Bacterial Dry Matter Content and Biomass Estimations

Vol. 48, No. 4

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1984, p. 755-757

0099-2240/84/100755-03$02.00/0 Copyright © 1984, American Society for Microbiology

Bacterial Dry Matter Content and Biomass Estimations GUNNAR BRATBAK* AND IAN DUNDAS Department of Microbiology and Plant Physiology, University of Bergen, N-SOOO Bergen, Norway Received 21 February 1984/Accepted 15

June

1984

were harvested by centrifugation. Intercellular water, as of the total water in the pellet, was determined with percent H20 (New England Nuclear) as marker for total water and [carboxyl-"4C]inulin (New England Nuclear) as marker for intercellular water in the pellet (4, 14, 16-19). The harvested cells were resuspended in glucose-free medium with 3H marker for 30 min or with 14C marker for 2 min and then centrifuged at 12,000 x g for 10 min. 14C marker trapped in the intercellular water and 3H marker trapped in both intercellular and intracellular water were washed out of the pellets by resuspending the cells in 10 ml of glucose-free medium and centrifuging. The amount of isotope marker in the supernatant was determined by liquid scintillation counting. The pellets formed after the last centrifugation were used for determination of pellet percent dry weight by weighing the pellet before and after drying at 105°C to constant weight. The salt content of the media was determined gravimetrically, drying 10 ml of glucose-free fresh medium at 105°C to constant weight. Carbon content of the dry pellets were determined with a CHN analyzer (Carlo Erba Strumentazione 1106). The weights of the dry pellets were corrected for the weight of the medium salts remaining in the pellets after drying, before calculating the carbon weight/dry weight ratio of the cells. Buoyant densities of individual bacterial cells and of bacterial pellets were determined with preformed density gradients of Percoll (Pharmacia Fine Chemicals) (12), with density marker beads (Pharmacia Fine Chemicals) (1) as references. RESULTS AND DISCUSSION The choice of B. subtilis, E. coli, and P. putida was motivated by the desire for comparing our results with previously published data and for working with a few bacteria with widely different properties. Using bacterial strains isolated from aquatic environments might have increased the relevance of the obtained results with respect to aquatic studies. However, isolating a few bacterial strains and cultivating them in the laboratory for experimental purposes will not by itself justify drawing general conclusions about the whole bacterial community or about the qualities of these strains in situ. Using bacteria known to have widely different properties will at least give an idea about the range of values one might expect to find for bacteria in general. The dry weight of the bacterial pellets as percent of the pellet wet weight ranged from 11 to 29%, averaging 20% (Table 1). These values for bacterial pellets are comparable

Reliable estimates of bacterial biomass (as carbon content) are essential to determine the quantitative importance of bacteria in many ecosystems. No reliable method for the direct determination of bacterial biomass is currently available. Useful estimates can be made, however, by converting bacterial biovolume into organic carbon (3, 10, 11, 21). The conversion factor involved may be calculated from values for the buoyant density, the dry weight/wet weight ratio, and the carbon weight/dry weight ratio of bacterial cells. These values are seldom determined for each case, use being made of literature data obtained with other organisms for other purposes. Luria (13) states that about 20% is a reasonable average estimate of bacterial dry weight. Similar values have later been assumed to be valid for bacteria in general (3, 10, 21). Percent dry matter of bacterial cells is usually determined by weighing a bacterial pellet before and after drying to constant weight. Available information on the water content in bacterial pellets, however, suggests that >60% of the total water in the pellets may be intercellular water (4, 17, 19). Thus, proper corrections for intercellular water in the pellets seem to be crucial when determining the dry weight/wet weight ratio to the bacterial cells. If a bacterial pellet contains 20% dry matter, and if 60% of the water in this pellet is assumed to be intercellular water, then the bacterial cells must have a dry-matter content of about 38%. This is nearly double the commonly assumed value of 20% dry matter for bacterial cells. To verify our hypothesis of a high dry-matter content of bacterial cells and to arrive at a reliable convertion factor for bacterial biomass, we have investigated the buoyant density, dry weight/wet weight ratio, and carbon weight/dry weight ratio of three bacterial species. MATERIALS AND METHODS Bacillus subtilis, Escherichia coli, and Pseudomonas putida were used in this study. B. subtilis and E. coli were grown in minimal medium (7) with glucose (8 mM), trace elements, and vitamins (9). P. putida was grown in aged seawater diluted to 70% with distilled water and enriched with glucose (33 mM), NaNO3 (12 mM), KH2PO4 (0.8 mM), trace elements, and vitamins (9). Both media were filtered through 0.2-p.m-pore size Nuclepore filters (Nuclepore Corp.) and autoclaved before use. All experiments were done with cells in an early stationary phase of growth. Cells *

