Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

Distribution of Heterotrophic Bacteria in Lake Shira

A study of the horizontal and vertical distribution of heterotrophic bacteria in brackish Lake Shira in summer periods showed that mesophilic bacteria dominated in all areas of the lake, whereas psychrotolerant bacteria dominated in the metalimnion and hypolimnion of its central part. Nonhalophilic bacteria were mostly mesophilic and dominated in coastal waters. Most psychrotolerant bacteria were able to grow in the presence of 5–10% NaCl. Heterotrophic bacteria isolated in different regions of the lake were identified to a generic level. The isolates were classified into autochthonous and allochthonous microorganisms on the bases of their distribution pattern in the lake water, halotolerance, and ability to grow at low temperatures.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

Inactivation of Bacteria in Seawater by Low-Amperage Electric Current

Seawater used in mariculture has been suspected of being a potential source of infection. In this study, the lethal effects of low-amperage electric treatment on microorganisms were examined in natural seawater and in seawater inoculated with Vibrio parahaemolyticus. In both cases, bacteria including V. parahaemolyticus in seawater were completely eliminated in 100 ms by a 0.5-A, 12-V direct current. Electron microscopic investigation of the electrically treated bacteria revealed substantial structural damage at the cellular level. In conclusion, our results indicate that low-amperage electric treatment is effective for rapid inactivation of microorganisms in seawater.     

Degradation of Putrescine and Cadaverine in Seawater Cultures by Marine Bacteria

Marine bacteria removed two diamines, putrescine and cadaverine, from coastal seawater supplemented only with these compounds. Batch cultures of natural bacterial communities were grown in filtered seawater (0.05 μm) supplemented with 500 μg of putrescine or cadaverine per liter. Increases in bacterial cell number were counted with an epifluorescence microscope after acridine orange staining. Removal of diamines from seawater was monitored by high-performance liquid chromatography. Diamines were removed from the seawater cultures within 48 h with no corresponding increase in bacterial yield, growth rate, or viability relative to control (unsupplemented) cultures. Shipboard experiments with open-ocean deep water (1,500 m) showed similar, if slower, removal of putrescine from seawater. Unlike uptake experiments with amino acids, labeled putrescine experiments indicated that most putrescine carbon is mineralized to CO₂ rather than assimilated by the bacteria. After growth in unsupplemented control cultures, the bacteria showed a significant potential to mineralize putrescine, indicating a general degradation potential for this compound by marine bacteria even if the compound was not present during growth. Indicators of metabolic activity such as glucose and glutamic acid uptake and mineralization were not affected by the presence of putrescine. This shows that at the concentrations added, the diamines are not toxic, and therefore detoxification was not the reason for degradation of the diamines by the bacteria.     

Metal-Resistant Heterotrophic Bacteria in Coastal Waters of Primorye

Colony-forming heterotrophic bacteria were isolated from several locations in coastal waters of Primorye that differ in the extent and character of heavy metal pollution. Statistically significant differences in the minimum inhibitory concentrations of some metals were found for bacteria isolated from the environments with different levels of pollution. Bacterial strains that were highly tolerant of several heavy metals also showed resistance to a wide spectrum of antibiotics, and some strains bore plasmids of varying numbers and sizes. Bacteria of the family Enterobacteriaceae and the genus Pseudomonas were the dominant metal-resistant bacterial forms in the polluted coastal wasters of Primorye. No strict correlation was found between metal resistance and taxonomic identity of bacteria.Original Russian Text Copyright © 2005 by Biologiya Morya, Bezverbnaya, Buzoleva, Khristoforova.     

Macroalgal Morphogenesis Induced by Waterborne Compounds and Bacteria in Coastal Seawater

