Enrichment of bacteria possessing catechol dioxygenase genes in the rhizosphere of Spirodela polyrrhiza: A mechanism of accelerated biodegradation of phenol

The bacterial community structure in bulk water and in rhizosphere fractions of giant duckweed, Spirodela polyrrhiza, was quantitatively and qualitatively investigated by PCR-based methods using 6 environmental water samples to elucidate the mechanisms underlying selective accumulation of aromatic compound-degrading bacteria in the rhizosphere of S. polyrrhiza. S. polyrrhiza selectively accumulated a diverse range of aromatic compound-degrading bacteria in its rhizosphere, regardless of the origin of water samples, despite no exposure to phenol. The relative abundances of the catechol 1,2-dioxygenase (C12O) gene (C12O DNA) and catechol 2,3-dioxygenase (C23O) gene (C23O DNA) were calculated as the ratios of the copy numbers of these genes to the copy number of 16S rDNA and are referred to as the rhizosphere effect (RE) value. The RE values for C12O DNA and C23O DNA were 1.0 × 10¹–9.3 × 10³ and 1.7 × 10²–1.5 × 10⁴ times as high, respectively, in rhizosphere fractions as in bulk water fractions, and these higher values were associated with a notably higher sequence diversity of C12O DNA and C23O DNA. The RE values during phenol degradation were 3.6 × 10⁰–4.3 × 10² and 2.2 × 10⁰–1.7 × 10², respectively, indicating the ability of S. polyrrhiza to selectively accumulate aromatic compound-degrading bacteria in its rhizosphere during phenol degradation. The bacterial communities in the rhizosphere fractions differed from those in the bulk water fractions, and those in the bulk water fractions were notably affected by the rhizosphere bacterial communities. S. polyrrhiza released more than 100 types of phenolic compound into its rhizosphere as root exudates at the considerably high specific release rate of 1520 mg TOC and 214 mg phenolic compounds/d/g root (wet weight). This ability of S. polyrrhiza might result in the selective recruitment and accumulation of a diverse range of bacteria harboring genes encoding C12O and C23O, and the subsequent accelerated degradation of phenol in the rhizosphere.     

Identification of important microbial populations in the mesophilic and thermophilic phenol-degrading methanogenic consortia

Active mesophilic and thermophilic phenol-degrading methanogenic consortia were obtained after an 18-month acclimation and enriching process in the serum bottles, and characterized using the rRNA-based molecular approach. As revealed by cloning, fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP), these two enrichments differed greatly in the community structures. The results for the first time suggest that group TA in the Deltaproteobacteria (88.0% of EUBmix FISH-detectable bacterial cell area) and Pelotomaculum spp. in the Desulfotomaculum family (81.2%) were the predominant fermentative bacteria under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions, respectively. These populations closely associated with mesophilic and thermophilic members of Methanosaetaceae, Methanobacteriaceae and Methanomicrobiales to mineralize phenol as the sole carbon substrate to carbon dioxide and methane. Moreover, these two enrichments could mineralize terephthalate and benzoate. During benzoate degradation in the mesophilic enrichment, a shift in the predominant bacterial population from Deltaproteobacteria group TA to Syntrophus spp. was observed, suggesting Syntrophus-related spp. could have a higher substrate affinity for benzoate. FISH further revealed that member of the Deltaproteobacteria group TA represented more than 68.3% of EUBmix FISH-detectable bacterial cell area in a full-scale mesophilic bioreactor treating phenol-containing wastewaters.     

