Abundance and diversity of anammox bacteria in a mainstream municipal wastewater treatment plant.

Among the factors that obstruct the application of anammox-based technology for nitrogen removal from mainstream municipal wastewater is the water's high organic loads. We hypothesized that some anammox species can adapt and grow in mainstream wastewater in which a minimal temperature of 13-15 °C is maintained. Using the AMX368F and AMX820R PCR-primers, anammox bacteria were detected in influent wastewater (COD/N ratio > 13) and in the anaerobic, anoxic, and aerobic chambers of a full-scale municipal wastewater treatment plant, reaching 107 copies/g VSS of the16S rRNA gene. Furthermore, anammox activity was demonstrated by 15N-isotopic tracing. The DNA sequences of clones randomly selected from a clone library were mainly clustered with Candidatus Brocadia flugida in addition to Ca. Brocadia sinica, Ca. Jettenia asiatica, and Ca. Anammoxoglobus propionicus. However, Ca. Brocadia was the only genus detected by high-throughput next-generation sequencing and denaturing gradient gel electrophoresis. The nitrite producers, ammonia-oxidizing archaea and bacteria, were both detected in the influent wastewater and the other chambers, while the nitrite consumers, Nitrospira nitrite oxidizers and the nirS-type denitrifiers, dominated all chambers. The results indicate the occurrence and potential activity of anammox bacteria in mainstream wastewater under certain conditions (proper temperature). The dominance of Brocadia flugida and Anammoxoglobus propionicus suggests a role for volatile fatty acids in selecting the anammox community in wastewater.

Viscoelastic Properties of Extracellular Polymeric Substances Can Strongly Affect Their Washing Efficiency from Reverse Osmosis Membranes.

The role of the viscoelastic properties of biofouling layers in their removal from the membrane was studied. Model fouling layers of extracellular polymeric substances (EPS) originated from microbial biofilms of Pseudomonas aeruginosa PAO1 differentially expressing the Psl polysaccharide were used for controlled washing experiments of fouled RO membranes. In parallel, adsorption experiments and viscoelastic modeling of the EPS layers were conducted in a quartz crystal microbalance with dissipation (QCM-D). During the washing stage, as shear rate was elevated, significant differences in permeate flux recovery between the three different EPS layers were observed. According to the amount of organic carbon remained on the membrane after washing, the magnitude of Psl production provides elevated resistance of the EPS layer to shear stress. The highest flux recovery during the washing stage was observed for the EPS with no Psl. Psl was shown to elevate the layer's shear modulus and shear viscosity but had no effect on the EPS adhesion to the polyamide surface. We conclude that EPS retain on the membrane as a result of the layer viscoelastic properties. These results highlight an important relation between washing efficiency of fouling layers from membranes and their viscoelastic properties, in addition to their adhesion properties.

Differential response of ammonia-oxidizing archaea and bacteria to the wetting of salty arid soil.

Ammonia-oxidizing archaea and bacteria (AOA, AOB) catalyze the first and rate-limiting step of nitrification. To examine their differential responses to the wetting of dry and salty arid soil, AOA and AOB amoA genes (encoding subunit A of the ammonia monooxygenase) and transcripts were enumerated in dry (summer) and wet (after the first rainfall) soil under the canopy of halophytic shrubs and between the shrubs. AOA and AOB were more abundant under shrub canopies than between shrubs in both the dry and wetted soil. Soil wetting caused a significant decrease in AOB abundance under the canopy and an increase of AOA between the shrubs. The abundance of the archaeal amoA gene transcript was similar for both the wet and dry soil, and the transcript-to-gene ratios were < 1 independent of niche or water content. In contrast, the bacterial amoA transcript-to-gene ratios were between 78 and 514. The lowest ratio was in dry soil under the canopy and the highest in the soil between the shrubs. The results suggest that the AOA are more resilient to stress conditions and maintain a basic activity in arid ecosystems, while the AOB are more responsive to changes in the biotic and abiotic conditions.

Integrated biological treatment of fowl manure for nitrogen recovery and reuse.

