth curves of strain 82, aerobically and modified BHI broth. anaerobically, at 35 C in modified BHI broth. Un wj mwll -J Lm < v > -Jo DAYS FIG. 2. Growth curves of strain 82, aerobically and anaerobically, at C in modified BHI broth. the maximal yield of cells (96 X 16), anaerobically, was obtained at 25 C. For ease of comparison and to provide some additional data on intermediate temperatures, the generation times for strain 82 at the various temperatures, aerobically and anaerobically, are plotted in Fig. 1. Also, the complete growth curves for the same organism, aerobically and anaerobically, at and 2 C are shown in Fig. 2 and 3. hj co w 2uz J < ( H > DISCUSSION The early investigations of Forster (1892), Schmidt-Nielsen (192), Muller (193), and others clearly showed that psychrophilic bacteria are widely distributed and can be found in soil, water, air, milk, fish, meats, vegetables, and other habitats. Our investigations indicate that facultatively anaerobic psychrophiles are commonly present in these same environments and include a variety of morphological types. This also suggests that under anaerobic conditions psychrophilic bacteria may be active in the important natural cycles of matter, and in the spoilage of foods. It is surprising, perhaps, that strictly anaerobic psychrophiles could not be isolated, since it seems likely that such bacteria exist in nature. Our enrichment techniques simply may not have been suitable for isolating strict anaerobes, despite variations in the composition of the enrichment medium, its initial ph, and the source of the introduced mixed microbial population. The data on the growth of the facultatively anaerobic strain 82, under aerobic conditions at various temperatures, are similar to those obtained by Hess (1934) with a strain of Pseudomonas fluorescens, presumably a strict aerobe. He also found that the lag phase and generation time lengthen with decrease in temperature, and that this is accompanied by an extended period of exponential growth and larger cell population Downloaded from on January 16, 219 by guest

6 1962] ANAEROBIC GROWTH OF PSYCHROPHILES 275 at the lower temperatures. Our experiments extend the data to growth under anaerobic conditions and show that, in the absence of 2, marked changes occur in the growth cycle. At C, growth is slower and less extensive anaerobically, but the cells survive much longer. At higher temperatures, death is more rapid anaerobically. Also, maximal cell yields occur at temperatures considerably above those of maximal aerobic growth. ACKNOWLEDGMENT This investigation was supported, in part, by a research grant from the National Science Foundation. LITERATURE CITED FORSTER, J Ueber die Entwickelung von Bakterien bei niederen Temperaturen. Centr. Bakteriol. Parasitenk. 12: HESS, E Effects of low temperatures on the growth of marine bacteria. Contribs. Can. Biol. and Fisheries, Ser. C, 8: INGRAHAM, J. L., AND J. L. STOKES Psychrophilic bacteria. Bacteriol. Rev. 23: M1LLER, M tber das Wachstum und die Lebenstatigkeit von Bakterien, sowie den Ablauf fermentativer Prozesse bei niederer Temperatur unter spezieller Berucksichtigung des Fleisches als Nahrungsmittel. Arch. Hyg. 47: SCHMIDT-NIELSEN, S Ueber einige psychrophile Mikroorganismen und ihr Vorkommen. Centr. Bakteriol. Parasitenk, Abt. 2, 9: STRAKA, R. P., AND J. L. STOKES Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol. 5: STRAKA, R. P., AND J. L. STOKES Psychrophilic bacteria from Antarctica. J. Bacteriol. 8: Downloaded from on January 16, 219 by guest