Bench-scale and field-scale evaluation of catechol 2,3-dioxygenase specific primers for monitoring BTX bioremediation

The objective of this work was to test a molecular genetic method for in situ monitoring of aerobic benzene, toluene, and xylene (BTX) biodegrading microorganisms. Catechol 2,3-dioxygenase (C23DO) genes occur in bacteria that biodegrade benzene, toluene, xylenes, phenol, biphenyl, and naphthalene. A competitive quantitative polymerase chain reaction (QC-PCR) technique using a single set of primers specific for an entire subfamily of C23DO genes was recently developed. To determine whether bacteria containing these C23DO genes actually exist in environments contaminated by BTX, aerobic microcosms containing previously uncontaminated soil were amended with different aromatic hydrocarbons and DNA extracts were analyzed by QC-PCR for C23DO genes. Anaerobic microcosms were established to confirm that oxygen was also necessary for the enrichment of C23DO genes. Field testing was done at two sites undergoing monitored natural attenuation. In microcosm experiments naphthalene, m-xylene, and p-xylene strongly enriched for C23DO genes while benzene, toluene, and o-xylene produced only transient, weakly detectable genes. In the field study, C23DO genes were detected in groundwater samples contaminated with either xylenes or naphthalene. The results of this study demonstrated that molecular genetic techniques can provide an accurate and rapid method to detect microorganisms capable of aromatic hydrocarbon biodegradation. Such a technique would be useful for monitoring the effectiveness of aeration technologies and for documenting microbial processes for monitored natural attenuation.     

Benzoate and salicylate degradation by Halomonas campisalis, an alkaliphilic and moderately halophilic microorganism

Alkaliphiles and halophiles have considerable potential to treat organic pollutants in industrial wastewaters having high pH and salinity. As model aromatic compounds, benzoate and salicylate at concentrations up to 380 mg/L were degraded as carbon and energy sources by Halomonas campisalis, an alkaliphile and moderate halophile. Aerobic batch experiments were performed at 50 and 100g/L NaCl and pH 9. Detected metabolites, catechol and cis, cis-muconate, indicated that H. campisalis used the ortho degradation pathway for both substrates. For benzoate concentrations up to 1600 mg/L, the intermediate 2-hydroxymuconic semialdehyde (2HMSA), characteristic of the meta pathway, was not detected. Improved understanding and characterization of these degradation processes in high pH, high salt systems may lead to improved application of haloalkaliphiles for industrial wastewater treatment.     

Seasonal and diel distributions of denitrifying and bacterial communities in a hypersaline microbial mat (Camargue, France)

Changes in spatio-temporal distribution of bacterial and denitrifying communities were qualitatively studied in a microbial mat from Camargue (France). During a diel and a seasonal cycle, patterns of 16S rRNA and nitrite reductase genes (nirS and nirK) were compared by denaturing gradient gel electrophoresis (DGGE). Statistical analysis of DGGE profiles showed a significant seasonal shift in the community structure of the nirS-containing bacteria with a winter superficial population that extended in summer, whereas the nirK-containing bacteria seemed more affected by vertical gradients rather than by month-to month-changes. Denitrifying activities remained stable during these sampling times. The bacterial community at the surface of the mat also changed according to season, but appeared stable over a day. Finally, during a diel cycle nirK populations were localized in zones with large fluctuations of environmental parameters (oxygen, pH, and sulfur levels) while nirS populations seemed more restricted to the permanent anoxic layer of the microbial mat.     

Monitoring thiocyanate-degrading microbial community in relation to changes in process performance in mixed culture systems near washout

