Inoculum size as a factor limiting success of inoculation for biodegradation

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Inoculum size as a factor limiting success of inoculation for biodegradation.

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Factors affecting the survival and growth of bacteria introduced into lake water

The populations of Pseudomonas sp. B4, Escherichia coli, Klebsiella pneumoniae, Micrococcus flavus, and Rhizobium leguminosarum biovar phaseoli declined rapidly in lake water. The initially rapid decline of the two pseudomonads and R. phaseoli was followed by a period of slow loss of viability, but viable cells of the other species were not found after 10 days. The rapid initial phase of decline was not a result of Bdellovibrio spp., bacteriophages, or toxins in the water since Bdellovibrio spp. were not present and passage of the lake water through filters that should not have removed bacteriophages or soluble toxins led to the elimination of the rapid phase of decline. The addition of 250 g of cycloheximide and 30 g of nystatin per ml eliminated viable protozoa form the lake water, and the population of Pseudomonas sp. B4 did not fall and the decline of E. coli and K. pneumoniae was delayed or slowed under these conditions. Pseudomonas sp. L2 proliferated rapidly in lake water amended with glucose, phosphate, and NH₄NO₃, but its numbers subsequently fell abruptly; however, in water amended with cycloheximide and nystatin, which killed indigenous protozoa, the population density was higher and the fall in numbers was delayed. Of the nutrients, the chief response was to carbon, but when glucose was added, phosphorus and nitrogen stimulated growth further. Removing other bacteria by filtering the lake water before inoculation with Pseudomonas sp. L2 suggested that competition reduced the extent of response of the pseudomonad to added nutrients. We suggest that the decline in lake water of bacteria that are resistant to starvation may be a result of protozoan grazing and that the extent of growth of introduced species may be limited by the supply of available carbon and sometimes of nitrogen and phosphorus, and by predation by indigenous protozoa.     

Bacterial Inhibitors in Lake Water

The populations of six bacterial genera fell rapidly after their addition to sterile lake water but not after their addition to buffer. The decline in numbers of two species that were studied further, Klebsiella pneumoniae and Micrococcus flavus, occurred even when the buffer was added to sterile lake water. The inhibition of K. pneumoniae by substances in lake water varied with the season of the year, and the rate and extent of decline of both species were different in sterile samples of different lakes. The extent of reduction in the density of K. pneumoniae was independent of initial population size and was diminished by the addition of 10 μg of glucose per ml of lake water. The toxin was removed from lake water by dialysis and by a cation-exchange resin but not by an anion-exchange resin, and it was destroyed by heating. The inhibition of K. pneumoniae was not evident in lake water buffered at a pH value above 8.0. We suggest that toxins may be important in determining the composition of the bacterial community of lakes.     

Alternative prey: a mechanism for elimination of bacterial species by protozoa.

Antibiotic-resistant strains of Salmonella typhimurium and Klebsiella pneumoniae died readily after their addition to raw sewage, but they grew in sterilized sewage. The decline was not a result of abiotic stresses, and because the bacteria were able to survive in large numbers for at least 15 days in solutions containing no organic nutrients, it was not a result of competition. Toxin production, bacteriophages, and Bdellovibrio sp. did not cause the disappearance of the two bacterial species. A decline was also evident if the sewage was first passed through a 3-micron (pore size) filter or treated with cycloheximide or cycloheximide plus nystatin, but protozoa developed under these conditions. Little or no decline occurred if the sewage was filtered and treated with the eucaryotic inhibitors before the addition of S. typhimurium or K. pneumoniae, and protozoa were not detected. S. typhimurium increased in abundance if cycloheximide, streptomycin, and erythromycin or large amounts of glucose were added to sewage. Tetrahymena thermophilus did not significantly reduce the population of S. typhimurium in buffer when the density of the bacterium was about 10(4)/ml. However, when more than 10(8) Enterobacter agglomerans cells per ml were added to the buffer, T. thermophilus reduced the abundance of E. agglomerans and S. typhimurium to 10(6) and 10/ml, respectively. The density of S. typhimurium was further decreased by a second increment of E. agglomerans cells. The disappearance of S. typhimurium and K. pneumoniae from sewage thus is the result of predation by protozoa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alternative prey: a mechanism for elimination of bacterial species by protozoa

