Thermophilic microbial cellulose decomposition and methanogenesis pathways recharacterized by metatranscriptomic and metagenomic analysis.

The metatranscriptomic recharacterization in the present study captured microbial enzymes at the unprecedented scale of 40,000 active genes belonged to 2,269 KEGG functions were identified. The novel information obtained herein revealed interesting patterns and provides an initial transcriptional insight into the thermophilic cellulose methanization process. Synergistic beta-sugar consumption by Thermotogales is crucial for cellulose hydrolysis in the thermophilic cellulose-degrading consortium because the primary cellulose degraders Clostridiales showed metabolic incompetence in subsequent beta-sugar pathways. Additionally, comparable transcription of putative Sus-like polysaccharide utilization loci (PULs) was observed in an unclassified order of Bacteroidetes suggesting the importance of PULs mechanism for polysaccharides breakdown in thermophilic systems. Despite the abundance of acetate as a fermentation product, the acetate-utilizing Methanosarcinales were less prevalent by 60% than the hydrogenotrophic Methanobacteriales. Whereas the aceticlastic methanogenesis pathway was markedly more active in terms of transcriptional activities in key genes, indicating that the less dominant Methanosarcinales are more active than their hydrogenotrophic counterparts in methane metabolism. These findings suggest that the minority of aceticlastic methanogens are not necessarily associated with repressed metabolism, in a pattern that was commonly observed in the cellulose-based methanization consortium, and thus challenge the causal likelihood proposed by previous studies.

Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach.

Antibiotic resistance has become a serious threat to human health. Sewage treatment plant (STP) is one of the major sources of antibiotic resistance genes (ARGs) in natural environment. High-throughput sequencing-based metagenomic approach was applied to investigate the broad-spectrum profiles and fate of ARGs in a full scale STP. Totally, 271 ARGs subtypes belonging to 18 ARGs types were identified by the broad scanning of metagenomic analysis. Influent had the highest ARGs abundance, followed by effluent, anaerobic digestion sludge and activated sludge. 78 ARGs subtypes persisted through the biological wastewater and sludge treatment process. The high removal efficiency of 99.82% for total ARGs in wastewater suggested that sewage treatment process is effective in reducing ARGs. But the removal efficiency of ARGs in sludge treatment was not as good as that in sewage treatment. Furthermore, the composition of microbial communities was examined and the correlation between microbial community and ARGs was investigated using redundancy analysis. Significant correlation between 6 genera and the distribution of ARGs were found and 5 of the 6 genera included potential pathogens. This is the first study on the fate of ARGs in STP using metagenomic analysis with high-throughput sequencing and hopefully would enhance our knowledge on fate of ARGs in STP.

Metagenomic analysis of sludge from full-scale anaerobic digesters operated in municipal wastewater treatment plants.

This study applied Illumina high-throughput sequencing to explore the microbial communities and functions in anaerobic digestion sludge (ADS) from two wastewater treatment plants based on a metagenomic view. Taxonomic analysis using SILVA SSU database indicated that Proteobacteria (9.52-13.50 %), Bacteroidetes (7.18 %-10.65 %) and Firmicutes (7.53 %-9.46 %) were the most abundant phyla in the ADS. Differences of microbial communities between the two types of ADS were identified. Genera of Methanosaeta and Methanosarcina were the major methanogens. Functional analysis by SEED subsystems showed that the basic metabolic functions of metagenomes in the four ADS samples had no significant difference among them, but they were different from other microbial communities from activated sludge, human faeces, ocean and soil. Abundances of genes in methanogenesis pathway were also quantified using a methanogenesis genes database extracted from KEGG. Results showed that acetotrophic was the major methanogenic pathway in the anaerobic sludge digestion.

Hydrophobicity of diverse bacterial populations in activated sludge and biofilm revealed by microbial adhesion to hydrocarbons assay and high-throughput sequencing.

Cell hydrophobicity is one of the key physicochemical properties of bacteria in activated sludge (AS) and biofilms can influence the efficient operation of wastewater treatment plants (WWTPs). In the present study the cell hydrophobicity of diverse bacterial populations in AS and biofilms from the Shatin and Stanley WWTPs of Hong Kong was characterized by combining the microbial adhesion to hydrocarbons (MATH) assay with the Illumina high-throughput sequencing. The results indicated that, at the phylum level, a majority of bacteria in AS and biofilms showed medium hydrophobicity. Most of the top 20 bacterial genera in the AS samples were hydrophilic. However, the top 20 genera in biofilms showed higher hydrophobicity than in the top 20 genera in AS samples, suggesting more hydrophobic bacteria existed in biofilms than in AS. Meanwhile, the hydrophobicity of two specific bacterial groups, including foaming and biosurfactant-producing bacteria, were also evaluated. The results demonstrated that, by combining the MATH assay with the Illumina sequencing approach, bacterial hydrophobicity could be evaluated with high efficiency and coverage in complex systems with high microbial diversity, e.g. AS and biofilms in WWTPs.

