Effects of gene-augmentation on the formation, characteristics and microbial community of 2,4-dichlorophenoxyacetic acid degrading aerobic microbial granules.

Development of 2,4-dichlorophenoxyacetic acid (2,4-D) degrading aerobic granular sludge was conducted in two sequencing batch reactors (SBR) with one bioaugmented with a plasmid pJP4 donor strain Pseudomonas putida SM1443 and the other as a control. Half-matured aerobic granules pre-grown on glucose were used as the starting seeds and a two-stage operation strategy was applied. Granules capable of utilizing 2,4-D (about 500 mg/L) as the sole carbon source was successfully cultivated in both reactors. Gene-augmentation resulted in the enhancement of 2,4-D degradation rates by the percentage of 65-135% for the granules on Day 18, and 6-24% for the granules on Day 105. Transconjugants receiving plasmid pJP4 were established in the granule microbial community after bioaugmentation and persisted till the end of operation. Compared with the control granules, the granules in the bioaugmented reactor demonstrated a better settling ability, larger size, more abundant microbial diversity and stronger tolerance to 2,4-D. The finally obtained granules in the bioaugmented and control reactor had a granule size of around 600 µm and 500 µm, a Shannon-Weaver diversity index (H) of 0.96 and 0.55, respectively. A shift in microbial community was found during the granulation process.

[Study on gfp labeling of a 2,4-D degrading strain and its detection in a wastewater biotreatment system].

A 2,4-dichlorophenoxyacetic acid (2,4-D) degrading special bacteria Achromobacter sp. was chromosomally labeled with a green fluorescent protein gene (gfp) using a mini-Tn7 transposon delivery system. The growth status, fluorescence expression and degradation ability of the strain before and after labeling were compared. Methods to quantify the strain in different biotreatment systems (activated sludge or granular sludge system) after inoculation were also investigated. Results showed that the labeled Achromobacter sp. and its control strain demonstrated a similar growth pattern and 2,4-D degradation ability: both of them could completely remove 2, 4-D of about 100 mg/L within 103-112 h. The labeled strain could express fluorescence stably during the course of growth and degradation with fluorescence intensity/D600 stabilized at about 4500. For an activated sludge system bioaugmented with this labeled strain, its abundance could determined through direct measuring fluorescence emitted by the sludge mixture, for it was linearly associated to the percentage of the strain in the range of 0-75% (R2 = 0.9952). For a granular sludge system bioaugmented with this strain, fluorescence of the sludge mixture could be measured after homogenous pretreatment, and the percentage of the strain in the range of 0-42% was also linearly related to the fluorescence intensity emitted by the sludge mixture (R2 = 0.9801). Overall, this gfp labeling method based on Tn7 delivery system can be used to monitor specific bacteria in a biotreatment system.

Bioaugmentation of aerobic sludge granules with a plasmid donor strain for enhanced degradation of 2,4-dichlorophenoxyacetic acid.

Aerobic sludge granules pre-grown on glucose were bioaugmented with a plasmid pJP4 carrying strain Pseudomonas putida SM1443 in a fed-batch microcosm system and a lab-scale sequencing batch reactor (SBR) to enhance their degradation capacity to 2,4-dichlorophenoxyacetic acid (2,4-D). The fed-batch test results showed that the bioaugmented aerobic granule system gained 2,4-D degradation ability faster and maintained a more stable microbial community than the control in the presence of 2,4-D. 2,4-D at the initial concentration of about 160 mg/L was nearly completely removed by the bioaugmented granule system within 62 h, while the control system only removed 26% within 66 h. In the bioaugmented SBR which had been operated for 90 days, the seeded aerobic granules pre-grown on glucose successfully turned into 2,4-D degrading granules through bioaugmentation and stepwise increase of 2,4-D concentration from 8 to 385 mg/L. The granules showed a compact structure and good settling ability with the mean diameter of about 450 microm. The degradation kinetics of 2,4-D by the aerobic granules can be described with the Haldane kinetics model with V(max)=31.1 mg 2,4-D/gVSS h, K(i)=597.9 mg/L and K(s)=257.3 mg/L, respectively. This study shows that plasmid mediated bioaugmentation is a feasible strategy to cultivate aerobic granules degrading recalcitrant pollutants.

[Effects of carbon sources changes on the property and morphology of 2,4-D degraded aerobic sludge granules].

This study investigated the changes of the morphology, structure, and capability of removing the target contamination of the aerobic granules pre-cultured with mixed substrates of glucose and 2,4-dichlorophenoxyacetic acid (2,4-D) in a long-time running sequence batch reactor (SBR), when the carbon source transformed into the sole carbon source of 2,4-D. Results showed that when the substrate turned to the sole carbon source of 2,4-D, the aerobic granules still maintained a strong degradation ability to the target contamination; a 2,4-D removal percentage of 99.2% -100% and an average COD removal rate of 85.6% were achieved at the initial 2,4-D concentration of 361-564 mg/L. Carbon source transformation caused certain damages to the original aerobic granule structure, made some parts of granules disintegrated, and led to granule size decline from 513 microm to 302 microm. However, those granules maintained the main body, re-aggregated and grew after a period of adaptation due to their strong resistance to toxicity. Aerobic granules capable of utilizing 2,4-D as the sole carbon source with a good settling ability (SYI 20-40 mL/g) and a mean diameter of 489 microm were finally obtained in this study. Scanning electron microscope (SEM) observation showed that the diversity of granule microbial species was declined when turned to the sole carbon source.