Microbial Population and Activity in Wetland Microcosms Constructed for Improving Treated Municipal Wastewater

The idea of using constructed wetlands for the treatment and improving of wastewater emerged in the second half of the last century. Despite relatively wide use of this environmentally friendly technology, relatively little is known about the microbial populations involved in biotransformation and removal of contaminants in this system. The aim of the current study was to investigate the assembly and function of microbial populations in vertical-flow constructed wetland microcosms designed to improve the quality of wastewater after activated sludge treatment. Also, the performance of 3-year-old wetland ponds was investigated. Even though the quality of the influent water was relatively high, improvement in water parameters such as coliform level, ammonia concentration, BOD, and TSS was observed. The performance of the wetland ponds was comparable to that of the microcosms. The microbial community composition of the biofilm formed on the surface of gravel particles in vegetated and plant-free microcosms was studied by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Highly complex bacterial diversity was observed in the biofilm. Cluster analysis of DGGE patterns demonstrated that depth within the wetland microcosm has a stronger effect on microbial community composition of the biofilm formed on wetland matrix than vegetation. Measurements of fluorescein diacetate hydrolysis activity and nitrification potential revealed that hydrolytic activity was affected by both microcosm depth and vegetation presence, whereas nitrification potential was mostly influenced by depth. Resolving the bacterial assemblage of wetland biofilm, which often is considered a black box, will help to understand the interactions involved in the development of diverse and mature biofilm and its function.     

Urea Transformation of Wetland Microbial Communities

Transformation of urea to ammonium is an important link in the nitrogen cycle in soil and water. Although microbial nitrogen transformations, such as nitrification and denitrification, are well studied in freshwater sediment and epiphytic biofilm in shallow waters, information about urea transformation in these environments is scarce. In this study, urea transformation of sedimentary, planktonic, and epiphytic microbial communities was quantified and urea transformation of epiphytic biofilms associated with three different common wetland macrophyte species is compared. The microbial communities were collected from a constructed wetland in October 2002 and urea transformation was quantified in the laboratory at in situ temperature (12°C) with the use of the ¹⁴C-urea tracer method, which measures the release of ¹⁴CO₂ as a direct result of urease activity. It was found that the urea transformation was 100 times higher in sediment (12–22 mmol urea-N m⁻² day⁻¹) compared with the epiphytic activity on the surfaces of the submerged plant Elodea canadensis (0.1–0.2 mmol urea-N m⁻² day⁻¹). The epiphytic activity of leaves of Typha latifolia was lower (0.001–0.03 mmol urea-N m⁻² day⁻¹), while urea transformation was negligible in the water column and on the submerged leaves of the emergent plant Phragmites australis. However, because this wetland was dominated by dense beds of the submerged macrophyte E. canadensis, this plant provided a large surface area for epiphytic microbial activity—in the range of 23–33 m² of plant surfaces per square meter of wetland. Thus, in the wetland system scale at the existing plant distribution and density, the submerged plant community had the potential to transform 2–7 mmol urea-N m⁻² day⁻¹ and was in the same magnitude as the urea transformation in the sediment.     

Diversity,Distribution and Hydrocarbon Biodegradation Capabilities of Microbial Communities in Oil-Contaminated Cyanobacterial Mats from a Constructed Wetland