Changes in microbial community structure, associated with changes in process performance, were investigated with respect to the sludge retention time (SRT) in bioreactors treating thiocyanate. Among the seven reactors operated at 0.8-3.0 d SRTs, respectively, the reactor at 2.0 d SRT displayed the maximal thiocyanate removal rate of 240.2mg/L/d. However, the thiocyanate removal efficiency suddenly decreased from 96.1% to 43.1% when the SRT was reduced from 2.0 to 1.8d, corresponding to a 50.1% drop in the removal rate. Microbial communities in the reactors operated at short SRTs, near washout, were analyzed by denaturing gradient gel electrophoresis (DGGE) based on bacterial 16S rRNA genes. All band sequences recovered were assigned to two phyla, Proteobacteria and Bacteriodetes. A Thiobacillus-like microorganism was commonly detected in all the reactors and is suggested to be the main organism responsible for thiocyanate decomposition. Several DGGE band sequences were closely related to the environmental clones detected in environments rich in sulfur and/or nitrogen compounds. Statistical analysis of the DGGE profiles demonstrated that the structure of thiocyanate-degrading communities, as well as the process performance, changed with change in SRT. The microbial community profiles were not always more closely related to those at similar SRT than those at less similar SRT on the non-metric multidimensional scaling (NMDS) map. This was also supported by clustering analysis. These results were contrary to the general notion that the community structures in continuous systems will be controlled by the washout of microbial populations. Our experimental results suggest that the structure of a microbial thiocyanate-degrading community at a given SRT would not be determined only by the washout effect.     

Natural organic matter (NOM) removal and structural changes in the bacterial community during artificial groundwater recharge with humic lake water

This study evaluated the removal of natural organic matter (NOM) and structural changes in the microbial community during infiltration of humic lake water at three artificial groundwater recharge (AGR) sites in Finland. The three sites were at waterworks in H?meenlinna, Jyv?skyl? and Tuusula, sites A, B and C, respectively. Site A used groundwater recharge by both basin and sprinkling infiltration, site B used only sprinkling infiltration, and site C used only basin infiltration. Reductions of total organic carbon at sites A, B and C were 91%, 84% and 74%, respectively, in the winter, and 88%, 77% and 73%, respectively, in the summer. The Finnish national recommended value of 2 mg/l for TOC was achieved at all sites and the TOC of natural groundwater at site C was much lower, at 0.6 mg/l. Large molecular fractions of NOM were removed more efficiently than the smaller ones. Total amount of DAPI-stained cells decreased during infiltration at sites A, B and C in winter by 94%, 94% and 75% and in summer by 96%, 97% and 94%, respectively. Bacterial communities in raw waters and extracted groundwaters were diverse with changes occurring during infiltration, which was shown by DNA extraction followed by PCR of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) fingerprinting. While the natural groundwater microbial community was diverse, it was different from that of the extracted groundwater in the AGR area. Simultaneous organic carbon removal and the decrease of bacterial counts during infiltration indicated biodegradation. In addition, the changing DGGE profiles during the process of infiltration, demonstrated that changing environmental conditions were reflected by changes in bacterial community composition.     

Microbial community analysis of thermophilic contact oxidation process by using ribosomal RNA approaches and the quinone profile method

Microbial community structure of a lab scale thermophilic aerobic wastewater treatment reactor was analyzed by a combination of culture-independent methods. Quinone profile method provides for chemical analysis of respiratory quinone molecular species, which corresponds to bacterial groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA partial sequences (PCR-DGGE) clarifies community changes at species level, as DGGE can separate DNA fragments of different sequences. Certain phvlogenetic groups of bacterial cells can be labeled by fluorescence in situ hybridization (FISH). Quinone profile showed a predominant presence of MK-7. PCR-DGGE revealed that constituents of the community were unchanged during the stable phase. FISH demonstrated the existence of the relatives of Bacillus lentus and B. thermocloacae in considerable proportions. The community was mainly composed of Bacillaceae, and obligate thermophilic and mesophilic Bacillus appeared in spite of the temperature fluctuation from 35 degrees C to 60 degrees C. The combination of these culture-independent methods revealed the community precisely enough to evaluate the reactor performance.     

