The road to Yellowstone--and beyond.

This memoir describes the professional life and times of Thomas D. Brock, with an emphasis on those aspects of his career relating to research in microbial ecology, and how this work led to field research in Yellowstone. The first discovery of extremely thermophilic bacteria is described, followed by a discussion of some of the consequences of this discovery for biotechnology and microbiology. Also covered briefly in this memoir are Brock's activities in textbook writing, publishing, computers, and the history of science.

Life at high temperatures.

Water environments with temperatures up to and above boiling are commonly found in association with geothermal activity. At temperatures above 60 degrees C, only bacteria are found. Bacteria with temperature optima over the range 65 degrees to 105 degrees C have been obtained in pure culture and are the object of many research projects. The upper temperature limit for life in liquid water has not yet been defined, but is likely to be somewhere between 110 degrees and 200 degrees C, since amino acids and nucleotides are destroyed at temperatures over 200 degrees C. Because bacteria capable of growth at high temperatures are found in many phylogenetic groups, it is likely that the ability to grow at high temperature had a polyphyletic origin. The macromolecules of these organisms are inherently more stable to heat than those of conventional organisms, but only small changes in sequence can lead to increases in thermostability. Because of their unique properties, thermophilic organisms and their enzymes have many potential biotechnological uses, and extensive research on industrial applications is under way.

Significance of algal excretory products for growth of epilimnetic bacteria.

Light-stimulated uptake of CO(2) and differential filtration through Nucleopore filters were used to estimate the significance of phytoplankton excretion as a source of bacterial carbon in water samples collected at different seasons of the year in Lake Mendota, Wis. On an annual basis, about 14% of the estimated bacterial production was accounted for by algal excretion, although at certain times of year the fraction of bacterial carbon derived from algal excretion was considerably higher. About 20% of the annual primary production was estimated to pass through the bacterial component.

How sensitive is the light microscope for observations on microorganisms in natural habitats?

Theoretical calculations based on the depth of field of standard microscope objectives and the visual acuity of normal observors show that direct microscopy of natural samples is rarely able to reveal the presence of small microorganisms at the densities found in natural systems. Over-estimation of the importance of bacterial aggregates is also likely from an uncritical use of light microscopy.

Assessing biomass and production of bacteria in eutrophic lake mendota, wisconsin.

Estimates were made of the biomass and production of heterotrophic bacteria in the epilimnion of Lake Mendota, Wis. Cell counts were done with epifluorescence microscopy and varied from 3 x 10 bacteria per ml in winter to 3 x 10 bacteria per ml in summer. Cell volumes were measured in scanning electron micrographs. The average cell volume was 0.159 mum. Annual variations and depth distribution were studied. Production was estimated from the frequency of dividing cells and from dark radioactive sulfate uptake. Annual productivity and daily average productivity were very close with both methods: 107 to 205 g of C per m per year for sulfate and 89 to 117 g of C per m per year for frequency of dividing cells. Zooplankton feeding removed 2 to 10% of the bacterial net production annually. When compared with biomass changes and losses due to zooplankton feeding, production values were very high. Therefore, it was suggested that other loss factors have to be more important than zooplankton feeding in controlling the bacterial population. Bacterial heterotrophic production was about 50% of gross primary production.

Dynamics of bacterial sulfate reduction in a eutrophic lake.