Various types of cyanobacterial mats were predominant in a wetland, constructed for the remediation of oil-polluted residual waters from an oil field in the desert of the south-eastern Arabian Peninsula, although such mats were rarely found in other wetland systems. There is scarce information on the bacterial diversity, spatial distribution and oil-biodegradation capabilities of freshwater wetland oil-polluted mats. Microbial community analysis by Automated Ribosomal Spacer Analysis (ARISA) showed that the different mats hosted distinct microbial communities. Average numbers of operational taxonomic units (OTUs_ARISA) were relatively lower in the mats with higher oil levels and the number of shared OTUs_ARISA between the mats was <60% in most cases. Multivariate analyses of fingerprinting profiles indicated that the bacterial communities in the wetland mats were influenced by oil and ammonia levels, but to a lesser extent by plant density. In addition to oil and ammonia, redundancy analysis (RDA) showed also a significant contribution of temperature, dissolved oxygen and sulfate concentration to the variations of the mats’ microbial communities. Pyrosequencing yielded 282,706 reads with >90% of the sequences affiliated to Proteobacteria (41% of total sequences), Cyanobacteria (31%), Bacteriodetes (11.5%), Planctomycetes (7%) and Chloroflexi (3%). Known autotrophic (e.g. Rivularia) and heterotrophic (e.g. Azospira) nitrogen-fixing bacteria as well as purple sulfur and non-sulfur bacteria were frequently encountered in all mats. On the other hand, sequences of known sulfate-reducing bacteria (SRBs) were rarely found, indicating that SRBs in the wetland mats probably belong to yet-undescribed novel species. The wetland mats were able to degrade 53–100% of C₁₂–C₃₀ alkanes after 6 weeks of incubation under aerobic conditions. We conclude that oil and ammonia concentrations are the major key players in determining the spatial distribution of the wetland mats’ microbial communities and that these mats contribute directly to the removal of hydrocarbons from oil field wastewaters.     

废水处理中的可溶性微生物产物

可溶性微生物产物（SMP）是污水生物处理中有机物质的主要成分,对出水水质有着重要影响。对污水生物处理中SMP的产生、分子质量分布、生物可降解性、毒性、离子螯合性、吸附性等进行了综述,分析了影响SMP产生的因子。分析表明,SRT、HRT、进水浓度、基质性质、有毒物质的存在以及温度都会对SMP的产生及浓度产生影响,控制系统运行的这些参数,可使出水中SMP量降到最少,从而提高出水质量。     

Comparison and Characterization of Microbial Communities in Sulfide-rich Wastewater with and without Propidium Monoazide Treatment

A 16S rRNA gene-based culture-independent approach was used to study the bacterial and archaeal communities in a sulfide-rich wastewater. Propidium Monoazide (PMA) treatment was applied to limit the analysis to the fraction of viable cells in environment. A total of 104 and 68 clones respective from bacterial clone library and archaeal library were picked and analyzed by restriction fragment length polymorphism (RFLP). 35 RFLP patterns from bacterial clone library and 10 RFLP patterns from archaeal clone library were unique and the respective clones were selected for sequencing. BLAST analysis and RFLP analysis showed that the bacterial clone library mainly consisted of Gammaproteobacteria (73%), Anaerolineae (6%), Bacilli (5%), Deltaproteobacteria (7%), Clostridia (4%), Bacteroidetes (1%), and Chlorobia (1%); Methanomicrobia (99%) and Thermococci (1%) were the only two lineages of the archaeal domains. This study gave a first insight into the overall microbial structure in a cloth printing and dyeing wastewater treatment plant with high concentration of sulfide and increased knowledge on the applicability of the PMA treatment in combination with PCR-based molecular techniques to analyze only viable cells in microbial ecology.     

Removal of Anionic Surfactants from Wastewater Using a Constructed Wetland

Removal of anionic surfactants from municipal wastewater using a constructed wetland with a horizontal subsurface flow was studied in 2007 and 2008. Extraction spectrophotometry with methylene blue served to determine the analyte concentrations in individual samples. The anionic surfactant‐removal efficiency depended on actual conditions, mostly the treated water flow intensity, its temperature, and a redox‐potential gradient in the longitudinal profile of the wetland bed. It increased with decreasing inflow and increasing temperature. The average efficiency was 83.7% in 2007 and 81.7% in 2008; however, values higher than 85% were often determined during the summer period. On the other hand, the efficiencies were usually lower than 80% in winter, especially in periods with intensive precipitation and inflows. The average concentration of anionic surfactants in water taken at the outflow was lower than 0.65 mg/l (expressed as sodium dodecyl sulfate). The most significant fraction of anionic surfactants (almost 50%) was degraded at the beginning (1 m from the inflow zone) of the wetland bed. The rhizosphere aeration via the vegetation roots strongly supported the anionic‐surfactant removal.     

