Highly efficient biodegradation of reactive blue 19 under the activation of tea residue by a newly screened mixed bacterial flora DDMY2

In this study, a newly screened mixed bacterial flora DDMY2 had high decolorization capacity for anthraquinone dye reactive blue 19 (RB19) and the decolorization efficiency of 300 mg L−1 RB19 could reach up to 98% within 48 h in the presence of tea residue. Results indicated that RB19 could be efficiently decolorized by flora DDMY2 in wide ranges of pH values (5.0–9.0), temperatures (30–40 °C) and initial dye concentrations (50–500 mg L−1) under the activation of tea residue. Concentration of tea residue had been proved to significantly impact the decolorization performance. UV-vis spectrophotometry, Fourier transform infrared spectrometry and liquid chromatography/time-of-flight/mass spectrometry analysis showed three identified degradation products and the possible degradation pathway of RB19 was speculated. High-throughput sequencing analysis revealed the community structures of bacterial flora before and after domestication by tea residue. Based on the result, it was inferred that unclassified_o_Pseudomonadales, Brevibacillus, Stenotrophomonas and Bordetella activated by tea residue were responsible for the excellent decolorization performance. Results of this research deepen our understanding of the biodegradation process of anthraquinone dyes by bacterial flora and broaden the knowledge of utilizing tea residue as a bioactivator in biological treatment.


Screening of RB19 decolorizing bacterial flora DDMY2
The CM medium (90 mL) was added in a 250 mL flask, and then inoculated 10 mL activated sludge suspension with 10% (v/v).The dye concentration gradually increased in sequences of 10, 20, 30, 50, 100, 200 and 300 mg L -1 .The culture was incubated at 37 ℃ under static condition for 48 h.When the decolorization rate at a certain dye concentration reached up to 80% or more after 48 h, it was transferred to a new medium with higher dye concentration, which was named gradient pressure acclimation method. 1 This process was repeated until dye concentration reached to 300 mg L -1 and the decolorization rate was 70%, the domestication process stopped and bacterial flora was collected, named as DDMY2.

Sample preparation for FTIR analysis
The culture medium containing RB19 and its decolorizing solution were harvested and centrifuged at 6200 × g for 10 min to obtain the supernatant.Afterwards, the achieved supernatant was extracted using dichloromethane (three times with 40, 30 and 30 mL, respectively) to obtain the extraction products.Subsequently, the extraction was transferred to an eggplant-shaped flask, placed in a water bath at 30 ℃ and subjected to rotary evaporation.
With the solution including extraction dried out, dichloromethane (2 mL) was used to dissolve the extraction.The new solution was concentrated to dry under nitrogen and the dried extraction was placed on the Attenuated Total Reflection instrument (Smart TR diamond).
The PCR products were examined on a 2% (w/v) agarose gel, purified with the AxyPrepDNA Gel (Axygen, CA, USA) and quantified using QuantiFluor ST (Promega, USA).The purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols. 2,3ter sequencing, raw fastq files were demultiplexed and quality-filtered using Quantitative Insights into Microbial Ecology (QIIME).Therefore, 34628 reads had been randomly picked from the sample, the reads were grouped into operational taxonomic units (OTUs) with 3% dissimilarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME.The rarefaction curve was generated using Mothur program, and the representative sequences were assigned at different taxonomic levels using RDP Classifier (http://rdp.cme.msu.edu/)against the silva (SSU115) 16S rRNA database using confidence threshold of 70%.

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Fig. S1 Degradation products of RB19 detected and identified by LC-TOF-MS: total ion

Fig. S2
Fig. S2 Sobs (a) and Shannon (b) rarefaction curves based on the 16S rDNA gene sequencing