Biowaste such as animal manure poses an environmental threat, due to among others, uncontrolled emissions of ammonia and additional hazardous gases to the atmosphere. This study presents a quantitative analysis of an alternative biowaste management approach aimed at nitrogen recovery and reduction of contamination risks. The suggested technology combines anaerobic digestion of nitrogen-rich biowaste with biofiltration of the resulting gaseous ammonia. A compost-based biofilter is used to capture the ammonia and convert it to nitrate by nitrifying microorganisms. Nitrogen mass balance was applied to quantify the system's capacity under various fowl manure-loading regimes and ammonia loading rates. The produced nitrate was recovered and its use as liquid fertilizer was evaluated with cucumber plant as a model crop. In addition, emissions of other hazardous gases (N(2)O, CH(4) and H(2)S) were monitored before and after biofiltration to evaluate the efficiency of the system for treating these gases. It was found that nitrate-rich liquid fertilizer can be continuously produced using the suggested approach, with an over 67 percentage of nitrogen recovery, under an ammonia loading rate of up to 40 g NH(3) per cubic meter biofilter per hour. Complete elimination of NH(3), H(2)S, CH(4) and N(2)O was achieved, demonstrating the potential of the suggested technology for mitigating emission of these gases from fowl manure. Moreover, the quality of the recovered fertilizer was demonstrated by higher yield performance of cucumber plant compared with control plants treated with a commonly applied organic liquid fertilizer.

Isolation and characterization of RDX-degrading Rhodococcus species from a contaminated aquifer.

Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel's coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 µM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l(-1)) while the T9N isolate's activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.

Persistence and spread of tetracycline resistance genes and microbial community variations in the soil of animal corrals in a semi-arid planted forest.

At the spring, goat and sheep herds are transferred to planted forests, in a semi-arid region in the northern Negev Desert, Israel, to reduce herbaceous biomass and, fire risk. The herds are held overnight in corrals for about 4 months, enriching the soil with organic matter and nitrogen. This research examined the effect of these enrichments on soil bacterial community structure (BCS) and the abundance of tetracycline resistance genes (TRGs) in active and abandoned corrals (1-10-years-old). Based on 16S rRNA gene sequences, the Proteobacteria and Actinobacteria phyla dominated the soil of all corrals. The Actinobacteria were less abundant in the active and 1-year-old corrals (23-26%) than in the other corrals and the control (33-38%). A principal component analysis showed that, the BCS in the active and the 1-year-old abandoned corrals was significantly different from that in the older corrals and the control. The Firmicutes phylum constituted 28% of the BCS in the active corrals, 12.5% in the 1-year-old corrals and 2% in the older corrals and the control. In contrast, the Acidobacteria phylum was hardly detected in the active and 1-year-old abandoned corrals and constituted 10% of the BCS in the older corrals. Genes conferring resistance to tetracycline were detected in high numbers. The tetG and tetW genes were detected in the active and abandoned corrals (1-10 years). The tetQ gene was detected only in the active and 1-year-old abandoned corrals. None of the genes were detected in the control soil. The three genes were detected outside an active corral, in the downstream section of an ephemeral tributary. The results prove that abandoned and unobserved periodic animal corrals are an environmental reservoir for TRGs.

Nitrogen transformations and diversity of ammonia-oxidizing bacteria in a desert ephemeral stream receiving untreated wastewater.

Levels of inorganic nitrogen species (ammonia, nitrite, and nitrate), ammonia oxidation potential (AOP), and diversity of ammonia-oxidizing bacteria (AOB) were studied in the sediments of a 50-km-long segment of an ephemeral stream in the Negev desert, receiving untreated wastewater. Water analysis in downstream sampling points showed reductions of 91.7% in biological oxygen demand, 87.7% in chemical oxygen demand, 73.9% in total nitrogen, and 72.8% in total ammonia nitrogen. Significant AOP levels in the sediment were detected mainly in the fall and spring seasons. Denaturing gradient gel electrophoresis of AOB 16S rRNA gene fragments showed that in most sampling points, the streambed was dominated by Nitrosospira cluster 3 strains similar to those dominating the stream bank's soils and sediments in nearby springs. Nitrosomonas strains introduced by discharged wastewater and others dominated some sections of the stream characterized by high organic carbon levels. The results suggest that climatic conditions in the Negev desert select for AOB belonging to Nitrosospira cluster 3, and these conditions dominate the aquatic environment effect along most of the stream sections. In addition, the nitrification-denitrification processes were not sufficient to reduce nitrogen levels in the sediment and prevent the eutrophication of some sections of the stream ecosystem. Thus, the discharge of high nitrogen wastewater into desert streams should be done carefully as it may endanger the already fragile ecosystem.