Biodegradation of triclosan by a wastewater microorganism

Triclosan, a synthetic antimicrobial agent, has been considered as an emerging environmental contaminant. Here we reported a triclosan-degrading wastewater bacterial isolate, Sphingopyxis strain KCY1, capable of dechlorinating triclosan with a stoichiometric release of chloride. The stain can degrade diphenyl ether but not 2,4,4′-tribromodiphenyl ether and 2,2′,4,4′-tetrabromodiphenyl ether, despite all these three compounds are structurally similar to triclosan. While strain KCY1 was unable to grow on triclosan and catechol, it could grow with glucose, sodium succinate, sodium acetate, and phenol. When grown with complex nutrient medium containing a trace amount of triclosan (as low as 5 μg/L), the strain could retain its degradation ability toward triclosan. The maximum-specific triclosan degradation rate (q_m) and the half-velocity constant (K_m) are 0.13 mg-triclosan/mg-protein/day and 2.8 mg-triclosan/L, respectively. As triclosan degradation progressed, five metabolites were identified and these metabolites continue to transform into non-chlorinated end products, which was supported by a sharp drop in androgenic potential. The activity of catechol 2,3-dioxygenase in the cell extract was detected. No triclosan degradation was observed in the presence of 3-fluorocatechol, an inhibitor of meta-cleavage enzyme, suggesting that triclosan degradation proceed via meta-cleavage pathway. Based on all the observations, a degradation pathway for triclosan by strain KCY1 was proposed.     

Relationship between phenol degradation efficiency and microbial community structure in an anaerobic SBR

Phenol is a common wastewater contaminant from various industrial processes, including petrochemical refineries and chemical compounds production. Due to its toxicity to microbial activity, it can affect the efficiency of biological wastewater treatment processes. In this study, the efficiency of an Anaerobic Sequencing Batch Reactor (ASBR) fed with increasing phenol concentrations (from 120 to 1200 mg L⁻¹) was assessed and the relationship between phenol degradation capacity and the microbial community structure was evaluated. Up to a feeding concentration of 800 mg L⁻¹, the initial degradation rate steadily increased with phenol concentration (up to 180 mg L⁻¹ d⁻¹) and the elimination capacity remained relatively constant around 27 mg phenol removed?gVSS⁻¹ d⁻¹. Operation at higher concentrations (1200 mg L⁻¹) resulted in a still efficient but slower process: the elimination capacity and the initial degradation rate decreased to, respectively, 11 mg phenol removed?gVSS⁻¹ d⁻¹ and 154 mg L⁻¹ d⁻¹. As revealed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis, the increase of phenol concentration induced level-dependent structural modifications of the community composition which suggest an adaptation process. The increase of phenol concentration from 120 to 800 mg L⁻¹ had little effect on the community structure, while it involved drastic structural changes when increasing from 800 to 1200 mg L⁻¹, including a strong community structure shift, suggesting the specialization of the community through the emergence and selection of most adapted phylotypes. The thresholds of structural and functional disturbances were similar, suggesting the correlation of degradation performance and community structure. The Canonical Correspondence Analysis (CCA) confirmed that the ASBR functional performance was essentially driven by specific community traits. Under the highest feeding concentration, the most abundant ribotype probably involved in successful phenol degradation at 1200 mg L⁻¹ was affiliated to the Anaerolineaceae family.     

Bioremediation of diesel oil in a co-contaminated soil by bioaugmentation with a microbial formula tailored with native strains selected for heavy metals resistance

The aim of the work is to assess the feasibility of bioremediation of a soil, containing heavy metals and spiked with diesel oil (DO), through a bioaugmentation strategy based on the use of a microbial formula tailored with selected native strains. The soil originated from the metallurgic area of Bagnoli (Naples, Italy). The formula, named ENEA-LAM, combines ten bacterial strains selected for multiple resistance to heavy metals among the native microbial community. The biodegradation process of diesel oil was assessed in biometer flasks by monitoring the following parameters: DO composition by GC-MS, CO₂ evolution rate, microbial load and composition of the community by T-RFLP, physiological profile in Biolog® ECOplates and ecotoxicity of the system. The application of this microbial formula allowed to obtain, in the presence of heavy metals, the complete degradation of n-C_12-20, the total disappearance of phenantrene, a 60% reduction of isoprenoids and an overall reduction of about 75% of the total diesel hydrocarbons in 42 days. Concurrently with the increase of metabolic activity at community level and the microbial load, the gradual abatement of the ecotoxicity was observed. The T-RFLP analysis highlighted that most of the ENEA-LAM strains survived and some minor native strains, undetectable in the soil at the beginning of the experiment, developed. Such a bioaugmentation approach allows the newly established microbial community to strike a balance between the introduced and the naturally present microorganisms. The results indicate that the use of a tailored microbial formula may efficiently facilitate and speed up the bioremediation of matrices co-contaminated with hydrocarbons and heavy metals. The study represents the first step for the scale up of the system and should be verified at a larger scale. In this view, this bioaugmentation strategy may contribute to overcome a critical bottleneck of the bioremediation technology.     