Antibiotic-resistant strains of Salmonella typhimurium and Klebsiella pneumoniae died readily after their addition to raw sewage, but they grew in sterilized sewage. The decline was not a result of abiotic stresses, and because the bacteria were able to survive in large numbers for at least 15 days in solutions containing no organic nutrients, it was not a result of competition. Toxin production, bacteriophages, and Bdellovibrio sp. did not cause the disappearance of the two bacterial species. A decline was also evident if the sewage was first passed through a 3-micron (pore size) filter or treated with cycloheximide or cycloheximide plus nystatin, but protozoa developed under these conditions. Little or no decline occurred if the sewage was filtered and treated with the eucaryotic inhibitors before the addition of S. typhimurium or K. pneumoniae, and protozoa were not detected. S. typhimurium increased in abundance if cycloheximide, streptomycin, and erythromycin or large amounts of glucose were added to sewage. Tetrahymena thermophilus did not significantly reduce the population of S. typhimurium in buffer when the density of the bacterium was about 10(4)/ml. However, when more than 10(8) Enterobacter agglomerans cells per ml were added to the buffer, T. thermophilus reduced the abundance of E. agglomerans and S. typhimurium to 10(6) and 10/ml, respectively. The density of S. typhimurium was further decreased by a second increment of E. agglomerans cells. The disappearance of S. typhimurium and K. pneumoniae from sewage thus is the result of predation by protozoa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either Cupriavidus necator, a potent bacterial predator, or one of its prey species, Micrococcus luteus. C. necator also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of Arthrobacter globiformis or Bacillus thuringiensis. Therefore, the autochthonous state of Arthrobacter species in soil and the survival of B. thuringiensis were possibly enhanced by the resistance of these species to protozoa. The addition of Bacillus mycoides and Escherichia coli cells caused specific responses by soil protozoa. The protozoa that responded to E. coli did not respond to B. mycoides or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as E. coli, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.     

Regulation of Predation by Prey Density: the Protozoan-Rhizobium Relationship

Tetramitus rostratus and strains of Hartmanella, Naegleria, and Vahlkampfia consumed large numbers of Rhizobium meliloti cells in a salt solution, but protozoan multiplication and the bacterial decline stopped when the prey density fell to about 10-6 to 10-7 cells/ml. At higher prey densities, the maximum numbers of Hartmanella sp. and Naegleria sp. were proportional to the quantity of R. meliloti initially provided to the amoebas. When supplemental rhizobia were supplied to Hartmanella sp. or Naegleria sp. after their active feeding had terminated, presumably because the remaining 10-6 or 10-7 bacteria/ml could not be captured, replication of the protozoa was initiated. The rate of elimination of rhizobia present in large populations was proportional to the initial abundance of Naegleria sp., but the final numbers of amoebas and surviving R. meliloti cells were independent of initial numbers of predators. The surviving bacteria were not intrinsically resistant to attack because 98% of the survivors, when concentrated, were consumed. It is suggested that large populations of bacteria in nature may be reduced in size by predatory protozoa, but many of the prey cells will not be eliminated.     

Role of sublethal injury in decline of bacterial populations in lake water.

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Role of sublethal injury in decline of bacterial populations in lake water

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Role of resistance to starvation in bacterial survival in sewage and lake water

A study was conducted to determine the significance of starvation resistance to the ability of a species to survive in sewage and lake water. Tests were conducted for periods of up to 14 days. Rhizobium meliloti and one fluorescent and one nonfluorescent strain of Pseudomonas were resistant to starvation because their population sizes did not fall appreciably in buffer and sterile lake water, and the first two maintained high numbers after being added to sterile sewage. Cell densities of these bacterial species dropped slowly in nonsterile sewage, and more cells of these three organisms than of the other test organisms remained in nonsterile lake water. Rhizobium leguminosarum was moderately resistant to starvation because its numbers fell slowly in buffer and sterile lake water and did not change appreciably in sterile sewage. The abundance of Micrococcus flavus added to buffer and sterile lake water did not change, but the density of M. flavus declined in nonsterile lake water. The abundance of R. leguminosarum fell in nonsterile lake water and nonsterile sewage. Streptococcus faecalis, Staphylococcus aureus, an asporogenous strain of Bacillus subtilis, and Streptococcus sp. were susceptible to starvation because their populations were markedly reduced in buffer. Populations of the last three species declined rapidly in nonsterile and sterile samples of lake water and sewage. S. faecalis declined rapidly when added to nonsterile lake water and sewage and sterile lake water but not when added to sterile sewage, the persistence in the last instance probably being associated with the availability of organic nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of resistance to starvation in bacterial survival in sewage and lake water.