Controlling nitritation in a continuous split-feed/aeration biofilm nitrifying bioreactor.

This study explored the stability of partial ammonium oxidation at low feed concentration (50 g N/m3), suitable for anammox process, in continuous fixed bed up-flow biofilm reactors with external recirculation-aeration. The reactors, filled with crushed basalt, were fed with synthetic medium at 20-25 °C at constant flow-rate with limiting dissolved oxygen concentration controlled by the recirculation ratio (R). Successful nitritation was achieved at R ≅ 4-6 with approx. 50% of NH4+ oxidized to NO2- with <5% NO3-accumulation. q-PCR analysis along the reactor showed ammonia oxidizing bacteria being the prevalent nitrifiers over the three-fourths of the bed in the flow direction, negligible denitrifiers and absent ammonium oxidizing archaea. A numerical model for predicting the concentration of the nitrogen species and DO was formulated. The model successfully predicted the experimental results and displayed good sensitivity to intrinsic oxygen uptake parameters. The proposed numerical model can serve both as an operational and design tool.

Seasonal and soil-type dependent emissions of nitrous oxide from irrigated desert soils amended with digested poultry manures.

Expansion of dryland agriculture requires intensive supplement of organic fertilizers to improve the fertility of nutrient-poor desert soils. The environmental impact of organic supplements in hot desert climates is not well understood. We report on seasonal emissions of nitrous oxide (N2O) from sand and loess soils, amended with limed and non-limed anaerobic digestate of poultry manure in the Israeli Negev desert. All amended soils had substantially higher N2O emissions, particularly during winter applications, compared to unammended soils. Winter emissions from amended loess (10-175mgN2Om-2day-1) were markedly higher than winter emissions from amended sand (2-7mgN2Om-2day-1). Enumeration of marker genes for nitrification and denitrification suggested that both have contributed to N2O emissions according to prevailing environmental conditions. Lime treatment of digested manure inhibited N2O emissions regardless of season or soil type, thus reducing the environmental impact of amending desert soils with manure digestate.

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils.

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.

Impact of streambed morphology on the abundance and activity of ammonia-oxidizing bacteria.

Ammonia oxidizers catalyze the first step of nitrification. Combined microbial nitrification-denitrification activities are essential for the removal of excess nitrogen from water bodies. In sandy streambeds, bed form structures are created by water flow and lead to the creation of heterogeneous microenvironments. The objective of this study, therefore, was to investigate the effect of bed form morphology on the abundance and activity of ammonia-oxidizing bacteria (AOB) within a benthic biofilm. An 8-month-old benthic biofilm was established in a recirculating laboratory flume under controlled flow conditions and frequent amendment with ammonium. The sand bed was arranged into bed form structures. The highest concentrations of chlorophyll a (indicative of algae) were measured on the upstream side of the bed forms. The biofilm was dominated by Nitrosospira species, and amoA gene abundance was higher on the downstream sides of the bed forms with no significant difference in oxygen consumption between the upstream and downstream sections of the bed form. In contrast, potential ammonium oxidation rates were higher on the upstream sides of the bed forms. The results suggest that bed form morphology can affect the spatial distribution and activity of AOB, possibly through the creation of distinct microhabitats. These results contribute to our understanding of nitrogen transformations and removal from streams.

Spatial and temporal diversity and abundance of ammonia oxidizers in semi-arid and arid soils: indications for a differential seasonal effect on archaeal and bacterial ammonia oxidizers.