Removal of Selected Metals from Wastewater Using a Constructed Wetland

Removal of selected metals from municipal wastewater using a constructed wetland with a horizontal subsurface flow was studied. The objective of the work was to determine the efficiency of Cu, Zn, Ni, Co, Sr, Li, and Rb removal, and to describe the main removal mechanisms. The highest removal efficiencies were attained for zinc and copper (89.8 and 81.5%, respectively). It is apparently due to the precipitation of insoluble sulfides (ZnS, CuS) in the vegetation bed where the sulfate reduction takes place. Significantly lower removal efficiencies (43.9, 27.7, and 21.5%) were observed for Li, Sr, and Rb, respectively. Rather, low removal efficiencies were also attained for Ni and Co (39.8 and 20.9%). However, the concentrations of these metals in treated water were significantly lower compared to Cu and Zn (e.g., 2.8 ± 0.5 and 1.7 ± 0.3 μg/l for Ni at the inflow and outflow from the wetland compared to 27.6 ± 12.0 and 5.1 ± 4.7 μg/l obtained for Cu, respectively). The main perspective of the constructed wetland is the removal of toxic heavy metals forming insoluble compounds depositing in the wetland bed. Metal uptake occurs preferentially in wetland sediments and is closely associated with the chemism of sulfur and iron.     

Characterization of Microbial Communities in Gas Industry Pipelines

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria. The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales. Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion.     

Impact of Coal-Coking Effluent on Sediment Microbial Communities: a Multivariate Approach

The functional response to and recovery from coal-coking waste effluent was evaluated for sediment microbial communities. Twenty estimates of microbial population density, biomass, and activity were measured five times during a 15-month period. Significant effects on microbial communities were observed in response to both wastewater contamination and diversion of the wastewater. Multivariate analysis of variance and discriminant analysis indicated that accurate differentiation between uncontaminated and contaminated sediments required a minimum of nine estimates of community response. Total viable population density, ATP, alkaline phosphatase, naphthalene, and phenanthrene mineralization rates were found to be highly weighted variables in site discrimination. Lipid and glucose mineralization, nitrogen fixation, and sediment protein also contributed significantly to explaining variation among sites. Estimates of anaerobic population densities and rates of methane production contributed little to discrimination among sites in the environment examined. In general, total viable population density, ATP, and alkaline phosphatase activity were significantly depressed in contaminated sediments. However, after removal of this contamination, the previously affected sites demonstrated greater temporal variability but a closer approximation of the mean response at the control site. Naphthalene and phenanthrene mineralization did not follow the general trend and were elevated at the contaminated sites throughout the investigation. Results of the investigation supported the hypothesis that multiple functional measures of microbial community response are required to evaluate the effect of and recovery from environmental contamination. In addition, when long-term effects are evaluated, select physiological traits, i.e., polyaromatic hydrocarbon mineralization, may not reflect population and biomass estimates of community response.     

Adaptation of Phytoplankton-Degrading Microbial Communities to Thermal Reactor Effluent in a New Cooling Reservoir

In water column and sediment inocula from a nuclear reactor cooling reservoir, natural phytoplankton substrate labeled with ¹⁴C was used to determine aerobic and anaerobic mineralization rates for a range of temperatures (25, 40, 55, and 70°C) expected during reactor operation. For experiments that were begun during reactor shutdown, aerobic decomposition occurred at temperatures of <55°C. After 2 months of reactor operation, aerobic rates increased substantially at 55 and 70°C, although maximum rates were observed at temperatures of ≤40°C. The temperature range for which maximum anaerobic mineralization (i.e., the sum of CH₄ and CO₂) was observed was 25 to 40°C when the reactor was off, expanding to 25 to 55°C during reactor operation. Increased rates at 55°C, but not 70°C, correlated with an increase in the ratio of cumulative methane to carbon dioxide produced over 21 days. When reduced reactor power lowered the maximum temperature of the reservoir to 42°C, aerobic decomposition at 70°C was negligible, but remained substantial at 55°C. Selection for thermophilic decomposers occurred rapidly in this system in both aerobic and anaerobic communities and did not require prolonged exposure to elevated temperatures.     

Functional Gene Composition,Diversity and Redundancy in Microbial Stream Biofilm Communities

We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.     

