Atrazine catabolism by a combined bacterial association (KRA30) under carbon- and nitrogen-limitations in a retentostat.

AIMS: Nutrient-limited atrazine catabolism study in continuous cultures with biomass retention to mimic in situ environmental conditions and thus gain insight of the efficacy of biosupplementation/biostimulation to eliminate reduced herbicide bioavailability. METHODS AND RESULTS: Carbon- and nitrogen-limited retentostat (1 and 5 l) cultivation of a combined atrazine (100 mg l-1)-catabolizing association KRA30 was made. As a nitrogen source, through citrate supplementation, increased herbicide catabolism resulted and was complete in the absence of NH4-N. Co-metabolism of the molecule in the presence of succinate was identified. Population characterization by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) indicated component species numerical dominance shifts in response to changes in nutrient limitation, mineral salts composition and biofilm formation, although the total species complement and catabolic potential were retained. CONCLUSIONS: Biomass and catabolic capacity maintenance, through cost-effective biosupplementation/biostimulation, should promote atrazine bioavailability and so ensure successful amelioration. SIGNIFICANCE AND IMPACT OF THE STUDY: All planning, implementation and monitoring of bioremediation programmes should be underpinned by a combination of molecular and (continuous) culture-based methods.

Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer.

Knowledge about the relationship between microbial community structure and hydrogeochemistry (e.g., pollution, redox and degradation processes) in landfill leachate-polluted aquifers is required to develop tools for predicting and monitoring natural attenuation. In this study analyses of pollutant and redox chemistry were conducted in parallel with culture-independent profiling of microbial communities present in a well-defined aquifer (Banisveld, The Netherlands). Degradation of organic contaminants occurred under iron-reducing conditions in the plume of pollution, while upstream of the landfill and above the plume denitrification was the dominant redox process. Beneath the plume iron reduction occurred. Numerical comparison of 16S ribosomal DNA (rDNA)-based denaturing gradient gel electrophoresis (DGGE) profiles of Bacteria and Archaea in 29 groundwater samples revealed a clear difference between the microbial community structures inside and outside the contaminant plume. A similar relationship was not evident in sediment samples. DGGE data were supported by sequencing cloned 16S rDNA. Upstream of the landfill members of the beta subclass of the class Proteobacteria (beta-proteobacteria) dominated. This group was not encountered beneath the landfill, where gram-positive bacteria dominated. Further downstream the contribution of gram-positive bacteria to the clone library decreased, while the contribution of delta-proteobacteria strongly increased and beta-proteobacteria reappeared. The beta-proteobacteria (Acidovorax, Rhodoferax) differed considerably from those found upstream (Gallionella, Azoarcus). Direct comparisons of cloned 16S rDNA with bands in DGGE profiles revealed that the data from each analysis were comparable. A relationship was observed between the dominant redox processes and the bacteria identified. In the iron-reducing plume members of the family Geobacteraceae made a strong contribution to the microbial communities. Because the only known aromatic hydrocarbon-degrading, iron-reducing bacteria are Geobacter spp., their occurrence in landfill leachate-contaminated aquifers deserves more detailed consideration.

Microorganisms with a taste for vanilla: microbial ecology of traditional Indonesian vanilla curing.

The microbial ecology of traditional postharvesting processing of vanilla beans (curing) was examined using a polyphasic approach consisting of conventional cultivation, substrate utilization-based and molecular identification of isolates, and cultivation-independent community profiling by 16S ribosomal DNA based PCR-denaturing gradient gel electrophoresis. At two different locations, a batch of curing beans was monitored. In both batches a major shift in microbial communities occurred after short-term scalding of the beans in hot water. Fungi and yeast disappeared, although regrowth of fungi occurred in one batch during a period in which process conditions were temporarily not optimal. Conventional plating showed that microbial communities consisting of thermophilic and thermotolerant bacilli (mainly closely related to Bacillus subtilis, B. licheniformis, and B. smithii) developed under the high temperatures (up to 65 degrees C) that were maintained for over a week after scalding. Only small changes in the communities of culturable bacteria occurred after this period. Molecular analysis revealed that a proportion of the microbial communities could not be cultured on conventional agar medium, especially during the high-temperature period. Large differences between both batches were observed in the numbers of microorganisms, in species composition, and in the enzymatic abilities of isolated bacteria. These large differences indicate that the effects of microbial activities on the development of vanilla flavor could be different for each batch of cured vanilla beans.