Spatial and temporal changes in Actinobacterial dominance in experimental artificial groundwater recharge

Artificial groundwater recharge (AGR) is used in the drinking water industry to supplement groundwater resources and to minimise the use of chemicals in water treatment. This study analysed the spatial and temporal changes of microbial communities in AGR using two test systems: a nutrient-amended fluidized-bed reactor (FBR) and a sand column. Structural changes in the feed lake water (Lake Roine), FBR, and sand column bacterial communities were determined by denaturing gradient gel electrophoresis (DGGE) and the length heterogeneity analysis of amplified 16S rRNA genes (LH-PCR). Two clone libraries were created to link the LH-PCR results to the dominant bacterial groups. The lake water bacterial community was relatively stable, with three bands dominating in all LH-PCR products. The most dominant fragment accounted for up to 72% and was derived from Actinobacteria. Based on the clone libraries and LH-PCR data, Actinobacteria also dominated in the unattached bacterial community of the FBR, whereas several Proteobacterial groups were more abundant on the FBR carrier particles. In the stabilised AGR system a major change in the community structure of the lake water bacteria took place during passage within the first 0.6 m in the sand column as the community composition shifted from Actinobacteria-dominated populations to a diverse, mainly Proteobacterial communities. Concurrently, most of the dissolved organic carbon (DOC) was removed at this stage. In summary, the study showed that the make-up of microbial communities in experimental AGR systems responded to changes in their environment. LH-PCR showed potential as a method to determine microbial community dynamics in long-term studies at real-scale AGR sites. This is the first step to provide data on microbial community dynamics in AGR for drinking water production.     

Biodegradation of pyridine using aerobic granules in the presence of phenol

Aerobic granules cultivated with 500mg/L phenol medium effectively degraded pyridine at a concentration of 250-2500mg/L; maximum degradation rate was 73.0mg pyridineg/VSS/h at 250mg/L pyridine concentration. Phenol concentrations of 500-2000mg/L limited pyridine degradation in a competitive inhibition pattern, as interpreted using Michaelis-Menten kinetics with corresponding parameters V(max), K(m) and K(I) of 63.7mg/Lh(-1), 827.8 and 1388.9mg/L, respectively. Fluorescent staining and confocal laser scanning microscopy (CLSM) tests suggested that an active biomass accumulated at the granule outer layer. Denaturing gradient gel electrophoresis (DGGE) fingerprint profile demonstrated that dominating microbial strains exist in phenol and pyridine-degrading aerobic granules.     

Improvement of oxidative decomposition of aqueous phenol by microwave irradiation in UV/H2O2 process and kinetic study