Mining of novel thermo-stable cellulolytic genes from a thermophilic cellulose-degrading consortium by metagenomics.

In this study, metagenomics was applied to characterize the microbial community and to discover carbohydrate-active genes of an enriched thermophilic cellulose-degrading sludge. The 16S analysis showed that the sludge microbiome was dominated by genus of cellulolytic Clostridium and methanogenesis Methanothermobacter. In order to retrieve genes from the metagenome, de novo assembly of the 11,930,760 Illumina 100 bp paired-end reads (totally 1.2 Gb) was carried out. 75% of all reads was utilized in the de novo assembly. 31,499 ORFs (Open Reading Frame) with an average length of 852 bp were predicted from the assembly; and 64% of these ORFs were predicted to present full-length genes. Based on the Hidden Markol Model, 253 of the predicted thermo-stable genes were identified as putatively carbohydrate-active. Among them the relative dominance of GH9 (Glycoside Hydrolase) and corresponding CBM3 (Carbohydrate Binding Module) revealed a cellulosome-based attached metabolism of polysaccharide in the thermophilic sludge. The putative carbohydrate-active genes ranged from 20% to 100% amino acid sequence identity to known proteins in NCBI nr database, with half of them showed less than 50% similarity. In addition, the coverage of the genes (in terms of ORFs) identified in the sludge were developed into three clear trends (112×, 29× and 8×) in which 85% of the high coverage trend (112×) mainly consisted of phylum of Firmicutes while 49.3% of the 29× trend was affiliated to the phylum of Chloroflexi.

Quantification and characterization of ß-lactam resistance genes in 15 sewage treatment plants from East Asia and North America.

The emerging antibiotic resistance genes in the aquatic environment have aroused public concern. As ß-lactam is the most widely used group of antibiotics, ß-lactam resistance genes were selected to investigate their distribution and diversity in the activated sludge from 15 geographically different sewage treatment plants (STPs) of China, Singapore, USA, and Canada. Specific PCR and quantitative real-time PCR (q-PCR) were used to investigate the occurrence and abundance of nine ß-lactam resistance genes. Five genes (OXA-1, OXA-2, OXA-10, ampC, and TEM-1) were detected in most of the sludge collected, while three genes (mecA, CTX-M-1, and SME) were not found in any sludge sample. The total abundances of the six detected ß-lactam resistance genes in the 15 STPs varied from 5.34 × 10(1) copies/ng DNA (ampC) to 5.49 × 10(4) copies/ng DNA (OXA-1). Overall, OXA-1 had the highest total concentration, followed by IMP and OXA-10. Noticeably, the abundances of TEM-1 in Chinese STPs were generally higher than those in the STPs of other countries, while the abundances of OXA-2 and IMP in the STPs of North America were much greater than those of East Asia. A total of 78 clones carrying ß-lactam resistance genes were randomly selected from six clone libraries for phylogenetic diversity analysis; the similarity of these cloned genes to known ß-lactam resistance genes with sequence identities ranged from 96% to 100%. Furthermore, OXA-1, ampC, and IMP were found to be more diverse than the other ß-lactam resistance genes.

Enhanced anoxic bioremediation of PAHs-contaminated sediment.

In this study, the biodegradation of 16 polycyclic aromatic hydrocarbons (PAHs) in marine sediment was investigated under three different anoxic conditions, i.e. sulfate-only, nitrate-only and mixed nitrate/sulfate as electron acceptors. All two-, three- and four-ring PAHs showed significant biodegradation with the removal efficiencies ranging from 42% to 77%, while five- and six-ring PAHs showed little degradation. The results illustrated that two- to three-ring PAHs could be degraded at a rate of 4.01×10(-2)-6.42×10(-2) d(-1) under nitrate-reducing condition, faster than that of under sulfate-reducing condition. Biodegradation of two- and three-ring PAHs followed first-order model well with the rate constants of 1.62×10(-2)-6.42×10(-2) d(-1). The biodegradation of four ring PAHs followed the zero-order kinetic model with the rate constants of 1.26×10(-2)-2.22×10(-2) mg/kg/d. Molecular analysis indicated that nahAc gene increased by two orders of magnitude during the biodegradation and served as a good indicator of PAHs-degrading bacterial population and biodegradation process.