Spatiotemporal Stability of an Ammonia-Oxidizing Community in a Nitrogen-Saturated Forest Soil.

Elevated levels of nitrogen input into various terrestrial environments in recent decades have led to increases in soil nitrate production and leaching. However, nitrifying potential and nitrifying activity tend to be highly variable over space and time, making broad-scale estimates of nitrate production difficult. This study investigates whether the high spatiotemporal variation in nitrate production might be explained by differences in the structure of ammonia-oxidizing bacterial communities in nitrogen-saturated coniferous forest soils. The diversity of ammonia-oxidizing bacteria of the b-subgroup Proteobacteria was therefore investigated using two different PCR-based approaches. The first targeted the 16S rRNA gene and involved temporal temperature gradient electrophoresis (TTGE) of specifically amplified PCR products, with subsequent band excision and nucleotide sequence determination. The second approach involved the cloning and sequencing of PCR-amplified amoA gene fragments. All recovered 16S rDNA sequences were closely related to the culture strain Nitrosospira sp. AHB1, which was isolated from an acid soil and is affiliated with Nitrosospira cluster 2, a sequence group previously shown to be associated with acid environments. All amoA-like sequences also showed a close affinity with this acid-tolerant Nitrosospira strain, although greater sequence variation could be detected in the amoA analysis. The ammonia-oxidizing bacterial community in the nitrogen-saturated coniferous forest soil was determined to be very stable, showing little variation between different organic layers and throughout the year, despite large differences in the total Bacterial community structure as determined by 16S rDNA DGGE community fingerprinting. These results suggest that environmental heterogeneity affecting ammonia oxidizer numbers and activity, and not ammonia oxidizer community structure, is chiefly responsible for spatial and temporal variation in nitrate production in these acid forest soils.

Analysis of Microbial Communities in a Landfill Leachate Polluted Aquifer using a New Method for Anaerobic Physiological Profiling and 16S rDNA Based Fingerprinting.

Databases containing information regarding presence and activity of microbial communities will be very useful for determination of the potential for intrinsic bioremediation in landfill leachate polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for inclusion into such databases. In this study we describe the development of a method for anaerobic CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the aquifer (10 degrees C) for 28 days was optimal for obtaining a specific, reproducible physiological profile. Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-dependent CLPP method and cultivation-independent DGGE were applied to groundwater and sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combination of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples yielded the best separating power for characterizing microbial communities in the aquifer. Communities in groundwater were significantly different from those in the corresponding sediment. Microbial communities present in subsamples from sediment cores usually were similar for the various sampling locations. Variation was observed for the heterogeneous sediment beneath the landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream and upstream of the landfill.

16S rDNA-based characterization of BTX-catabolizing microbial associations isolated from a South African sandy soil.

In the presence of different selection pressures, particularly pH and electron donor concentration, indigenous microbial associations which catabolize selected petroleum hydrocarbon components (benzene, toluene and o-, m- and p-xylene (BTX)) were enriched and isolated from a petroleum hydrocarbon-contaminated KwaZulu-Natal sandy soil. Electron microscopy revealed that, numerically, rods constituted the majority of the populations responsible for BTX catabolism. Molecular techniques (polymerase chain reaction (PCR) and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE)) were employed to explore the diversities and analyze the structures of the isolated microbial associations. Pearson product-moment correlation indicated that the different, but chemically similar, petroleum hydrocarbon molecules, effected the isolation of different associations. However, some similar numerically-dominant bands characterized the associations. A 30% similarity was evident between the m- and o-xylene-catabolizing associations regardless of the molecule concentration and the enrichment pH. PCR-DGGE was also used to complement conventional culture-based microbiological procedures for environmental parameter optimization. Band pattern differences indicated profile variations of the isolated associations which possibly accounted for the growth rate changes recorded in response to pH and temperature perturbations.

Maintenance energy demand and starvation recovery dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi cultivated in a retentostat with complete biomass retention.

Nitrosomonas europaea and Nitrobacter winogradskyi (strain "Engel") were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25 degrees C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 micromol of O2 mg-1 of biomass-C x h-1 for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 microg x ml-1) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514-1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.

