A study of deep-sea natural microbial populations and barophilic pure cultures using a high-pressure chemostat

Abstract

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.

FIG. 1

Direct cell counts for a deep-sea NSW sample grown at 450 × 10⁵ Pa and 3°C at a dilution rate of 0.02 h⁻¹ (retention time, 50 h) before and after the introduction at 160 h (solid arrow) of 1.0 mg of yeast extract per liter to the seawater reservoir. Growth under these conditions was continued until 560 h, and then the flow was stopped and the pressurized population was held as a batch culture until 617 h (dotted arrow). This was followed by a return to a NSW flow with no yeast extract supplement at a dilution rate of 0.035 h⁻¹ (retention time, 29 h). (Insets) Exponential curve fits for cell numbers at actual and theoretical washout rates in NSW that was not supplemented.

FIG. 2

Direct cell counts for a deep-sea NSW sample grown at 450 × 10⁵ Pa and 3°C at a dilution rate of 0.028 h⁻¹ (retention time, 35 h) before and after the introduction at 160 h (solid arrow) of 10 mg of glucose per liter to the seawater reservoir. Growth under these conditions was continued until 427 h (dotted arrow), and then the chemostat was decompressed to 10⁵ Pa with the flow continuing at the same dilution rate. (Inset) Exponential curve fit for cell numbers at the actual and theoretical washout rates in NSW that was not supplemented.

FIG. 3

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barophilic isolate F1 grown at 3°C at different pressures in the presence of different yeast extract concentrations at different growth rates. (A) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.044 h⁻¹ (60% of the maximum growth rate). (B) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.066 h⁻¹ (90% of the maximum growth rate). (C) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.044 h⁻¹ (60% of the maximum growth rate). (D) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.066 h⁻¹ (90% of the maximum growth rate). Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.

FIG. 4

Steady state-direct counts (mean ± standard deviation) and viable cell counts for barophilic isolate F1 grown at 8°C at different pressures in the presence of different yeast extract concentrations at different growth rates. (A) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.044 h⁻¹ (60% of the maximum growth rate). (B) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.066 h⁻¹ (90% of the maximum growth rate). (C) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.044 h⁻¹ (60% of the maximum growth rate). (D) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.066 h⁻¹ (90% of the maximum growth rate). Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.

FIG. 5

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barophilic isolate 27AB grown at 3°C at different pressures in the presence of different yeast extract concentrations at different growth rates. (A) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.06 h⁻¹ (60% of the maximum growth rate). (B) Culture grown in the presence of 10 mg of yeast extract per liter at a growth rate of 0.09 h⁻¹ (90% of the maximum growth rate). (C) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.06 h⁻¹ (60% of the maximum growth rate). (D) Culture grown in the presence of 1 mg of yeast extract per liter at a growth rate of 0.09 h⁻¹ (90% of the maximum growth rate). Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.

FIG. 6

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barophilic isolate O-96-2 grown at 3°C in the presence of 10 mg of yeast extract per liter at different pressures and different growth rates. (A) Culture grown at a growth rate of 0.06 h⁻¹ (60% of the maximum growth rate). (B) Culture grown at a growth rate of 0.09 h⁻¹ (90% of the maximum growth rate). Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.

FIG. 7

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barophilic isolate O-96-12 grown at 3°C in the presence of 1 mg of C per liter (AGL medium) at a growth rate of 0.031 h⁻¹ (90% of the maximum growth rate) at different pressures. Stippled bars, direct counts; shaded bars, viable counts on 2216 marine agar; wave pattern bars, viable counts on 25× AGL medium. One bar is equal to 10⁵ Pa.

FIG. 8

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barotolerant isolate K-4 grown at 3°C in the presence of 1 mg of sodium glutamate per liter at different pressures and growth rates. (A) Culture grown at a growth rate of 0.017 h⁻¹ (60% of the maximum growth rate). (B) Culture grown at a growth rate of 0.028 h⁻¹ (90% of the maximum growth rate). Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.

FIG. 9

Steady-state direct counts (mean ± standard deviation) and viable cell counts for barotolerant isolate 82 grown at 3°C in the presence of 100 mg of sodium glutamate per liter at a growth rate of .037 h⁻¹ (60% of the maximum growth rate) at different pressures. Stippled bars, direct counts; shaded bars, viable counts. One bar is equal to 10⁵ Pa.