Effects of Mo(VI) on phototrophic hydrogen production by Rhodobacter sphaeroides.

Effects of Mo(6+) concentration on phototrophic hydrogen production of Rhodobacter sphaeroides were investigated using lactate as the sole carbon source. Results showed that an increase of Mo(6+) from nil to 1000 microg l(-1) led to increases in hydrogen yield, maximum production rate, conversion efficiency, biomass yield and lactate removal. At 100 microg-Mo l(-1), the maximum rate was 12.0 ml h(-1) with a conversion efficiency of 36.1%, the cell yields were 1.11 g-cell g(-1) -lactate and 2.4 g-cell g(-1)-TOC removed. Further increase of Mo(6+) improved hydrogen production only marginally. Degradation of lactate by R. sphaeroides produced not just hydrogen but also acetate, butyrate, i-valerate, i-caproate, hexanoate and some unidentified organic intermediates, but did not produce propionate and alcohols. Nitrogenase activity, as measured by the acetylene reduction method, had no clear correlation with either Mo(6+) concentration or hydrogen yield.

Quantification of the lateral detachment force for bacterial cells using atomic force microscope and centrifugation.

To determine the lateral detachment force for individual bacterial cells, a quantitative method using the contact mode of an atomic force microscope (AFM) was developed in this study. Three key factors for the proposed method, i.e. scan size, scan rate and cantilever choice, were evaluated and optimized. The scan size of 40×40 µm² was optimal for capturing sufficient number of adhered cells in a microscopic field and provide adequate information for cell identification and detachment force measurement. The scan rate affected the measurement results significantly, and was optimized at 40 µm/s considering both force measurement accuracy and experimental efficiency. The hardness of applied cantilevers also influenced force determination. The proposed protocol for cantilever selection is to use those with the lowest spring constant first and then step up to a harder cantilever until all cells are detached. The lateral detachment force of Escherichia coli cells on polished stainless steel and a glass-slide coated with poly-l-lysine were measured as 0.763±0.167 and 0.639±0.136 nN, respectively. The results showed that the established method had good repeatability and sensitivity to various bacteria/substrata combinations. The detachment force quantified by AFM (0.639±0.136 nN) was comparable to that measured by the centrifugation method (1.12 nN).

Class 1 integronase gene and tetracycline resistance genes tetA and tetC in different water environments of Jiangsu Province, China.

Class 1 integronase gene (intI1) and tetracycline resistance genes (tetA and tetC) from various environmental sites in Jiangsu Province (China) were detected using qualitative PCR (polymerase chain reaction) and quantified with SYBR Green-based qRT-PCR (quantitative real-time PCR) in this study. Qualitative PCR assays demonstrated that intI1, tetA and tetC occurred in the water environments of Taihu Lake, the Nanjing section of the Yangtze River, a sewage treatment plant (STP) in Nanjing City, and two drinking water treating bioreactors. qRT-PCR results showed that abundance of intI1 in lake water and sediments was lower than the tet genes, for a given sample site and date (P < 0.05). On a volumetric basis, lake sediments contained higher concentrations of the three genes by four to five orders of magnitude than water samples, and lake water and sediments sampled in April contained fewer copies of all the genes than the samples collected in June and August (P < 0.05). The levels of intI1, tetA and tetC in the Yangtze River water increased significantly after the river flowed through Nanjing City (P < 0.05). 94.1% integron, 97.2% tetA and 98.3% tetC were removed by the activated sludge process in the STP, and more than 80% of each gene was removed in both of the two biofilters in terms of relative concentration based on sample volume. However, on the basis of DNA mass, lower removals were obtained for both the activated sludge and biofiltration processes.

Autotrophic denitrification and its effect on metal speciation during marine sediment remediation.