Bacterial sulfate reduction in the surface sediment and the water column of Lake Mendota, Madison, Wis., was studied by using radioactive sulfate (SO(4)). High rates of sulfate reduction were observed at the sediment surface, where the sulfate pool (0.2 mM SO(4)) had a turnover time of 10 to 24 h. Daily sulfate reduction rates in Lake Mendota sediment varied from 50 to 600 nmol of SO(4) cm, depending on temperature and sampling date. Rates of sulfate reduction in the water column were 10 times lower than that for the surface sediment and, on an areal basis, accounted for less than 18% of the total sulfate reduction in the hypolimnion during summer stratification. Rates of bacterial sulfate reduction in the sediment were not sulfate limited at sulfate concentrations greater than 0.1 mM in short-term experiments. Although sulfate reduction seemed to be sulfate limited below 0.1 mM, Michaelis-Menten kinetics were not observed. The optimum temperature (36 to 37 degrees C) for sulfate reduction in the sediment was considerably higher than in situ temperatures (1 to 13 degrees C). The response of sulfate reduction to the addition of various electron donors metabolized by sulfate-reducing bacteria in pure culture was investigated. The degree of stimulation was in this order: H(2) > n-butanol > n-propanol > ethanol > glucose. Acetate and lactate caused no stimulation.

Characterization of an acetate-decarboxylating, non-hydrogen-oxidizing methane bacterium.

A methanogenic bacterium, commonly seen in digested sludge and referred to as the "fat rod" or Methanobacterium soehngenii, has been enriched to a monoculture and is characterized. Cells are gramnegative, non-motile and appear as straight rods with flat ends. They form filaments which can grow to great lengths. The structure of the outer cell envelop is similar to Methanospirillum hungatii. The organism grows on a mineral salt medium with acetate as the only organic component. Acetate is the energy source, and methane is formed exclusively from the methyl group. Acetate and carbon dioxide act as sole carbon source and are assimilated in a molar ratio of about 1.9:1. The reducing equivalents necessary to build biomass from these two precursors are obtained from the total oxidation of some acetate. Hydrogen is not used for methane formation and is not needed for growth. Formate is cleaved into hydrogen and carbon dioxide. Coenzyme M was found to be present at levels of 0.35 nmol per mg of dry cells and F420 amounted to 0.55 microgram per mg protein. The mean generation time was 9 days at 33 degrees C.

Anaerobic methane oxidation: occurrence and ecology.

Anoxic sediments and digested sewage sludge anaerobically oxidized methane to carbon dioxide while producing methane. This strictly anaerobic process showed a temperature optimum between 25 and 37 degrees C, indicating an active microbial participation in this reaction. Methane oxidation in these anaerobic habitats was inhibited by oxygen. The rate of the oxidation followed the rate of methane production. The observed anoxic methane oxidation in Lake Mendota and digested sewage sludge was more sensitive to 2-bromoethanesulfonic acid than the simultaneous methane formation. Sulfate diminished methane formation as well as methane oxidation. However, in the presence of iron and sulfate the ratio of methane oxidized to methane formed increased markedly. Manganese dioxide and higher partial pressures of methane also stimulated the oxidation. The rate of methane oxidation in untreated samples was approximately 2% of the CH(4) production rate in Lake Mendota sediments and 8% of that in digested sludge. This percentage could be increased up to 90% in sludge in the presence of 10 mM ferrous sulfate and at a partial pressure of methane of 20 atm (2,027 kPa).

Lytic organisms and photooxidative effects: influence on blue-green algae (cyanobacteria) in lake mendota, wisconsin.

The effects of exposure to high light intensities on blue-green algal (cyanobacterial) populations were examined in Lake Mendota, Wis. The algal populations were shown to be susceptible to inhibition of photosynthetic activity and pigment bleaching as a result of exposure. These effects generally influence only a small percentage of the lake population and thus are probably not important in causing major declines in chlorophyll a. Lytic organisms were shown to increase in numbers in the lake in response to the seasonal development of blue-green algae, reaching values of greater than 1,000 plaque-forming units per ml in midsummer. Both bacteria and protozoa were observed in plaque zones, but it could not be determined whether these lytic organisms had a major role in algal biomass declines.

Decomposition of blue-green algal (cyanobacterial) blooms in lake mendota, wisconsin.