Corresponding author. 755

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Approximately 20% dry-matter content appears to be an accepted standard value for bacterial cells. We have found that the dry-matter content of bacteria may be more than twice as high as generally assumed. The main reason for the low estimates seems to be that proper corrections for intercellular water have not been made when estimating the wet weight of the cells. Using three different bacterial strains, we detetmined a drymatter content of cells ranging from 31 to 57%, suggesting not only that the accepted standard value is much too low but also that it is far from standard. To convert bacterial biovolume into biomass (carbon content), we suggest that 0.22 g of C cm-3 should be used as a conversion factor.

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APPL. ENVIRON. MICROBIOL.

BRATBAK AND DUNDAS

TABLE 1. Calculated values for percent dry weight of bacterial cells based on experimental values for percent dry weight of bacterial pellets and percent intercellular water and experimentally determined values for percent carbon in dry cellsa % Dry wt of % Dry wt of %% Salt in % Carbon in Bacteria

Expt

bacterial pellet

Intercellular water

media

B. subtilis

1 2

10.7 ± 0.3 12.0 ± 0.2

91.2 ± 2.7 86.5 ± 1.7

1.35 ± 0.02

1 2 3

22.5 ± 0.1 23.2 ± 0.1 23.7 ± 0.04

38.8 ± 0.8 34.5 ± 1.9 32.0 ± 0.6

1.21 ± 0.02

E. coli

bacterial cells 54.9 + 7.7

47.2 ± 1.5

48.1 ± 3.1

50.4 ± 1.7

31.8 ± 0.3

47.8 47.9 ± 0.2 48.3 ± 0.003

31.3 ± 0.6 31.1 + 0.2

dry cells

2.90 ± 0.04 36.4 ± 0.8 47.8 ± 0.1 62.3 ± 1.3 18.9 ± 0.1 71.6 ± 3.7 44.7 ± 3.1 46.9 ± 0.7 20.1 ± 0.1 2.75 ± 0.03 45.7 ± 0.8 57.3 7.5 70.9 ± 8.9 3b 29.1 ± 1.1 55.1 ± 15 4C 83.0 ± 10 45.3 18.8 ± 0.3 The precision limits given are standard error of the mean (SEM). Five to eight replicas were used for determination of percent dry weight in bacterial pellets, four to seven were used for determination of percent salt in the media, and one to four were used for determination of percent carbon in the dry cells. SEM for per-

P. putida

1 2

cent intercellular water was calculated as the error propagated from the determination of radioactivity in the supernatants, two to four replicas for each radioactivity determination. SEM for percent dry weight of cells was calculated as the error propagated from the determination of percent dry weight of the pellets, percent intercellular water, and percent salt in the media. b Phosphate-starved cells; phosphate concentration in medium 1/10 of standard. c Nitrogen-starved cells; nitrogen concentration in medium 1/10 of standard.