Besides water, nitrogen is the limiting factor for biomass production in arid ecosystems. Global climatic changes are exacerbating aridity levels, and the response of nitrogen-transforming microorganisms to these changes is not clear yet. Using semi-arid and arid ecosystems as surrogates for conditions of increased aridity, we investigated the activity, abundance, and diversity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in arid and semi-arid soils. Ammonia oxidation potentials were higher during the winter in both sites than in the summer, and higher nitrate concentrations were measured in the arid soil than in the semi-arid soil. Denaturing gradient gel electrophoresis (DGGE) patterns of AOB 16S rRNA gene fragments were similar for the arid and semi-arid soils with no seasonal variations. In contrast, the DGGE patterns of the AOA amoA gene fragments differed between the sites and a soil transfer experiment suggested that these differences are possibly associated with soil type. AOB numbers were higher during the winter than in the summer, while AOA numbers were higher during the summer. The results indicate the resistance of AOB and AOA community structure to arid conditions, albeit with seasonal variations in their abundance. Together, the results suggest the resilience of nitrification activity to increased aridity level.

Ammonia transformations and abundance of ammonia oxidizers in a clay soil underlying a manure pond.

Unlined manure ponds are constructed on clay soil worldwide to manage farm waste. Seepage of ammonia-rich liquor into underlying soil layers contributes to groundwater contamination by nitrate. To identify the possible processes that lead to the production of nitrate from ammonia in this oxygen-limited environment, we studied the diversity and abundance of ammonia-transforming microorganisms under an unlined manure pond. The numbers of ammonia-oxidizing bacteria and anammox bacteria were most abundant in the top of the soil profile and decreased significantly with depth (0.5 m), correlating with soil pore-water ammonia concentrations and soil ammonia concentrations, respectively. On the other hand, the numbers of ammonia-oxidizing archaea were relatively constant throughout the soil profile (10(7) amoA copies per g(soil)). Nitrite-oxidizing bacteria were detected mainly in the top 0.2 m. The results suggest that nitrate accumulation in the vadose zone under the manure pond could be the result of complete aerobic nitrification (ammonia oxidation to nitrate) and could exist as a byproduct of anammox activity. While the majority of the nitrogen was removed within the 0.5-m soil section, possibly by combined anammox and heterotrophic denitrification, a fraction of the produced nitrate leached into the groundwater.

Assessment of the spatial distribution of soil microbial communities in patchy arid and semi-arid landscapes of the Negev Desert using combined PLFA and DGGE analyses.

Arid and semi-arid ecosystems are often characterized by vegetation patchiness and variable availability of resources. Phospholipid fatty acid (PLFA) and 16S rRNA gene fragment analyses were used to compare the bulk soil microbial community structure at patchy arid and semi-arid landscapes. Multivariate analyses of the PLFA data and the 16S rRNA gene fragments were in agreement with each other, suggesting that the differences between bulk soil microbial communities were primarily related to shrub vs intershrub patches, irrespective of climatic or site differences. This suggests that the mere presence of a living shrub is the dominant driving factor for the differential adaptation of the microbial communities. Lipid markers suggested as indicators of Gram-positive bacteria were higher in soils under the shrub canopies, while markers suggested as indicators of cyanobacteria and anaerobic bacteria were elevated in the intershrub soils. Secondary differences between soil microbial communities were associated with intershrub characteristics and to a lesser extent with the shrub species. This study provides an insight into the multifaceted nature of the factors that shape the microbial community structure in patchy desert landscapes. It further suggests that these drivers not only act in concert but also in a way that is dependent on the aridity level.

Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1.

We hypothesized that biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)--a widely used explosive contaminating soil and groundwater--by Rhodococcus strain YH1 is controlled by the presence of external nitrogen sources. This strain is capable of degrading RDX while using it as sole nitrogen source under aerobic conditions. Both inorganic and organic nitrogen sources were found to have a profound impact on RDX-biodegradation activity. This effect was tested in growing and resting cells of strain YH1. Nitrate and nitrite delayed the onset of RDX degradation by strain YH1, while ammonium inhibited it almost completely. In addition, 2,4,6-trinitrotoluene (TNT) inhibited RDX degradation and growth of strain YH1. On the other hand, tetrahydrophthalamide did not influence biodegradation or growth. Growth on RDX induced the expression of a cytochrome P-450 enzyme that is suggested to be involved in the first step in the aerobic pathway of RDX degradation, as identified by SDS-PAGE analysis. Ammonium and nitrite strongly repressed cytochrome P-450 expression. Our findings suggest that effective RDX bioremediation by strain YH1 requires the design of a treatment scheme that includes initial removal of ammonium, nitrite, nitrate and TNT before RDX degradation can take place.

