Polyphosphate-Accumulating and Denitrifying Bacteria Isolated from Anaerobic-Anoxic and Anaerobic-Aerobic Sequencing Batch Reactors

In this study, phosphate-accumulating bacteria achieved complete phosphate removal in two different systems: an anaerobic-anoxic sequencing batch reactor and an anaerobic-aerobic sequencing batch reactor. This result shows that phosphate-accumulating bacteria in the A₂ SBR can use nitrate as terminal electron acceptor instead of oxygen. Phosphate-accumulating bacteria accumulated phosphate with a rates between 30 and 70 mg P/L/h in the A/O SBR and between 15 and 32 mg P/L/h in the A₂ SBR. Twenty denitrifying isolates were screened from A₂ SBR and nine from A/O SBR. Identification of these isolates by the Biolog system and the API 20 NE identification kit revealed that the most active denitrifiers in both SBRs reactors were species of Ochrobactrum, Pseudomonas, Corynebacterium, Agrobacterium, Aquaspirillum, Haemophilus, Xanthomonas, Aeromonas, and Shewanella. The most active phosphate accumulating and denitrifying bacteria were identified as Agrobacterium tumefaciens B, Aquaspirillum dispar, and Agrobacterium radiobacter. This study showed that the active phosphate accumulating-bacteria were also the most efficient denitrifying bacteria in both reactors. Received: 24 February 1998 / Accepted: 21 July 1998     

Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

Growth of Bacteroides fragilis in Continuous Culture and in Batch Cultures at Controlled pH

Bacteroides fragilis NCTC 9343 has been grown in continuous cultures with glucose as growth-limiting factor. At pH 7.0 and at a dilution rate of 0.07 per h, glucose limited growth in concentrations up to 0.6%. Maximal cell yield and productivity were obtained with 0.87% glucose in the inflowing medium. A pH of 7.0 was optimal for growth. With 0.6% glucose in the fresh medium and at pH 7.0, cell yield and productivity were highest at a dilution rate of 0.07 per h and 0.11 per h, respectively. At dilution rates higher than 0.07 per h, glucose was no longer growth limiting, and at dilution rates above 0.11 per h, another compound seemed to have replaced glucose also as energy source. When grown in batch cultures at pH 7.0, the best yields of B. fragilis was achieved with 0.6% glucose in the fresh medium. The highest specific growth rate (mum) determined from viable counts was 0.45, corresponding to a mean generation time of 92 min.     

Correlation of Functional Instability and Community Dynamics in Denitrifying Dispersed-Growth Reactors

Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing Delftia- and Achromobacter-like T-RFs. The isolate representing the Acidovorax-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The Acidovorax-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.     

海水反硝化细菌富集培养及性能研究

目的：建立一种海水反硝化细菌的富集培养方法,为海水反硝化微生物在实际应用中处理硝酸盐氮做基础工作。方法：选取污水处理厂缺氧池活性污泥,利用人工海水富集培养反硝化细菌。结果：经过25d,可以培养出反硝化速率为4.83mg/（gMLSS·h）、反硝化强度为110mg/（L·h）的海水反硝化细菌培养物。结论：该培养物可利用柠檬酸钠、葡萄糖、甲醇、乙醇、可溶性淀粉作为唯一碳源进行反硝化,其中以柠檬酸钠和乙醇作碳源时硝酸盐氮去除效果最佳,经过12h,去除率即可达到100%。pH在7～8范围内时,改变pH对去除率的影响不大,但当pH〈6时,去除率明显下降。温度在35℃时,去除效果最好,并且随着温度的降低,去除率也降低。     

利用可生物降解聚合物同时作为反硝化微生物的碳源和附着载体研究

饮用水源水中硝酸盐污染已引起世界各国的普遍关注,异养反硝化是去除水中硝酸盐的主要技术之一.利用可生物降解聚合物(biodegradable polymers,BDPs)同时作为反硝化微生物的碳源和附着生长的载体,近年来受到了人们的关注.该系统对进水水质的波动具有良好的适应能力;BDPs对人体无毒无害,不会污染出水水质.随着各种新型BDPs材料的不断涌现以及BDPs材料生产成本的降低,BDPs材料在饮用水源水生物脱氮中会得到越来越广泛的应用.对利用可生物降解聚合物进行反硝化的研究进展进行了综述.     