A study was conducted to determine the significance of starvation resistance to the ability of a species to survive in sewage and lake water. Tests were conducted for periods of up to 14 days. Rhizobium meliloti and one fluorescent and one nonfluorescent strain of Pseudomonas were resistant to starvation because their population sizes did not fall appreciably in buffer and sterile lake water, and the first two maintained high numbers after being added to sterile sewage. Cell densities of these bacterial species dropped slowly in nonsterile sewage, and more cells of these three organisms than of the other test organisms remained in nonsterile lake water. Rhizobium leguminosarum was moderately resistant to starvation because its numbers fell slowly in buffer and sterile lake water and did not change appreciably in sterile sewage. The abundance of Micrococcus flavus added to buffer and sterile lake water did not change, but the density of M. flavus declined in nonsterile lake water. The abundance of R. leguminosarum fell in nonsterile lake water and nonsterile sewage. Streptococcus faecalis, Staphylococcus aureus, an asporogenous strain of Bacillus subtilis, and Streptococcus sp. were susceptible to starvation because their populations were markedly reduced in buffer. Populations of the last three species declined rapidly in nonsterile and sterile samples of lake water and sewage. S. faecalis declined rapidly when added to nonsterile lake water and sewage and sterile lake water but not when added to sterile sewage, the persistence in the last instance probably being associated with the availability of organic nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of a role for lytic microorganisms in the decline of bacteria andSaccharomyces introduced into soil

The populations ofKlebsieila pneumoniae, Escherichia coli, Enterobacter aerogenes, andPseudomonas sp. fell following their addition to soil, but species lysing these gram-negative bacteria were not detected. The numbers ofStaphylococcus aureus andMicrococcus flavus fell by more than four orders of magnitude and ofSaccharomyces cerevisiae by more than two orders after their addition to soil. Organisms lysing these gram-positive bacteria were present in soil, but their numbers did not increase as a result of the additions. Lytic activity againstS. aureus was detected in soil filtrates, but this activity was not enhanced by inoculation of soil with the bacterium. Addition of cycloheximide to soil suspensions delayed the fall in abundance ofM. flavus but did not suppress the lytic populations. We conclude that lysis is not responsible for the decline of bacteria orS. cerevisiae added to soil.     

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.     

Role of Predatory Bacteria in the Termination of a Cyanobacterial Bloom

Changes in cyanobacterial abundance and in the occurrence of bacteria of bacteria capable of lysing cyanobacteria were monitored over a period of 6 months (May to October 1998) in eutrophic Brome Lake (Quebec, Canada), in which dense cyanobacterial blooms recur regularly. By screening lake water, we isolated two strains of lytic bacteria, from the family Cytophagaceae. When tested on 12 cyanobacteria and 6 heterotrophic bacteria, strain 1 lysed only Anabaena flos-aquae and strain 2 lysed only Synechococcus cedorum, Synechococcus leopoliensis, Synechococcus elongatus, and Anacystic nidulans: both liquid and agar-grown cultures of these cyanobacteria were lysed. The number of plaque forming units of bacteria increased dramatically during the decline of the bloom. The results are consistent with an important role for these host-specific lytic bacteria in control and elimination of cyanobacterial blooms in this lake.     

Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters.

The removal of Escherichia coli from estuarine water was investigated. The survival of E. coli was dependent on the presence of protozoan predators and not on the presence of lytic bacteria. When indigenous protozoa were removed from estuarine water by filtration, the destruction of coliform populations was negligible. In studies designed to prevent the growth of indigenous bacterial populations without affecting protozoan populations, coliform destruction was significant.     