2.5GHz of microwave irradiation can cause a considerable improvement of oxidative decomposition of aqueous phenol in a UV/H2O2 system. The experimental results showed that the microwave irradiation can raise both the phenol conversion and the TOC removal efficiency up to or above 50%. Also, the microwave irradiation could considerably enhance the oxidative degradation of phenol in the UV/H2O2 system even under a suppression of thermal effect. Addition of hydrogen peroxide by more than a stoichiometric amount was critical to mineralize aqueous phenol to create a short reaction time. Also, microwave irradiation can accelerate the degradation rate of intermediates, hydroquinone and catechol, produced in the course of phenol oxidative decomposition. From the kinetic study, the disappearance rate of phenol can be expressed as dX/dt = kPH[M]0(alpha - X)(1 - X), where alpha equivalent [H2O2]0/[M]0 + kOH[OH*]/kPH[M]0, shows a good correlation with the experimental data. The kinetic analysis showed that an indirect reaction of phenol with OH radical might be dominant in the absence of microwave irradiation, meanwhile a direct reaction of phenol with hydrogen peroxide might be dominant in the presence of microwave irradiation except for low concentrations of hydrogen peroxide.     

Aerobic Biological Treatment of a Pharmaceutical Wastewater: : Effect of Temperature on COD Removal and Bacterial Community Development

The effect of temperature was studied on the efficiency of soluble COD removal and bacterial community development during the aerobic biological treatment of a pharmaceutical wastewater. Using wastewater and bacterial inoculum obtained from the full-scale facility treating this wastewater, batch laboratory cultures were operated at 5°C intervals from 30°C to 70°C. Following four culture transfers to allow for bacterial acclimation, residual soluble COD levels were measured and bacterial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. Soluble COD removal efficiency declined as temperature increased from 30°C (62%) to 60°C (38%). Biological treatment of this wastewater failed to occur at temperatures higher than 60°C. Gradual shifts in bacterial community structure were detected as temperature increased, including a concomitant reduction in the number of different bacterial populations. The impact of temperature on a two-stage biological treatment process was also compared. Better soluble COD removal was achieved when both reactors were operated at 30°C compared to a system where the two stages were consecutively operated at 55°C and 30°C. These results indicate that operation of aerobic biological wastewater treatment reactors at elevated temperatures can have adverse effects on process performance.     

Strategies for targeting marker bacterial oxygenases involved in transformation of hydrocarbons in contaminated soil

A variety of activities leads to release of petroleum products in soil. In this study, DNA extracted from oil and grease contaminated soil by a simple technique was used as a template for Polymerase Chain Reaction based assays. Target gene sequences of certain key bacterial oxygenases involved in transformation of aromatic hydrocarbons viz. naphthalene dioxygenase, catechol 2,3‐dioxygenase, cytochrome P‐450cam monooxygenase as well as eubacterial 16S rRNA gene, were evaluated for determining the presence of microbial genotypes in soil. It was concluded that duplex, nested and multiplex PCR assay strategies in combination with residue analysis can play a crucial role in the selection of treatment processes as well as monitoring the efficacy of bioremedial technique.     

Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles

Cork manufacturing is a traditional industry in Southern Europe, being the main application of this natural product in wine stoppers and insulation. Cork processing begins at boiling the raw material. As a consequence, great volumes of dark wastewaters, with elevated concentrations of chlorophenols, are generated, which must be depurated through costly physicochemical procedures before discarding them into public water courses. This work explores the potential of bacteria, isolated from cork-boiling waters storage ponds, in bioremediation of the same effluent. The bacterial population present in cork-processing wastewaters was analysed by DGGE; low bacterial biodiversity was found. Aerobic bacteria were isolated and investigated for their tolerance against phenol and two chlorophenols. The most tolerant strains were identified by sequencing 16S rDNA. The phenol-degrading capacity was investigated by determining enzyme activities of the phenol-degrading pathway. Moreover, the capacity to form biofilms was analysed in a microtitre plate assay. Finally, the capacity to form biofilms onto the surface of residual small cork particles was evaluated by acridine staining followed by epifluorescence microscopy and by SEM. A low-cost bioremediation system, using phenol-degrading bacteria immobilised onto residual cork particles (a by-product of the industry) is proposed for the remediation of this industrial effluent (self-bioremediation).     