Denitrification-based remediation has been proved as a cost-effective approach for organic removal in sediment. However, little attention has been drawn on the concomitant autotrophic denitrification process and its impacts during such treatment. In this study, a contaminated marine sediment sample was treated with nitrate in a series of experiments to characterize the autotrophic denitrification and its impacts on metal speciation. Through treatment, as the consequence of autotrophic denitrification which accounts for 73.9% of nitrate reduction, approximately 98.8% acid volatile sulfide (AVS) was oxidized to sulfate, causing changes of Zn, Cu and Pb speciation in the sediment. Their oxidizable fractions decreased by 71.7%, 13% and 71% respectively while the bound-to-carbonate fractions increased by 52.0%, >700% and >40%, and the reducible fractions also increased by 276%, >280% and 140%. Thus, the relatively stable oxidizable phase of Zn, Cu and Pb was generally transferred to the more mobile bound-to-carbonate and reducible phases. According to SEM (simultaneously extracted metal) analysis, most of extractable Zn and Pb were no longer present in the form of metal sulfides after denitrification. The (Zn+Pb)/AVS ratio increased from 0.030 to 3.1. Both sequential extraction and AVS-SEM suggested a possible increase of heavy metal mobility and, thus, toxicity. Two major species responsible for autotrophic denitrification were identified to be phylogenetically related with Sulfurimonas paralvinellae and Thiohalophilus thiocyanoxidans.

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants.

Class 1 integrons and gene cassettes containing antibiotic resistance genes (ARGs) in five different sewage treatment plants (STPs) were characterized and quantified using polymerase chain reaction (PCR), sequencing, and quantitative real-time PCR (qRT-PCR) in this study. Class 1 integronase gene (intI1) was found commonly occurring in all of activated sludge samples from the five STPs, as well as in influent and effluent of two STPs at Hong Kong. One hundred and nine lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from activated sludge of Shatin STP. Among them, 36 strains (33.0%) were found to carry class 1 integrons. PCR assays showed that 11 of the 36 intI1-carrying isolates harbored a common type of gene cassette array of about 1,600 bps, as well as the static genes (sulI and qacEDelta1) on class 1 integrons. This gene cassette array was found phylogenetically close to antibiotic resistance genes dfr17 and aadA5, encoding dihydrofolate reductase conferring resistance to trimethoprim and adenylyltransferase conferring resistance to spectinomycin/streptomycin, respectively. Antimicrobial susceptibility analysis demonstrated that all the 11 LFEs carrying gene cassette were multi-resistant, especially having common resistance to trimethoprim and streptomycin. qRT-PCR assay showed that genes copies of both class 1 integron and the gene cassette varied significantly among the activated sludge sampled from different STPs, at different time points or different treatment steps. More than 90% of class 1 integrons and the gene cassette were removed by activated sludge processes in two STPs, while the disinfection process removed 94% integron and 77% gene cassette in one STP.

Antibiotic resistance genes in water environment.

The use of antibiotics may accelerate the development of antibiotic resistance genes (ARGs) and bacteria which shade health risks to humans and animals. The emerging of ARGs in the water environment is becoming an increasing worldwide concern. Hundreds of various ARGs encoding resistance to a broad range of antibiotics have been found in microorganisms distributed not only in hospital wastewaters and animal production wastewaters, but also in sewage, wastewater treatment plants, surface water, groundwater, and even in drinking water. This review summarizes recently published information on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs, including specific and multiplex PCR (polymerase chain reaction), real-time PCR, DNA sequencing, and hybridization based techniques.

Phthalates biodegradation in the environment.

Phthalates are synthesized in massive amounts to produce various plastics and have become widespread in environments following their release as a result of extensive usage and production. This has been of an environmental concern because phthalates are hepatotoxic, teratogenic, and carcinogenic by nature. Numerous studies indicated that phthalates can be degraded by bacteria and fungi under aerobic, anoxic, and anaerobic conditions. This paper gives a review on the biodegradation of phthalates and includes the following aspects: (1) the relationship between the chemical structure of phthalates and their biodegradability, (2) the biodegradation of phthalates by pure/mixed cultures, (3) the biodegradation of phthalates under various environments, and (4) the biodegradation pathways of phthalates.

Use of TaqMan gene probe for real-time monitoring of acidophilic hydrogen-producing bacteria.

Using a TaqMan gene probe targeting the 16S rDNA of acidophilic hydrogen-producing bacteria (HPB), the abundance of this HPB in the biomass was found to increase from 0.02 to 72% in a single batch treating rice slurry waste at pH 4.5 over 130 h. The corresponding abundances were 4.4% in the batch operated at pH 5.0 and 0.01-0.02% at pH 5.5-6.5. During the growth phase, the generation time for the acidophilic HPB at pH 4.5 averaged 3.5 h.

Anaerobic degradation of dimethyl phthalate in wastewater in a UASB reactor.