Biodegradation of 2,4,6-tribromophenol during transport in fractured chalk.

The effect of physicochemical conditions (residence time, oxygen concentrations, and chalk characteristics) on the biodegradation of 2,4,6-tribromophenol (TBP) during transport was investigated in low-permeability fractured-chalk cores. Long-term (approximately 600 d) biodegradation experiments were conducted in two cores (approximately 21 cm diameter, 31 and 44 cm long, respectively), intersected by a natural fracture. TBP was used as a model contaminant and as the sole carbon source for aerobic microbial activity. Bacterial isolates were recovered and identified by both Biolog identification kit and 16S rDNA sequences from batch enrichment cultures. One of the strains, with 98% similarity (based on the 16S rDNA data) to Achromobacter xylosoxidans, was shown to have the ability to degrade TBP in the presence of chalk. The decrease in TBP concentration along the fracture due to biodegradation was not affected by reducing the residence time from 49 to 8 min. In contrast, adding oxygen to the water at the inlet and increasing the flow rates improved TBP removal. Although the matrix pore-size distribution limits microbial activity to the fracture void, the chalk appears to provide an excellent environment for biodegradation activity. Approximately 90% of TBP removal occurred within 10 cm of the TBP source, indicating that in-situ bioremediation can be used to remove organic contaminants in low-permeability fractured rocks if nutrient-delivery pathways within the aquifer are secured.

Molecular characterization of a gender-linked DNA marker and a related gene in Mercurialis annua L.

The dioecious Mercurialis annua L. was used as a model plant to study some aspects of the molecular basis of sex determination in plants. We report in this paper the characterization of a previously identified male specific DNA marker, OPB01-1562, from diploid dioecious M. annua. The marker co-segregated with male sex in the progeny of hormonally feminized males. Sequence analysis showed the presence of approximately 0.6 kb retrotransposon-like sequence at its 3' end. Homologous sequences were isolated from diploid female, hexaploid male and monoecious plants. These sequences contained RNaseH and integrase domains of reverse transcriptase and were most similar to pineapple retrotransposon dea1, hence were named M. annua retrotransposon-like sequences (MARL-1 to MARL-5). A 771 bp fragment isolated from a diploid female, named fem771, was homologous to the 5' end of OPB01-1562. Results from DNA blot hybridization suggested OPB01-1562 and fem771 to be from the same locus and MARL-1 from a different one. RNA blot hybridization with OPB01-1562 and MARL-1 detected an approximately 2.8 kb transcript which was expressed strongly in stems and flowers of females but not males. This transcript was named M. annua female expressed (Mafex). Sex linkage of OPB01-1562 and expression of Mafex detected by OPB01-1562 strongly suggested Mafex to be a candidate gene involved in sex determination in M. annua.

Alterations in protein synthesis and levels of heat shock 70 proteins in response to salt stress of the halotolerant yeast Rhodotorula mucilaginosa.

Responses of the halotolerant yeast Rhodotorula mucilaginosa YRH2 to salt stress was studied. Strain YRH2 was isolated from chemical industry park wastewater evaporation ponds that are characterized by large fluctuations in salinity and pH. Upon shift to high salt medium there is a shutdown of protein synthesis. Radiolabeling and separation of proteins from salt stressed and non-stressed cells identified down-regulated heat shock 70 proteins Ssb1/2p, by N-terminal sequencing and Western blotting. Ssb's role in salt stress in both R. mucilaginosa and S. cerevisiae was examined and we show that its response to salt stress and amino acid limitation is similar. Other proteins such as the heat shock 70 protein Kar2p/BiP and Protein Disulfide Isomerase were strongly induced in response to a shift to high salt in R. mucilaginosa and reacted in a manner similar to the effect of tunicamycin, a known unfolded protein response inducer. Also, assaying carboxypeptidase Y, we showed that high salt medium reduces the specific activity of the enzyme in R. mucilaginosa. It is suggested that the changes in the expression of the heat shock 70 proteins is a part of a mechanism which alleviates the damaging effects of high salt on protein folding in the yeast Rhodotorula mucilaginosa.