ALBERT

Notes: Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881–891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a ‘model oligotroph’ and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable ‘low-nutrient-conditioned’ phenotype.

Notes: In nature a significant part of the microbial activity is concentrated at or near oxic/anoxic interfaces, where oxygen concentrations are often low. Bacteria possessing different kinetic characteristics for oxygen and employing distinct metabolic pathways for the degradation of (halo)aromatic substrates for which oxygen is needed as co-substrate may have to compete with each other in such environments. In this study the competitiveness of Pseudomonas sp. strain A3 relative to Alcaligenes sp. strain L6 was tested in batch and in continuous cultures. While both of these strains are able to metabolise 3-chlorobenzoate (3CBA), the former was isolated under air saturating conditions and employs the catechol pathway, whereas the latter was isolated under reduced partial pressures of oxygen and was capable of metabolising 3CBA via the gentisate pathway. Competition experiments in batch culture resulted in pure cultures of Pseudomonas sp. strain A3 under air saturating conditions. However, if reduced partial pressures of oxygen (2%) were used, Alcaligenes sp. strain L6 remained present in substantial numbers after three transfers. Continuous culture experiments demonstrated that Alcaligenes sp. strain L6 was able to outcompete Pseudomonas sp. strain A3 under oxygen- as well as under carbon-limiting conditions as long as the dilution rate remained below 0.136 h−1 (low oxygen) and below 0.178 h−1 (high oxygen). These results support the hypothesis that organisms metabolising chlorobenzoate via the gentisate pathway may play a significant role in natural ecosystems where xenobiotic compounds and naturally produced aromatics occur at very low concentrations and in combination with limiting oxygen tensions.

Notes: Three strains of Rhodopseudomonas palustris were isolated from phototrophic enrichment cultures containing 3-chlorobenzoate (3-CBA) and benzoate (BA). These new strains as well as several previously described strains of R. palustris were tested in this study and shown to degrade 3-CBA if grown in media that contained BA as a co-substrate. All of the pure cultures that originally required BA for the degradation of 3-CBA acquired the ability to degrade 3-CBA as the sole carbon source after long periods of incubation that ranged from 1 to 3 months. After this adaptation period, the 3-CBA-degrading capabilities of all variants were stable, and the rates of 3-CBA degradation were significantly enhanced as compared to the parental strains. Furthermore, the variants had also acquired the ability to metabolize 2- and 4-CBA as sole carbon sources indicating that the enhanced ability to metabolize 3-CBA was accompanied by an expanded ability to metabolize chlorinated benzoates. These data indicate that acquisition of the ability to degrade 3-CBA may be rather common among strains of R. palustris and mutations that confer the ability to metabolize 3-CBA may provide a selective advantage to R. palustris under specific environmental conditions.

Notes: Abstract: Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)−1), but the growth rate with this substrate was very low (0.015 h−1). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O2 min−1 (mg protein)−1). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.

Notes: The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O2) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μmax values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.

Notes: Abstract Bacteria from an anaerobic enrichment reductively removed chlorine from the ortho- position of 2,3,6-trichlorobenzoic acid (2,3,6-TBA) producing 2,5-dichlorobenzoate (2,5-DBA). The strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-DBA as a growth substrate in the presence of oxygen. The anaerobic dechlorinating microbial population was grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on a stainless-steel support, contained vermiculite particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent of oxygen input into the reactor. Under strictly anaerobic conditions 2,5-DBA and Cl− were produced stoichiometrically through the reductive dechlorination of 2,3,6-TBA. This process of reductive dechlorination was not inhibited by (moderate) aeration resulting in an O2-concentration of 0.3–0.5 μM in the culture liquid.

Notes: In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized cells occurred during extended periods of starvation. These changes were observed both in batch cultures grown and starved aerobically in the dark, and in cultures grown phototrophically and starved anaerobically in the dark. Aerobic growth in batch culture and subsequent starvation resulted in a complete lack of detectable hybridization after 20 days of starvation. In contrast, even after 30 days of starvation, 50% of all cells were still detectable in cultures grown aerobically at growth rates 〈0.06 h−1 and then starved aerobically in the dark. The same was true for phototrophically grown cells that were starved anaerobically in the light. During starvation there was a clear, though non-linear, positive correlation between the percentage of hybridized cells and the RNA content. In contrast, no direct correlation was observed between the number of hybridized cells in a culture and the viability of this culture. Thus, in habitats with growing, non-growing, and starving bacteria, data on quantitative detection of populations based on 16S rRNA-targeted probing should be used with extreme caution as the detectability of the individual cells is strongly influenced by their physiological history and current physiological state.

Notes: A new inulin-fermenting strain of Clostridium thermoautotrophicum was isolated through enrichment on dahlia tubers, and subsequent plating on agar media with undissolved inulin. Both the cell-bound and cell-free inulinase(s) functioned optimally at 60°C and atneutral pH. This new strain I1 is readily distinguished from the type strain of C. thermoautotrophicum with respect to fermentation products, substrate spectrum and optimum temperature for growth.

Notes: Abstract A sulfate-reducing bacterium capable of growth with methanol as sole source of energy was isolated from an anaerobic waste-water treatment plant via enrichment in an ethanol/sulfate medium. The cells were curved, Gram-negative, nonmotile rods. No spore formation was observed. Glycerol could be fermented to 1,3-propanediol and 3-hydroxypropionate. In the presence of sulfate, glycerol was stoichiometrically converted to 3-hydroxypropionate. The cells contained stacked intracellular membranes during growth with ethanol or lactate but not with methanol. The doubling time during growth on methanol was approx. 75 h. Methanol was metabolized according to the following equation:〈displayedItem type="mathematics" xml:id="mu1" numbered="no"〉〈mediaResource alt="image" href="urn:x-wiley:03781097:FML125:FML_125_mu1"/〉 The possible role of sulfate-reducing bacteria in the anaerobic mineralization of methanol is discussed.