Over 99% of dimethyl phthalate (DMP) and 93% of chemical oxygen demand (COD) were effectively removed in a continuous upflow anaerobic sludge blanket (UASB) reactor from a wastewater containing 600 mg/L DMP at 8h of hydraulic retention time (HRT), corresponding to a loading rate of 3g-COD/(Ld). Each gram of sludge, expressed as volatile suspended solids (VSS), had a maximum specific methanogenic activity (SMA) of 24 mg-CH(4)/(g-VSSd) using DMP as the sole carbon source. The sludge yield was estimated as 0.08 g-VSS/g-COD. During anaerobic degradation, DMP was de-esterified, first to mono-methyl phthalate (MMP) and then to phthalate, before being de-aromatized and subsequently converted to CH(4) and CO(2). The maximum specific degradation rates of DMP, MMP and phthalate were 415, 88 and 36 mg/(g-VSSd), respectively. Analysis based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) showed a gradual shift of microbial population with the increase of DMP loading.

Inhibition of heavy metals on fermentative hydrogen production by granular sludge.

This study was conducted to investigate the toxicity of six electroplating metals on the H(2)-producing activity of a granular sludge sampled from an H(2)-producing upflow reactor treating sucrose-containing wastewater. The H(2) production activities of the sludge were measured in serum vials using wastewater containing not just sucrose and proper nutrient, but also individual heavy metals at concentrations ranging 0-5000 mg l(-1). The relative toxicity to H(2) production was found in the following order: Cu (most toxic)>>Ni approximately Zn>Cr>Cd>Pb (least toxic). The C(I,50) values, at which the bioactivity of the sludge was reduced to 50% of the control, for individual heavy metals were Cu 30 mg l(-1), Ni and Zn 1600 mg l(-1), Cr 3000 mg l(-1), Cd 3500 mg l(-1), and Pb >5000 mg l(-1). Compared with the literature data, H(2)-producing sludge exhibited in general higher resistance to metal toxicity than methanogenic granular sludge.

Denitrifying degradation of dimethyl phthalate.

Results of batch experiments on the denitrifying degradation of dimethyl phthalate (DMP) was most favorable at pH 7-9 and 30-35 degrees C. DMP was first degraded to monomethyl phthalate (MMP), which was in turn degraded to phthalate before complete mineralization. There was no fatty acid residue in the mixed liquor throughout the experiments. The maximum specific degradation rates were 0.32 mM/(gVSS x h) for DMP, 0.19 mM/(gVSS x h) for MMP, and 0.14 mM/(gVSS x h) for phthalate. About 86% of available electron in DMP was utilized for denitrification; the remaining 14% was presumable conserved in the new biomass with an estimated yield of 0.17 mg/mg DMP. Based on 16S rDNA analysis, the denitrifying sludge was mainly composed of beta-subdivision and alpha-subdivision of Proteobacteria (33 and 5 clones out of a total of 43 clones, respectively), plus some Acidobacteria. Using a primer set specifically designed to amplify the denitrification nirK gene, 10 operational taxonomy units (OTUs) were recovered from the clone library. They clustered into a group in the alpha-subdivision of Proteobacteria most closely related to denitrifier Bradyrhizobium japonicum USDA110 and several environmental clones.

Aerobic degradation of diethyl phthalate by Sphingomonas sp.

An aerobic diethyl phthalate (DEP) degrading bacterium, DEP-AD1, was isolated from activated sludge. Based on its 16S rDNA sequence, this isolate was identified belonging to Sphingomonas genus with 99% similarity to Sphingomonas sp. strain C28242 and 98% similarity to S. capsulate. The specific degradation rate of DEP was concentration dependent with a maximum of 14 mg-DEP/(Lh). Results of degradation tests showed that DEP-AD1 could also degrade monoethyl phthalate (MEP), dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethylhexyl phthalate (DEHP), but not phthalate and benzoate.

Bacterial survival in evaporating deposited droplets on a teflon-coated surface.

Understanding of bacterial survival in aerosols is crucial for controlling infection transmission via airborne aerosols and/or large droplets routes. The cell viability changes of four bacteria species (Escherichia coli K12 JM109; Acinetobacter sp. 5A5; Pseudomonas oleovorans X5; and Staphylococcus aureus X8), three Gram-negative and one Gram-positive, in a large evaporating droplet of size 1,800 microm in diameter on teflon-coated slides were measured using the LIVE/DEAD BacLight solution and a microscope. Droplets of three levels of salinity (0, 0.9, and 36% w/v) were tested. All four species survived well during the droplet evaporation process, but died mostly at the time when droplets were dried out at 40-45 min. The final bacteria survival rate after droplets were completely dried was dependent on bacteria species and the salinity of the suspension solution. Droplet evaporation over the first 35-40 min had no adverse effect on bacterial survival for the droplets tested. The lethal effect of desiccation was found to be the most important death mechanism.