to published estimates of the percent dry matter of bacterial cells themselves (1, 13). The percent intercellular water of our pellets ranged from 32 to 91%, which, taking into account our rather low centrifugation speed and the natural variability between bacterial types, is comparable to the range of 40 to 69% intercellular water reported by others (4, 17, 19). When the average percent dry matter of the bacterial cells is calculated from the percent dry weight of the pellets and the percent intercellular water, and salt content of the

media is corrected for, we find that B. subtilis cells contain 51.5%, E. coli cells contain 31.4%, and P. putida cells contain 48.4% dry matter (Table 1). Intracellular water comprises approximately 80% of the mass of most living cells (8). Cells with considerably lower water content do, however, function normally. Cysts of Artemia salina (brine shrimp) with as much as 60% dry matter exhibit "conventional metabolism" (5). Corresponding results have also been reported for mammalian cells (6). Caution is called for in comparing procaryotic and eucaryotic cells, but our estimate of 50% dry matter for bacterial cells does not seem incompatible with normal metabolism. From literature data on bacterial cell content of protein, DNA, RNA, lipid, carbohydrate, and ash (13, 20), and data on the specific gravity of these compounds (1, 2, 20), one may estimate the specific gravity of bacterial dry matter to be in the range of 1.35 to 1.6 g cm-3. Based on these estimates and on our experimentally determined values for percent dry weight of cells, we have calculated the theoretical buoyant densities of bacterial cells. These calculated values were, in general, slightly higher than the buoyant density values determined by direct experimentation (Table 2). Determination of cell buoyant density implies a definition of cell volume. The densities determined by direct experimentation depend on cell volumes as defined by the exclusion of the Percoll silica particles in the density gradient, whereas the theoretically calculated buoyant densities depend on cell volumes as defined by the exclusion of the inulin molecules in the pellet intercellular water. The differences between experimental and calculated buoyant densities shown in Table 2 may be explained by the differences in the defined cell volumes, if cellular adjuncts (flagella, fimbri-

pili, slime layers, electrical charges, hydration layer) exclude inulin molecules from a smaller volume than the volume from which the Percoll silica particles are excluded. To verify this hypothesis, we have compared the calculated buoyant densities of the bacterial pellets with the densities of the pellets as measured directly in Percoll (Table 2). For both calculated and directly measured buoyant densities, cell volumes will in this case be defined by inulin molecules and pellet volumes will be defined by Percoll silica particles. With all three bacterial strains the calculated cell buoyant densities are within the range of the directly measured values (Table 2), as would be the case if the experimentally determined values for the percent dry matter of the cells were ae,

correct. To convert bacterial biovolume into carbon biomass, a

conversion factor of 0. 121 g of C cm-3 has been used (11, 15, 21). This factor was estimated assuming a bacterial cell buoyant density of 1.1 g cm-3, a dry weight/wet weight ratio of 0.22, and a carbon weight/dry weight ratio of 0.5 (21). Using our experimental data, we may calculate a conversion factor ranging from 0.16 to 0.29 g of C cm-3. The range of our estimate is mainly due to differences in the percenit dry matter of the cells of the three bacterial strains we used. The dry-matter content of the cells of a given strain may also vary with the growth conditions (see P. putida, Table 1).

TABLE 2. Calculated and measured values for buoyant densities of bacterial cells and bacterial pelletsa Buoyant density (g cm-3) Calculated (range) Measured directly Bacteria Cells Pellet Cells Pellet B. subtilis 1.15-1.24 1.03-1.05 1.13 1.04 E. coli 1.09-1.13 1.07-1.11 1.09 1.07 P. putida 1.14-1.22 1.07-1.10 1.12 1.07 a The cells and pellets used for determination of buoyaht densities were prepared under conditions identical to those used for determination of percent dry weight of the pellets and percent intercellular water in the pellets. Average values for percent dry weight of cells and pellets and percent intercellular water in the pellets (from Table 1) were used for calculation of buoyarit densities.


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VOL. 48, 1984

Our work indicates that bacterial cells generally have a dry weight/wet weight ratio closer to 0.4 than to the currently assumed value of 0.22. We would thus suggest that, if a general factor for the conversion of bacterial biovolume into biomass is called for, a value of 0.22 g of C cm-3 will give more realistic estimates than a value of 0.121 g of C cm3. The implication is that many published values for bacterial biomass in natural ecosystems may be considerable underestimates. 1.

3. 4. 5.

6.

7. 8.

9.