Fluorimetric Technique for Monitoring Changes in the Level of Reduced Nicotinamide Nucleotides in Continuous Cultures of Microorganisms

A metabolite fluorimeter was modified to monitor nicotinamide nucleotides in cells growing in a chemostat culture. By illuminating and collecting fluorescence over a relatively large area of the side of the culture vessel, the noise level was kept low even in highly turbulent cultures. Calibration of the system was by enzymatic assay of samples. Changes in nicotinamide nucleotide of concentration less than 0.1 nmoles/ml could be detected [i.e., less than 10% aerobic/anaerobic response for a cell concentration of 5 mg (dry wt) per ml].     

Treating Urban Wastewater: Nutrient Removal by Using Immobilized Green Algae in Batch Cultures

Essential roles of microalgae during the tertiary treatment of municipal wastewater have been proven. In order to avoid wash out of the biomass and also modify the quality of the treated wastewater; some techniques such as cell immobilization have been developed. To do so, in this study four samples from two species of microalgae (Chlorella vulgaris and Chlamydomonas sp.) were determined and confirmed by taxonomic identification. The samples were immobilized in calcium alginate beads. Within 10 days the amounts of nitrate (No₃^?-N) and orthophosphate (Po₄^3?-P) were measured to calculate the removal efficacy for each individual sample. Based on the standard methods, the amount of nitrate and orthophosphate were determined every 3 days within 10 days. The results indicate that immobilized microalgae are able to remove the nutrients and reduce the amount of nitrate and orthophosphate, significantly. Furthermore, the C. vulgaris (YG02) was the best species in this experience with 72% and 99% of reduction in the amount of nitrate and orthophosphate, respectively.     

Mineralization of Trichloroethylene by Heterotrophic Enrichment Cultures

Microbial consortia capable of aerobically degrading more than 99% of exogenous trichloroethylene (TCE) (50 mg/liter) were collected from TCE-contaminated subsurface sediments and grown in enrichment cultures. TCE at concentrations greater than 300 mg/liter was not degraded, nor was TCE used by the consortia as a sole energy source. Energy sources which permitted growth included tryptone-yeast extract, methanol, methane, and propane. The optimum temperature range for growth and subsequent TCE consumption was 22 to 37°C, and the pH optimum was 7.0 to 8.1. Utilization of TCE occurred only after apparent microbial growth had ceased. The major end products recovered were hydrochloric acid and carbon dioxide. Minor products included dichloroethylene, vinylidine chloride, and, possibly, chloroform.     

Maleic Acid Utilization by Mixed Cultures of Microorganisms

In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and B. subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions of water.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 10⁵ CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum

Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.Atmospheric concentrations of methane have risen 2.6-fold since preindustrial times (10). After several years of stagnation, there was again a clear increase in the methane concentration in 2007 (29). Currently, it is uncertain whether an increase in the number of sources and production or a decrease in the number of sinks and consumption is responsible for this reversal of the trend.Freshwater habitats like natural wetlands and rice fields are a major source (38% [9]) of atmospheric methane. In the absence of other documented electron donors, aerobic methane oxidation is assumed to be the most important sink in these habitats, but the role of alternative electron donors is not well understood (19, 30). Anaerobic methane oxidation coupled to denitrification is energetically favorable, but evidence that it occurs is scarce. In marine, methane-containing sediments, nitrate and nitrite are usually not quantitatively important electron acceptors; in freshwater sediments the denitrifying and aerobic zones are in close proximity (3, 22, 35), possibly masking the process from detection. To our knowledge, concomitant methane and nitrate profiles of sediments have never been published.So far, methane oxidation coupled to denitrification has received the most attention in the field of hydrogeology. In groundwater, contamination with nitrate and nitrite occurs frequently, whereas electron donors are limiting. Methane plumes often form around landfills, and their attenuation has sometimes been attributed to denitrification (2, 37). So far, a single previous study unambiguously demonstrated anaerobic oxidation of methane coupled to denitrification in a contaminated freshwater aquifer (32). The first in vitro observation of anaerobic methane oxidation coupled to denitrification came from a laboratory-scale sludge digestor (11). The use of a laboratory enrichment culture also eventually resulted in identification of the organisms involved; bacteria of the NC10 phylum and archaea of the order Methanosarcinales dominated a mixed culture carrying out anaerobic methane oxidation coupled to denitrification (27). This culture was enriched from a freshwater canal sediment after 1 year of continuous supply of methane and nitrite. Subsequently, the archaea were shown to be dispensable, as they disappeared after prolonged incubation of the same culture (7). Mass balance calculations showed that methane oxidation was coupled to the reduction of nitrite with a 3:8 stoichiometry, in accordance with theoretical expectations. The bacteria that dominated the mixed culture and apparently oxidized methane anaerobically are members of the NC10 phylum, one of the many phyla having no members in pure culture (8, 28). The 16S rRNA gene sequences of such organisms, however, have been found in a number of environmental surveys of aquatic environments; e.g., the most closely related sequences have been found in aquifers (1, 23) and lake sediments (13, 17). Sequence similarity and phylogenetic affiliation may indicate similar metabolic capacities of organisms, but by itself this is not sufficient to infer similar metabolism (5). This is especially true for denitrifying methanotrophs, because only a single enrichment culture has been described so far (7, 27).The objective of the present study was to generalize the previous finding that NC10 bacteria were associated with anaerobic methane oxidation, a necessary step forward in addressing the significance of this poorly understood process as a methane sink in freshwater habitats.     