The inoculum effect on the ammonia-oxidizing bacterial communities in parallel sequential batch reactors

Three identical sequential batch reactors (SBRs) were each inoculated with sludge from a full-scale wastewater treatment plant (WWTP) treating a waste stream of different origin, i.e. a hospital, a meat processing company, and a municipal WWTP. The SBRs were run in parallel for 84 consecutive days to investigate whether the reactors would become more phylogenetically similar or stay separated concerning their functionality and microbial communities. Overall, the nitrification functionality was high throughout the experiment, and the size and structure of the sludge flocs were very similar. The total bacterial and ammonia-oxidizing bacterial (AOB) communities were analyzed by PCR-DGGE. Cluster analysis demonstrated very distinct bacterial communities in the three SBRs, not showing any trend becoming more similar. The carrying capacity, dynamics and functional organization of the communities were assessed by DGGE analysis and based on these patterns the range-weighted richness, moving window analysis, and constructing Pareto-Lorenz evenness distribution curves were calculated. Between the SBRs, highly comparable internal structure and dynamics of the AOB communities were observed, although they had only one AOB DGGE band in common. These observations indicate that community characteristics such as the extent of biodiversity and dynamics are more important indicators of good microbial functionality than the presence of certain specific species.     

Co-variations of bacterial composition and catabolic genes related to PAH degradation in a produced water treatment system consisting of successive anoxic and aerobic units

This paper reports on the investigation of concentration levels of PAHs, community structure, as well as the abundance of PAH-related catabolic genes including upper-pathway dioxygenase genes (nahAc and phnAc) and down-pathway catechol dioxygenase genes (C12O and C23O) in a successive anoxic and aerobic treatment of produced water from the Jidong Oilfield, China. 93% of total PAHs were removed, almost equally contributed by the anoxic and aerobic units. However, PAHs of more than 3 benzene rings remained almost unchanged. The signals for phnAc and C12O were undetectable in this biological system, whereas the existence of nahAc and C23O was confirmed in the system and the copies of the two genes in the aerobic tank were 2 or 3 orders higher than those in the influent water sample. The different behavior of C23O demonstrated that mineralization of PAHs might mainly occur in the aerobic unit. The existence of nahAc and C23O genes in the influent and the high similarity of genotype between the influent and the two sludge samples suggested that bacteria existing in the influent contributed to PAH removal and bacteria harboring PAH catabolic genes were enriched in the sludge.     

The effect of inoculum on the performance of sulfate-reducing columns treating heavy metal contaminated water

Sulfate-reducing permeable reactive zones (SR-PRZs) are a passive means of immobilizing metals and neutralizing the pH of mine drainage through microbially mediated reactions. In this bench-scale study, the influence of inoculum on the performance of columns simulating SR-PRZs was investigated using chemical and biomolecular analyses. Columns inoculated from two sources (bovine dairy manure (DM) and a previous sulfate-reducing column (SRC)) and uninoculated columns (U) were fed a simulated mine drainage and compared on the basis of pH neutralization and removal of cadmium, zinc, iron, and sulfate. Cadmium, zinc, and sulfate removal was significantly higher in SRC columns than in the DM and U columns, while there was no significant difference between the DM and U columns. Denaturing gradient gel electrophoresis (DGGE) analysis revealed differences in the microbial community composition among columns with different inocula, and indicated that the microbial community in the SRC columns was the first to reach a pseudo-steady state. In the SRC columns, a higher proportion of the DGGE band DNA sequences were related to microorganisms that carry out cellulose degradation, the rate-limiting step in SR-PRZ energy flow, than was the case in the other columns. The proportion of sulfate-reducing bacteria of the genus Desulfobacterium was monitored using real-time quantitative PCR and was observed to be consistently higher in the SRC columns. The results of this study suggest that the inoculum plays an important role in SR-PRZ performance. This is the first report providing a detailed analysis of the effect of different microbial inocula on the remediation of acid mine drainage.