Decomposition of natural populations of Lake Mendota phytoplankton dominated by blue-green algae (cyanobacteria) was monitored by using oxygen uptake and disappearance of chlorophyll, algal volume (fluorescence microscopy), particulate protein, particulate organic carbon, and photosynthetic ability (CO(2) up-take). In some experiments, decomposition of C-labeled axenic cultures of Anabaena sp. was also measured. In addition to decomposition, mineralization of inorganic nitrogen and phosphorus were followed in some experiments. Decomposition could be described as a first-order process, and the rate of decomposition was similar to that found by others using pure cultures of eucaryotic algae. Nitrogen and phosphorus never limited the decomposition process, even when the lake water was severely limited in soluble forms of these nutrients. This suggests that the bacteria responsible for decomposition can obtain all of their key nutrients for growth from the blue-green algal cells. Filtration of lake water through plankton netting that removed up to 90% of the algal biomass usually did not cause a similar decrease in oxygen demand, suggesting that most of the particulate organic matter used for respiration of the decomposing bacteria was in a small-particle fraction. Short-term oxygen demand correlated well with the particulate chlorophyll concentration of the sample, and a relationship was derived that could be used to predict community respiration of the lake from chlorophyll concentration. Kinetic analysis showed that not all analyzed components disappeared at the same rate during the decomposition process. The relative rates of decrease of the measured parameters were as follows: photosynthetic ability > algal volume > particulate chlorophyll > particulate protein. Decomposition of C-labeled Anabaena occurred at similar rates with aerobic epilimnetic water and with anaerobic sediment, but was considerably slower with anaerobic hypolimnetic water. Of the various genera present in the lake, Aphanizomenon and Anabaena were more sensitive to decomposition than was Microcystis. In addition to providing a general picture of the decomposition process, the present work relates to other work on sedimentation to provide a detailed picture of the fate of blue-green algal biomass in a eutrophic lake ecosystem.

Measuring radioactive methane with the liquid scintillation counter.

Although a gas proportional counter is the most convenient method of measuring the radioactivity of fixed gases such as methane, it cannot be used when high nonradioactive concentrations of methane are present in the gas phase, due to quenching. If only methane and carbon dioxide are present in radioactive form in the gas phase, a liquid scintillation method for measuring these substances can be used. The procedure is described in detail, and the solubility of methane in liquid scintillation cocktails is determined.

Methane formation and methane oxidation by methanogenic bacteria.

Methanogenic bacteria were found to form and oxidize methane at the same time. As compared to the quantity of methane formed, the amount of methane simultaneously oxidized varied between 0.3 and 0.001%, depending on the strain used. All the nine tested strains of methane producers (Methanobacterium ruminantium, Methanobacterium strain M.o.H., M. formicicum, M. thermoautotrophicum, M. arbophilicum, Methanobacterium strain AZ, Methanosarcina barkeri, Methanospirillum hungatii, and the "acetate organism") reoxidized methane to carbon dioxide. In addition, they assimilated a small part of the methane supplied into cell material. Methanol and acetate also occurred as oxidation products in M. barkeri cultures. Acetate was also formed by the "acetate organism," a methane bacterium unable to use methanogenic substrates other than acetate. Methane was the precursor of the methyl group of the acetate synthesized in the course of methane oxidation. Methane formation and its oxidation were inhibited equally by 2-bromoethanesulfonic acid. Short-term labeling experiments with M. thermoautotrophicum and M. hungatii clearly suggest that the pathway of methane oxidation is not identical with a simple back reaction of the methane formation process.

Effect of temperature on blue-green algae (cyanobacteria) in lake mendota.

The temperature optimum for photosynthesis of natural populations of blue-green algae (cyanobacteria) from Lake Mendota was determined during the period of June to November 1976. In the spring, when temperatures ranged from 0 to 20 degrees C, there were insignificant amounts of blue-green algae in the lake (less than 1% of the biomass). During the summer and fall, when the dominant phytoplankton was blue-green algae, the optimum temperature for photosynthesis was usually between 20 and 30 degrees C, whereas the environmental temperatures during this period ranged from 24 degrees C in August to 12 degrees C in November. In general, the optimum temperature for photosynthesis was higher than the environmental temperature. More importantly, significant photosynthesis also occurred at low temperature in these samples, which suggests that the low temperature alone is not responsible for the absence of blue-green algae in Lake Mendota during the spring. Temperature optima for growth and photosynthesis of laboratory cultures of the three dominant blue-green algae in Lake Mendota were determined. The responses of the two parameters to changes in temperature were similar; thus, photosynthesis appears to be a valid index of growth. However, there was little photosynthesis by laboratory cultures at low temperatures, in contrast to the natural samples. Evidence for an interaction between temperature and low light intensities in their effect on photosynthesis of natural samples is presented.