Axenic gametes of the marine green macroalga Ulva mutabilis Føyn (Ria Formosa, locus typicus) exhibit abnormal development into slow-growing callus-like colonies with aberrant cell walls. Under laboratory conditions, it was previously demonstrated that all defects in growth and thallus development can be completely abolished when axenic gametes are inoculated with a combination of two specific bacterial strains originally identified as Roseobacter sp. strain MS2 and Cytophaga sp. strain MS6. These bacteria release diffusible morphogenetic compounds (= morphogens), which act similar to cytokinin and auxin. To investigate the ecological relevance of the waterborne bacterial morphogens, seawater samples were collected in the Ria Formosa lagoon (Algarve, Southern Portugal) at 20 sampling sites and tidal pools to assess their morphogenetic effects on the axenic gametes of U. mutabilis. Specifically the survey revealed that sterile-filtered seawater samples can completely recover growth and morphogenesis of U. mutabilis under axenic conditions. Morphogenetic activities of free-living and epiphytic bacteria isolated from the locally very abundant Ulva species (i.e., U. rigida) were screened using a multiwell-based testing system. The most represented genera isolated from U. rigida were Alteromonas, Pseudoalteromonas and Sulfitobacter followed by Psychrobacter and Polaribacter. Several naturally occurring bacterial species could emulate MS2 activity (= induction of cell divisions) regardless of taxonomic affiliation, whereas the MS6 activity (= induction of cell differentiation and cell wall formation) was species-specific and is probably a feature of difficult-to-culture bacteria. Interestingly, isolated bacteroidetes such as Algoriphagus sp. and Polaribacter sp. could individually trigger complete Ulva morphogenesis and thus provide a novel mode of action for bacterial-induced algal development. This study also highlights that the accumulation of algal growth factors in a shallow water body separated from the open ocean by barrier islands might have strong implications to, for example, the wide usage of natural coastal seawater in algal (land based) aquacultures of Ulva.     

Survival of Lactic Acid Bacteria in Seawater: A Factorial Study

A feasibility study of lactic bacteria as potential probiotics in larval cultures of marine fish was performed by investigating the survival of five strains of lactic bacteria in seawater by readily standardized procedures at different temperatures and salinities. These conditions were chosen in such a way that their combinations define a complete first-order factorial design. Depending on the strain and the ambient conditions, the survival adhered to first-order kinetics in some cases, and to the Gompertz equation in others. The half lives (t _0.5) calculated from these models were subsequently introduced as responses to the factorial designs, estimating the coefficients of empirical equations that describe the group effect of temperature and salinity on t _0.5. Simply additive effects were found in two cases, a negative first-order interaction in another case, while another two required second-order models.     

Changes in Bacterial Numbers and Leucine Assimilation during Estimations of Microbial Respiratory Rates in Seawater by the Precision Winkler Method

During incubation of seawater in bottles, the decrease in dissolved oxygen is often nonlinear over time scales frequently used to measure respiration. Numbers of bacteria always increase, and rates of assimilation of dissolved leucine often increase exponentially. This suggests that sample handling disrupts the previously existing food web, leading to shifts of trophic state and unbalanced growth. Potential errors in measuring respiratory rate can be minimized by documenting these variables.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: II. Method of Characterization of Activated Sludge Bacteria

The replica-plating technique and Lochhead's nutritional method were combined in exploratory experiments to test their feasibility as useful means for characterizing the aerobic heterotrophic flora of activated sludge and to minimize the burdensome process of isolation, purification, and testing of isolates. In the test run, the method was about 86% reliable at the 0.05 level of significance. About 40% of the total number of bacteria able to grow on an aqueous extract of activated sludge did not grow on media containing glucose, amino acids, growth factors, and inorganic salts. The requirement for activated sludge extract suggested the existence of a requirement for unidentified nutrients contained in the activated sludge extract.     

DNA Synthesis and Tritiated Thymidine Incorporation by Heterotrophic Freshwater Bacteria in Continuous Culture

Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. The bacteria were grown on a yeast extract medium with generation times of 0.25 to 3.7 days. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 × 10¹⁸ to 1.31 × 10¹⁸ cells mol of tritiated thymidine^-1 (mean, 0.60 × 10¹⁸ cells mol of tritiated thymidine^-1). In addition, DNA concentrations were measured by fluorometry with Hoechst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell^-1. Theoretical conversion factors, which can be derived from DNA content under several assumptions, were between 0.26 × 10¹⁸ and 0.34 × 10¹⁸ cells mol of thymidine^-1 (mean, 0.30 × 10¹⁸ cells mol of thymidine^-1). Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. Control experiments indicated maximum incorporation at this concentration. It was therefore concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.     

Clustering of Marine Bacteria in Seawater Enrichments

Seawater enrichments of marine bacteria clustered in 20- to 50-(mu)m-wide bands near air-water interfaces. The cells within the band travelled at up to 212 (mu)m s(sup-1) and at an average speed of 163 (mu)m s(sup-1). Mean cell speeds peaked mid-run at 187 (mu)m s(sup-1). At the end of the run, bacteria reversed direction rather than randomly reorienting. The duration of the stops during reversal was estimated at 18 ms, six to seven times shorter than that found in enteric bacteria. Cells hundreds of micrometers from the band travelled at half the speed of the bacteria in the band. The fastest isolate from the seawater enrichment was identified as Shewanella putrefaciens and had an average speed of 100 (mu)m s(sup-1) in culture. Air-water interfaces produced no clustering or speed changes in isolates derived from enrichments. Salinity and pH, however, both influenced speed. The speed and reversal times of the seawater enrichments indicate that the bacteria in them are better adapted for clustering around small point sources of nutrients than are either enteric or cultured marine bacteria.     