Two-Stage Mineralization of Phenanthrene by Estuarine Enrichment Cultures

The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring ¹⁴CO₂ production from low concentrations (0.5 mg liter⁻¹) of radiolabeled phenanthrene. Here, mineralization of ¹⁴C-labeled 1H2NA could explain the incremental ¹⁴CO₂ produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.     

Habitat Specialization Along a Wetland Moisture Gradient Differs Between Ammonia-oxidizing and Denitrifying Microorganisms

Gradients in abiotic parameters, such as soil moisture, can strongly influence microbial community structure and function. Denitrifying and ammonia-oxidizing microorganisms, in particular, have contrasting physiological responses to abiotic factors such as oxygen concentration and soil moisture. Identifying abiotic factors that govern the composition and activity of denitrifying and ammonia-oxidizing communities is critical for understanding the nitrogen cycle. The objectives of this study were to (i) examine denitrifier and archaeal ammonia oxidizer community composition and (ii) assess the taxa occurring within each functional group related to soil conditions along an environmental gradient. Soil was sampled across four transects at four locations along a dry to saturated environmental gradient at a restored wetland. Soil pH and soil organic matter content increased from dry to saturated plots. Composition of soil denitrifier and ammonia oxidizer functional groups was assessed by terminal restriction fragment length polymorphism (T-RFLP) community analysis, and local soil factors were also characterized. Microbial community composition of denitrifiers and ammonia oxidizers differed along the moisture gradient (denitrifier: ANOSIM R?=?0.739, P?<?0.001; ammonia oxidizers: ANOSIM R?=?0.760, P?<?0.001). Individual denitrifier taxa were observed over a larger range of moisture levels than individual archaeal ammonia oxidizer taxa (Wilcoxon rank sum, W?=?2413, P value?=?0.0002). Together, our data suggest that variation in environmental tolerance of microbial taxa have potential to influence nitrogen cycling in terrestrial ecosystems.     

Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures

Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-μm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis.     

Effects of Continuous and Interrupted Radiation on Microorganisms

Various bacterial spores exhibited a wide range of radiation resistance to doses of 0.25 to 2.5 Mrad from a cobalt-60 radiation facility. Bacillus pumilus and Clos-tridium tetani were shown to have the highest degree of resistance when compared with other bacterial sporeformers. B. subtilis E163 was the least resistant of the bacterial spores studied. Dried spores contained on cellulose discs were more readily destroyed by gamma-rays than were wet spores under similar conditions. Mycobacterium tuberculosis was destroyed by radiation doses much lower than that required by the least resistant bacterial spores. Interrupted dosimetry tests performed with materials of various types showed that sutures and other similar materials were effectively sterilized when the total radiation dose was given in two separate exposures with periods of interruption of 1 to 19 days. When "agar dosimeters" were employed in similar interrupted dosimetry series, B. pumilus spores were recovered in a few tests after administration of a total combined dosage of 2.5 Mrad with interruption periods of 2 to 19 days. When the experiment was repeated with interruption for 14 days, no survivors were found after a total dose of 2.0 to 2.8 Mrad.