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The protein coding genes (rbcL/cbbL/cbbM) for RuBisCO large subunit, the most abundant protein on earth that drives biological CO2 fixation, were considered as useful marker genes in characterizing CO2-assimilating plankton. However, their community specificity has hindered comprehensive screening of genetic diversity. In this study, six different rbcL/cbbL/cbbM primers were employed to screen clone libraries to identify CO2-assimilating plankton in Jiaozhou Bay. The following community compositions were observed: the community components in Form I A/B rbcL/cbbL clone library mainly comprised Chlorophyta and Proteobacteria, Form ID2 and ID3 libraries consisted of Bacillariophyta, Form II cbbM library consisted of Proteobacteria and Alveolata, and both Form I green and red libraries included Proteobacteria, respectively. At the genus taxonomic level, no overlaps among these clone libraries were observed, except for ID2 and ID3. Overall, the phytoplankton in Jiaozhou Bay mainly consists of Bacillariophyta, Chlorophyta, Cryptophyta, Haptophyceae and Alveolata. The CO2-assimilating prokaryotes mainly consist of Proteobacteria. Considering the high-sequence specificities of these marker genes, we propose that the joint use of multiple primers may be utilized in unveiling the diversity of CO2-assimilating organisms. In addition, designing novel RuBisCO gene primers that generate longer amplicons and have broader phylogenetic coverage may be necessary in the future.

Pseudomonas sp. strain WBC-3 mineralizes the priority pollutant para-nitrophenol (PNP) and releases nitrite (NO2 (-)), which is probably involved in the nitrification. In this study, the rate of PNP removal in soil bioaugmented with strain WBC-3 was more accelerated with more NO2 (-) accumulation than in uninoculated soils. Strain WBC-3 survived well and remained stable throughout the entire period. Real-time polymerase chain reaction (real-time PCR) indicated a higher abundance of ammonia-oxidizing bacteria (AOB) than ammonia-oxidizing archaea (AOA), suggesting that AOB played a greater role in nitrification in the original sampled soil. Real-time PCR and multivariate analysis based on the denaturing gradient gel electrophoresis showed that PNP contamination did not significantly alter the abundance and community structure of ammonia oxidizers except for inhibiting the AOB abundance. Bioaugmentation of PNP-contaminated soil showed a significant effect on AOB populations and community structure as well as AOA populations. In addition, ammonium (NH4 (+)) variation was found to be the primary factor affecting the AOB community structure, as determined by the correlation between the community structures of ammonia oxidizers and environmental factors. It is here proposed that the balance between archaeal and bacterial ammonia oxidation could be influenced significantly by the variation in NH4 (+) levels as caused by bioaugmentation of contaminated soil by a pollutant containing nitrogen.

The Gram-negative strain of Pseudomonas plecoglossicida NyZ12 isolated from soil has the ability to degrade cyclohexylamine (CHAM). The genes encoding CHAM degradation by gram-negative bacteria, however, have not been reported previously. In this study, ORFs predicted to encode CHAM degradation by NyZ12 were identified by bioinformatics analysis. Differential expression of the proposed ORFs was analyzed via RNA-seq and quantitative reverse transcription-PCR (qRT-PCR), using RNA extracted from NyZ12 cultured with or without CHAM addition. One CHAM-inducible ORF, RK21_02867 predicted to encode a cyclohexanone monooxygenase (ChnB) was disrupted, as were five ORFs, RK21_00425, RK21_02631, RK21_04207, RK21_04637 and RK21_05539, that had weak homology to the only known cyclohexylamine oxidase (CHAO encoded by chaA) found in Brevibacterium oxydans IH-35A. We also found that a tandem array of five ORFs (RK21_02866-02870) shared homology with those in an operon responsible for oxidation of cyclohexanone to adipic acid, although the ORFs in strain NyZ12 were arranged in a different order with previously found in cyclohexane, cyclohexanol or cyclohexanone degradation strains. The ORFs in this cluster were all up-regulated when CHAM was supplied as the sole carbon source. When one of these five genes, RK21_02867 encoding cyclohexanone (CHnone) monooxygenase, was knocked out, NyZ12 could not grow on CHAM, but it accumulated equimolar amounts of CHnone. Our results show that strain NyZ12 metabolized CHAM directly to CHnone which was then further metabolized to adipate. Despite clearly identifying genes encoding the steps for metabolism of CHAM metabolites, not every one of the putative chaAs was differentially expressed in the presence of CHAM and deletion of each one individually did not completely eliminate the capacity of NyZ12 to degrade CHAM, though it did reduce its growth in several instances. Our results suggest that there is genetic redundancy encoding the initial step in the oxidation of CHAM to CHnone in NyZ12 and that its CHAOs differ considerably from the ChaA, originally described in Brevibacterium oxydans IH-35A.

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (MP) and para -nitrophenol as the sole source of carbon, nitrogen and energy. In this study, strain WBC-3 was inoculated into lab-scale MP-contaminated soil for bioaugmentation. Accelerated removal of MP was achieved in bioaugmentation treatment compared to non-bioaugmentation treatment, with complete removal of 0.536 mg g −1 dry soil in bioaugmentation treatment within 15 days and without accumulation of toxic intermediates. The analysis of denaturing gradient gel electrophoresis and real-time PCR showed that strain WBC-3 existed stably during the entire bioaugmentation period. Simultaneously, redundancy analysis for evaluating the relationships between the environmental factors and microbial community structure indicated that the indigenous bacterial community structure was significantly influenced by strain WBC-3 inoculation ( P = 0.002).

A strain utilizing cyclohexylamine as the sole source of carbon and nitrogen, designated NyZ12, was isolated from soil and identified by 16S rDNA sequencing as Pseudomonas plecoglossicida. This bacterium released ammonia into the medium when grown on cyclohexylamine, and also grows readily on cyclohexanone as the sole carbon source, suggesting that degradation involves an initial deamination step.

A consortium consisting of para -nitrophenol utilizer Pseudomonas sp. strain WBC-3, meta -nitrophenol utilizer Cupriavidus necator JMP134 and ortho -nitrophenol utilizer Alcaligenes sp. strain NyZ215 was inoculated into soil contaminated with three nitrophenol isomers for bioaugmentation. Accelerated removal of all nitrophenols was achieved in inoculated soils compared to un-inoculated soils, with complete removal of nitrophenols in inoculated soils occurring between 2 and 16 days. Real-time polymerase chain reaction (PCR) targeting nitrophenol-degradation functional genes indicated that the three strains survived and were stable over the course of the incubation period. The abundance of total indigenous bacteria (measured by 16S rRNA gene real-time PCR) was slightly negatively impacted by the nitrophenol contamination. Denaturing gradient gel electrophoresis profiles of total and group-specific indigenous community suggested a dynamic change in species richness occurred during the bioaugmentation process. Furthermore, Pareto–Lorenz curves and Community organization parameters indicated that the bioaugmentation process had little impact on species evenness within the microbial community.