Dimethyl sulphoxide reduction by micro-organisms.

Dimethyl sulphoxide (DMSO) was reduced to dimethyl sulphide by a wide variety of micro-organism, including prokaryotes and eukaryotes, aerobes and anaerobes. Dimethyl sulphone was not reduced by any of the organisms tested. Cell-free extracts of Escherichia coli reduced DMSO using reduced pyridine nucleotides as electron donors. Activity was greater in anaerobically grown cells than in those grown aerobically. Two other sulphoxides, methionine sulphoxide and tetramethylene sulphoxide, substantially inhibited DMSO reduction by extracts. Mutants of E. coli, which were unable to reduce biotin sulphoxide to biotin, were tested for their ability to reduce DMSO in whole cells and extracts. These mutants were in four different gene loci, bisA to bisD. DMSO reductase activity of the mutants was generally less than that of the wild-type strain, and activity depended upon the gene locus involved, the growth medium and the growth conditions. Only the bisA mutant had very low activity under all conditions. All of the bis mutants were able to grow using methionine sulphoxide as a sulphur source, indicating that biotin sulphoxide and methionine sulphoxide are reduced by different enzyme systems. DMSO may be reduced by both of these enzyme systems.

Changes in photosynthetic rate and pigment content of blue-green algae in Lake Mendota.

Blue-green algal blooms were present in Lake Mendota (Dane County, Wis.) from June to November 1976. Concentrations of total algal biomass and of particular algal species were monitored and compared with the pigment contents (chlorophyll a and phycocyanin) and photosynthetic rate of the algal populations. The specific photosynthetic rate (micrograms of C fixed per microgram of chlorophyll a per hour) was a good measure of the physiological state of the algae because this quantity increased just before each population increase and decreased before algal densities diminished. Since the quantity of light in the epilimnion which was available for photosynthesis by algal cells decreased in summer when the high algal densities attenuated incoming radiation, we investigated the possibility that the organisms would utilize lower light intensities more efficiently by increasing their pigment contents. Although some evidence of enhanced utilization of low light levels was found in the period from July to October, this result was not due to increasing chlorophyll and phycocyanin contents. There was a decrease in the phycocyanin content of the algae during this period, perhaps related to the availability of inorganic nitrogen.

Methane, carbon dioxide, and hydrogen sulfide production from the terminal methiol group of methionine by anaerobic lake sediments.

A significant portion of the sulfide in lake sediments may be derived from sulfur-containing amino acids. Methionine degradation in Lake Mendota (Wisconsin) sediments was studied with gas chromatographic and radiotracer techniques. Temperature optimum and inhibitor studies showed that this process was biological. Methane thiol and dimethyl sulfide were produced in sediments when 1-mumol/ml unlabeled methionine was added. When chloroform (an inhibitor of one-carbon metabolism) was added to the sediments, methane thiol, carbon disulfide, and n-propane thiol were produced, even when no methionine was added. When S-labeled methionine was added to the sediments in tracer quantities (1.75 nmol/ml), labeled hydrogen sulfide was produced, and a roughly equal amount of label was incorporated into insoluble material. Methane and carbon dioxide were produced from [methyl-C]methionine. Evidence is given favoring methane thiol as an intermediate in the formation of methane, carbon dioxide, and hydrogen sulfide from the terminal methiol group of methionine. Methionine may be an important source of sulfide in lake sediments.