Control of temperature-dependent synthesis of K99 fimbriae.

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.

Isolation and characterization of ubiquinol oxidase complexes from Paracoccus denitrificans cells cultured under various limiting growth conditions in the chemostat.

To obtain more information about the composition of the respiratory chain under different growth conditions and about the regulation of electron-transfer to several oxidases and reductases, ubiquinol oxidase complexes were partially purified from membranes of Paracoccus denitrificans cells grown in carbon-source-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. The isolated enzymes consisted of cytochromes bc1, c552 and aa3. In comparison with the aerobic ubiquinol oxidase complex, the oxygen- and nitrate-limited ones contained, respectively, less and far less of the cytochrome aa3 subunits and the anaerobic complex also contained lower amounts of cytochrome c552. In addition, extra haem-containing polypeptides were present with apparent Mr of 14,000, 30,000 and 45,000, the former one only in the anaerobic and the latter two in both the anaerobic and oxygen-limited preparations. This is the first report describing four different membrane-bound c-type cytochromes. The potentiometric and spectral characteristics of the redox components in membrane particles and isolated ubiquinol oxidase fractions were determined by combined potentiometric analysis and spectrum deconvolution. Membranes of nitrate- and oxygen-limited cells contained extra high-potential cytochrome b in comparison with the membranes of aerobically grown cells. No difference was detected between the three isolated ubiquinol oxidase complexes. Aberrances with already published values of redox potentials are discussed.

Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat.

Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials. (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV. The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550. In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones. Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present. Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions.

Modeling of microbial substrate conversion, growth and product formation in a recycling fermentor.

Paracoccus denitrificans and Bacillus licheniformis were grown in a carbon- and energy source-limited recycling fermentor with 100% biomass feedback. Experimental data for biomass accumulation and product formation as well as rates of carbon dioxide evolution and oxygen consumption were used in a parameter optimization procedure. This procedure was applied on a model which describes biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of the biomass growth rate. The fitting procedure yielded two growth domains for P. denitrificans. In the first domain the values for the maximal growth yield and the maintenance coefficient were identical to those found in a series of chemostat experiments. The second domain could be described best with linear biomass increase, which is equal to a constant growth yield. Experimental data of a protease producing B. licheniformis also yielded two growth domains via the fitting procedure. Again, in the first domain, maximal growth yield and maintenance requirements were not significantly different from those derived from a series of chemostat experiments. Domain 2 behaviour was different from that observed with P. denitrificans. Product formation halts and more glucose becomes available for biomass formation, and consequently the specific growth rate increases in the shift from domain 1 to 2. It is concluded that for many industrial production processes, it is important to select organisms on the basis of a low maintenance coefficient and a high basic production of the desired product. It seems less important that the maximal production becomes optimized, which is the basis of most selection procedures.

Eubacteria have 3 growth modes keyed to nutrient flow. Consequences for the concept of maintenance and maximal growth yield.

Aerobic growth of Escherichia coli and Paracoccus denitrificans has been studied in chemostat, fed batch, and recycling fermentor modes under carbon and energy limitation. Two abrupt drops or discontinuities in molar growth yield, Y, have been found that occur over relatively short ranges in the value of specific growth rate. Before the first discontinuity, Y is constant and maximal. After the first discontinuity, at a doubling time of 33 h, Y becomes constant again and independent of mu until the second discontinuity appears at a doubling time of about 50 h, corresponding to a mu of about 0.014. At this point, Y drops to a lower value that is constant at doubling times longer than 100 h, corresponding to a mu of about 0.007. The second discontinuity is associated in Paracoccus with elevated levels of guanosine tetraphosphate (ppGpp) that impose stringent regulation as has been found previously with Bacillus and Escherichia species. It is thus likely that the stringent response generally occurs in bacteria in vivo at a doubling time of about 50 h. The cause of the first discontinuity is unknown. All experiments indicate that Pirt-type calculations relating mu, Y, and maintenance energy demand are no longer valid. In chemostat experiments, the intercept of the relationship between specific substrate utilization and specific growth rate is defined as maintenance. However, this intercept most probably is caused by stringent regulation at low dilution rates. Three regions of bacterial growth rates are defined by this study, corresponding to doubling times of 0.5 to 15 h, 33 to 50 h, and greater than 100 h. Some growth behavior in each region is unique to that region.