Glucose Catabolism in Strains of Acidophilic, Heterotrophic Bacteria

Pathways of glucose catabolism, potentially operational in six strains of obligately aerobic, acidophilic bacteria, including Acidiphilium cryptum strain Lhet2, were investigated by short-term radiorespirometry and enzyme assays. Short-term radiorespirometry was conducted at pH 3.0 with specifically labeled [¹⁴C]glucose. The high rate and yield of C-1 oxidized to CO₂ indicated that the Entner-Doudoroff, pentose phosphate, or both pathways were operational in all strains. Apparent nonequivalent yields of CO₂ from C-1 and estimated CO₂ from C-4 (C-1 > C-4) were suggestive of simultaneous glucose catabolism by both pathways in all strains tested. Variation in the relative contribution of the two pathways of glucose catabolism appears to account for observed strain differences. Calculation of the actual percent pathway participation was not feasible. Enzyme assays were completed with crude extracts of glucose-grown cells to substantiate the results obtained by radiorespirometry. The key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase) and the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrase) were present in all strains examined (PW2, Lhet2, KLB, OP, and QBP). However, none of the strains exhibited detectable levels of the key enzyme of the Embden-Meyerhof-Parnas pathway, 6-phosphofructokinase. All strains contained glucose-6-phosphate dehydrogenase and fructose bisphosphate aldolase. The results of the enzyme study supported the contention that the pentose phosphate and Entner-Doudoroff pathways are operational for glucose catabolism in the acidophilic heterotrophs, and that the Embden-Meyerhof-Parnas pathway is apparently absent.     

Growth of Heterotrophic Bacteria and Algal Extracellular Products in Oligotrophic Waters

The unexpected observation of 200 to 400 coliform bacteria per 100 ml in an unpolluted pristine stream was studied within Grand Teton National Park, Wyo. The high numbers of waterborne bacteria occurred in mid- to late summer at a location where there was a coincidental bloom of an algal mat community. Periphyton samplers were used to measure the algal growth that coincided with the increase in number of bacteria. Laboratory studies followed the growth of various coliform bacteria in the supernatant obtained from a Chlorella culture isolated from the mat community. Mixed natural bacterial populations from the stream and pure cultures of water-isolated fecal and nonfecal coliforms increased by two to three orders of magnitude at 13°C when grown in the algal supernatant. Radioactive algal products were obtained by feeding an axenic Chlorella culture ¹⁴C-labeled bicarbonate under laboratory cultivation at 13°C with illumination. Radioactive organic material from the algae became incorporated into the particulate fraction of pure cultures of coliform bacteria as they reproduced and was later released as they died.     

Patchiness in the Distribution of Planktonic Heterotrophic Bacteria in Lakes

By using conventional dilution plating procedures, the heterotrophic planktonic bacteria in Lake Washington were found to be distributed in small-scale (<1.0 ml) and large-scale (>1 m in horizontal distance) patches. Recommendations are made on sampling and enumeration procedures to minimize the effect of patchiness on obtaining an accurate estimate of bacterial numbers.     

Marine Heterotrophic Bacteria in Continuous Culture,the Bacterial Carbon Growth Efficiency,and Mineralization at Excess Substrate and Different Temperatures

To model the physiological potential of marine heterotrophic bacteria, their role in the food web, and in the biogeochemical carbon cycle, we need to know their growth efficiency response within a matrix of different temperatures and degrees of organic substrate limitation. In this work, we present one part of this matrix, the carbon growth efficiencies of marine bacteria under different temperatures and nonlimiting organic and inorganic substrate supply. We ran aerobic turbidostats with glucose enriched seawater, inoculated with natural populations of heterotrophic marine bacteria at 10, 14, 18, 22, and 26°C. The average cell-specific growth rates increased with temperature from 1.17 to 2.6 h−1. At steady-state total CO₂ production, biomass production [particulate organic carbon (POC) and nitrogen (PON)], and viruslike particle abundance was measured. CO₂ production and specific growth rate increased with increasing temperature. Bacterial carbon growth efficiency (BCGE), the particulate carbon produced per dissolved carbon utilized, varied between 0.12 and 0.70. Maximum BCGE values and decreased specific respiration rates occurred at higher temperatures (22 and 26°C) and growth rates. This trend was largely attributable to an increase in POC per cell abundance; when the BCGE was recalculated, parameterizing the biomass as the product of cell concentration and a constant cellular carbon content, the opposite trend was observed.