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10. Ferguson, R. L., and P. Rublee. 1976. Contribution of bacteria to standing crop of coastal plankton. Limnol. Oceanogr. 22:141145. 11. Fuhrman, J. A., and F. Azam. 1980. Bacterioplankton secondary production estimates for coastal waters of British Columbia, Antarctica, and California. Appl. Environ. Microbiol. 39:10851095. 12. Kubitschek, H. E., W. W. Baldwin, and R. Graetzer. 1983. Buoyant density constancy during cell cycle of Escherichia coli J. Bacteriol. 155:1027-1032. 13. Luria, S. E. 1960. The bacterial protoplasm: composition and organization, p. 1-34. In I. C. Gunsalus and R. Y. Stainer (ed.), The bacteria, vol. 1. Academic Press, Inc., New York. 14. Matheson, A. T., G. D. Sprott, I. J. McDonald, and H. Tessier. 1976. Some properties of an unindentified halophile: growth characteristics, internal salt concentration, and morphology. Can. J. Microbiol. 22:780-786. 15. Riemann, B., J. Fuhrman, and F. Azam. 1982. Bacterial secondary production in freshwater measured by 3H-thymidine incorporation method. Microb. Ecol. 8:101-114. 16. Shindler, D. B., R. M. Wydro, and D. J. Kushner. 1977. Cellbound cations of the moderately halophilic bacterium Vibrio costicola. J. Bacteriol. 130:698-703. 17. Sprott, G. D., J. P. Drozdowski, E. L. Martin, and R. A. MacLeod. 1975. Kinetics of Na+-dependent amino acid transport using cells and membrane vesicles of a marine pseudomonad. Can. J. Microbiol. 21:43-50. 18. Takacs, F. P., T. I. Matula, and R. A. MacLeod. 1964. Nutrition and metabolism of marine bacteria. XIII. Intracellular concentrations of sodium and potassium ions in a marine pseudomonad. J. Bacteriol. 87:510-518. 19. Thompson, J., and R. A. MacLeod. 1971. Functions of Na+ and K+ in the active transport of a-aminoisobutyric acid in a marine pseudomonad. J. Biol. Chem. 264:4066-4074. 20. van Veen, J. A., and E. A. Paul. 1979. Conversion of biovolume measurements of soil organisms, grown under various moisture tensions, to biomass and their nutrient content. Appl. Environ. Microbiol. 37:686-692. 21. Watson, S. W., T. J. Novitsky, H. L. Quinby, and F. W. Valois. 1977. Determination of bacterial number and biomass in the marine environment. Appl. Environ. Microbiol. 33:940-946.


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LITERATURE CITED Bakken, L. R., and R. A. Olsen. 1983. Buoyant densities and dry-matter contents of microorganisms: conversion of a measured biovolume into biomass. Appl. Environ. Microbiol. 45:1188-1195. Birnie, G. D., D. Rickwood, and A. Hell. 1973. Buoyant densities and hydration of nucleic acids, proteins and nucleoprotein complexes in metrizamide. Biochim. Biophys. Acta 331:283294. Bowden, W. B. 1977. Comparison of two direct-count techniques for enumerating aquatic bacteria. Appl. Environ. Microbiol. 33:1229-1232. Buckmire, F. L. A., and R. A. MacLeod. 1970. Penetrability of a marine pseudomonad by inulin, sucrose, and glycerol and its relation to the mechanism of lysis. Can. J. Microbiol. 16:75-81. Clegg, J. S. 1979. Metabolism and the intracellular environment: the vicinal-water network model, p. 363-413. In W. DrostHansen and J. S. Clegg (ed.), Cell-associated water. Academic Press, Inc., New York. Clegg, J. S., and J. L. Mansell. 1982. Cellular and molecular consequences of reduced cell water content. Cryobiology 19:672-673. Clowes, R. C., and W. Hayes (ed.). 1968. Experiments in microbial genetics, p. 184-185. Blackwell Scientific Publications, Oxford. Cooke, R., and I. D. Kuntz. 1974. The properties of water in biological systems. Annu. Rev. Biophys. Bioeng. 3:95-126. Eppley, R. W., R. W. Holmes, and J. D. H. Strickland. 1967. Sinking rates of marine phytoplankton measured with a fluorometer. J. Exp. Mar. Biol. Ecol. 1:191-208.

BACTERIAL BIOMASS