Activity of Nitrifying Biofilms Constructed on Low-Density Polyester Enhances Bioremediation of a Coastal Wastewater Effluent

Summary Nitrifying biofilms were constructed on low density polyester Dacron for the bioremediation of nitrogen from wastewater effluent of a municipal treatment plant. Dacron disks were inoculated with wastewater sludge enriched for 15 days for either ammonia- or nitrite-oxidizing bacteria (AOB or NOB, respectively) and packed into glass bioreactors. Wastewater effluent containing high levels of ammonia, nitrite, and phosphate was collected and fed to inoculated and uninoculated bioreactors. Both inoculated bioreactors showed stable nitrification efficiencies, removing 96 and 76% of the ammonia and 12 and 35% of the nitrite for AOB- and NOB-inoculated bioreactors, respectively. Efficiencies of phosphate removal were similar in both inoculated and uninoculated bioreactors, indicating that nitrifiers were not required for this process. AOB-inoculated bioreactors accumulated nitrite mid-way through the experiment and had low rates of conversion to nitrate, suggesting slow nitrite oxidizer growth. DGGE and sequence analysis of AOB 16S rRNA genes showed enrichment of Nitrosomonas spp. in both inoculated bioreactors, and a dominance of Nitrosospira spp. in non-inoculated bioreactors. This study describes an inexpensive and efficient technology for removing ammonia and nitrite from wastewater effluents of municipal treatment plants before its release to the environment.     

Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.     

Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.     

The Influence of Tropical Plant Diversity and Composition on Soil Microbial Communities

There is growing interest in understanding the linkages between above- and belowground communities, and very little is known about these linkages in tropical systems. Using an experimental site at La Selva Biological Station, Costa Rica, we examined whether plant diversity, plant community composition, and season influenced microbial communities. We also determined whether soil characteristics were related to differences in microbial communities. Phospholipid fatty acid (PLFA) composition revealed that microbial community composition differed across a plant diversity gradient (plots contained 1, 3, 5, or over 25 species). Plant species identity also was a factor influencing microbial community composition; PLFA composition significantly varied among monocultures, and among three-species combinations that differed in plant species composition. Differences among treatments within each of these comparisons were apparent in all four sampling dates of the study. There was no consistent shift in microbial community composition between wet and dry seasons, although we did see significant changes over time. Of all measured soil characteristics, soil C/N was most often associated with changes in microbial community composition across treatment groups. Our findings provide evidence for human alteration of soil microbial communities via the alteration of plant community composition and diversity and that such changes are mediated in part by changes in soil carbon quality.     

Assessment of the Microbial Community in a Constructed Wetland that Receives Acid Coal Mine Drainage

Constructed wetlands are used to treat acid drainage from surface or underground coal mines. However, little is known about the microbial communities in the receiving wetland cells. The purpose of this work was to characterize the microbial population present in a wetland that was receiving acid coal mine drainage (AMD). Samples were collected from the oxic sediment zone of a constructed wetland cell in southeastern Ohio that was treating acid drainage from an underground coal mine seep. Samples comprised Fe(III) precipitates and were pretreated with ammonium oxalate to remove interfering iron, and the DNA was extracted and purified by agarose gel electrophoresis prior to amplification of portions of the 16S rRNA gene. Amplified products were separated by denaturing gradient gel electrophoresis and DNA from seven distinct bands was excised from the gel and sequenced. The sequences were matched to sequences in the GenBank bacterial 16S rDNA database. The DNA in two of the bands yielded matches with Acidithiobacillus ferrooxidans and the DNA in each of the remaining five bands was consistent with one of the following microorganisms: Acidithiobacillus thiooxidans, strain TRA3-20 (a eubacterium), strain BEN-4 (an arsenite-oxidizing bacterium), an Alcaligenes sp., and a Bordetella sp. Low bacterial diversity in these samples reflects the highly inorganic nature of the oxic sediment layer where high abundance of iron- and sulfur-oxidizing bacteria would be expected. The results we obtained by molecular methods supported our findings, obtained using culture methods, that the dominant microbial species in an acid receiving, oxic wetland are A. thiooxidans and A. ferrooxidans.     

藏灵菇微生物种群结构的分子特性研究

用PCR—DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78％-84％,酵母菌种群结构的相似性为80％-92％。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带（条带E）的序列与乳酸乳球菌的相似性为100％。     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.