A Note on the Flora and Fauna in the Rumen of Steers Fed a Feedlot Bloat-provoking Ration and the Effect of Penicillin

A study was made of the predominant culturable bacteria and ciliate protozoa present in the rumen of two steers that were regularly bloating on a pelleted ratio containing 22% alfalfa meal, 16% soybean oil meal, 61% barley, and 1% common salt. The ruminal microorganisms in the two animals differed as indicated by a high total culture count of bacteria, an almost complete absence of ciliate protozoa, a low pH, and a difference in the proportions of presumptively identified predominant bacterial groups in one animal (steer 26) as compared with the other (steer 32). The first exposure of the animals to procaine penicillin (75 or 150 mg per day on 2 successive days) resulted in an abnormal ruminal flora 31 hr after the first treatment as indicated by drastic drops in total and cellulolytic bacterial counts and a change in the proportions of predominant bacterial groups. The animals refused feed for 32 to 48 hr after the first treatment. After feed consumption resumed, further treatment with 75 mg penicillin on 4 successive days did not appear to greatly alter the flora and did not result in feed refusal in animal 32. In animal 26, amounts of penicillin progressing from 50 to 200 mg per day did not result in feed refusals and observations on rumen ingesta samples during this period indicated a decrease in total bacterial count, a great increase in numbers of ciliate protozoa, a higher pH, and a change in the proportions of predominant bacterial groups so that the ruminal picture was much more similar to that of animal 32 than to its own during the pre-penicillin period. Bloat was not relieved except during the period of feed refusal.

The results indicate that the ruminal flora rapidly adapts to penicillin and that bloat of the feedlot type can occur in animals with widely differing numbers and kinds of bacteria and protozoa. Feedlot bloat does not appear to be correlated with the occurrence or numbers of any of the individual predominant groups of bacteria cultured.

     

Protozoan grazing of bacteria in soil—impact and importance

Interactions between bacteria and protozoa in soil were studied over 2-week periods in the field and in a pot experiment. Under natural conditions the total biological activity was temporarily synchronized by a large rainfall, and in the laboratory by the addition of water to dried-out soil, with or without plants. In the field, peaks in numbers and biomass of bacteria appeared after the rain, and a peak of naked amoebae quickly followed. Of the three investigated groups—flagellates, ciliates, and amoebae—only populations of the latter were large enough and fluctuated in a way that indicated a role as bacterial regulators. The bacterial increase was transient, and the amoebae alone were calculated to be able to cause 60% of the bacterial decrease. The same development of bacteria and protozoa was observed in the pot experiment: in the presence of roots, amoebic numbers increased 20 times and became 5 times higher than in the unplanted soil. In the planted pots, the amoebic increase was large enough to cause the whole bacterial decrease observed; but in the unplanted soil, consumption by the amoebae caused only one-third of the bacterial decrease.     

Growth Response of Soda Lake Bacterial Communities to Simulated Rainfall

Moderately saline soda lakes harbor extremely abundant and fast growing bacterial communities. An interesting phenomenon of an explosive bacterial growth in shallow soda lakes in Eastern Austria after dilution with rainwater, concomitantly with a significant decrease in temperature was observed in a former study. In the present study, we tried to identify the factors being responsible for this enhanced bacterial growth in laboratory batch cultures. Three experiments were performed with water taken from two different lakes at different seasons. Natural soda lake water was diluted with distilled water, artificial lake water, sterile filtered soda lake water, and grazer-free water to test (1) for the influence of compatible solutes released to the environment and reduced salt stress after osmotic down-shock, (2) for the influence of nutrients, which may be washed in from the dry areas of the lake bottom after rainfall and (3) for the decrease of grazing pressure due to dilution. The potential influence of (4) viruses was indirectly deduced. The response of the bacterial community to the manipulations was measured by changes in bacterial numbers, the incorporation of ³H-leucine and the concomitant determination of the amount of ³H-leucine uptaking bacteria by microautoradiography. The influence of the environmental factors enhancing bacterial growth after a simulated rainfall event showed variations between the lakes and over the seasons. The addition of nutrients was, in all experiments, the main factor triggering bacterial growth. The decrease in grazing pressure and viral lysis after dilution was of significant importance in two of three experiments. In the experiment with the highest salinity, we could show that either compatible solutes released after osmotic down-shock and used as a source of nutrients for the soda lake bacterial populations or reduced salt stress were most probably responsible for the observed marked enhancement of bacterial growth.     

Bacterial Growth in Mixed Cultures on Dissolved Organic Carbon from Humic and Clear Waters

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 μm of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.