Bacterial community analysis of anoxic/aeration (A/O) system in a combined process for gibberellin wastewater treatment

Gibberellin wastewater cannot be directly discharged without treatment due to its high concentrations of sulfate and organic compounds and strong acidity. Therefore, multi-stage anaerobic bioreactor + micro-aerobic+ anoxic/aeration (A/O) + biological contact oxidation combined processes are used to treat gibberellin wastewater. However, knowledge of the treatment effects of the A/O process and bacterial community structure in the aeration tank reactors of such systems is sparse. Therefore, this study was conducted to investigate the treatment effects and operation of the A/O process on gibberellin wastewater, as well as changes in the bacterial community structure of activated sludge in the aeration tank during treatment. Moreover, removal was examined based on evaluation of effluent after A/O treatment. Although influent chemical oxygen demand (COD), NH3-N and total phosphorus (TP) fluctuated, effluent COD, NH3-N and TP remained stable. Moreover, average COD, NH3-N and TP removal efficiency were 68.41%, 93.67% and 45.82%, respectively, during the A/O process. At the phylum level, Proteobacteria was the dominant phylum in all samples, followed by Chloroflexi, Bacteroidetes and Actinobacteria. Proteobacteria played an important role in the removal of organic matter. Chloroflexi was found to be responsible for the degradation of carbohydrates and Bacteroidetes also had been found to be responsible for the degradation of complex organic matters. Actinobacteria are able to degrade a variety of environmental chemicals. Additionally, Anaerolineaceae_uncultured was the major genus in samples collected on May 25, 2015, while Novosphingobium and Nitrospira were dominant in most samples. Nitrosomonas are regarded as the dominant ammonia-oxidizing bacteria, while Nitrospira are the main nitrite-oxidizing bacteria. Bacterial community structure varied considerably with time, and a partial Mantel test showed a highly significant positive correlation between bacterial community structure and DO. The bacterial community structure was also positively correlated with temperature and SO42-.


Introduction
Plant hormones are essential intrinsic regulators of plant development and growth [1].Gibberellins not only promote internode elongation and cambial activity of stems [2], but also participate in the regulation of plant growth and development [3], including seed germination, stem elongation, flowering development, fruit growth, and root development.Gibberellin wastewater is generated by biological fermentation processes.The raw materials and methods for production of gibberellin result in gibberellin wastewater containing high concentrations of sulfate, organic compounds, and NH 3 -N, as well as strong acidity.These features make wastewater difficult to treat; therefore, a highly efficient treatment is required before discharge to prevent pollution.Fig 1 shows the production process of gibberellin.
High concentrations of sulfate have been found to have an inhibitory effect on microorganisms during biological treatment [4].Nevertheless, Li. et al. [5] investigated the treatment of chemical synthesis-based pharmaceutical wastewater containing rich organic sulfur compounds and sulfate using an Upflow Anaerobic Sludge Blanket (UASB) reactor.They found that this method removed a considerable amount of COD and sulfate.The anaerobic/oxic (A/ O) process is consists of a sequential anaerobic and aerobic stage for the biological phosphorus removal [6].The A/O process has been widely used for sewage treatment in both urban and rural areas and is favored for its high efficiency and low energy consumption [7].In this study, the concentrations of organic materials and sulfate were both high, and the composition of wastewater was very complex.The process for treatment of gibberellin production wastewater with a multi-stage anaerobic bioreactor combined with micro oxygen, A/O, and contact oxidation was established herein, while also applying chemical dosing to reduce the sulfate.
The specific purpose of this study was to investigate the impacts and operational conditions of the A/O process when applied for nutrient removal from gibberellin wastewater and to investigate changes in the bacterial community structure and diversity in activated sludge during treatment.The results demonstrated stability and effectiveness of the A/O process based on eight months of observation.Moreover, the bacterial community diversity in activated sludge was studied by Illumina MiSeq sequencing.

Ethics statement
Permission for the samples was granted by the Jiangxi Ruifeng Biochemical Co. Ltd. (Jiangxi, China).The field studies also did not involve endangered or protected species.

Raw water
The wastewater was obtained from a biochemical company located in Jiangxi province, China (115.478584˚E,27.900485˚N).This company can produce 110 tons of gibberellin per year, accounting for more than 39% of the world's total gibberellin production.The gibberellin wastewater consists of three parts: raffinate wastewater, high concentration wastewater and comprehensive wastewater.In addition, the gibberellin wastewater contained high concentrations of sulfate and organic compounds, as well as strong acidity.The quality of gibberellin wastewater was shown in Table 1.

Start-up and operation of A/O system
The A/O system could remove most of the NH 3 -N and a portion of the organics.The volume of A/O system was 2200m 3 and the hydraulic retention time was 26.52h.The inoculum of in the A/O system is activated sludge and it was obtained from a previous company which produces gibberellin.The quantity of the sludge inoculation was 30 t.In the preliminary stage of the setup, the A/O system took in water once a day in the first day and the COD was kept at about 300 mg/L.The influent was stopped over the next two days and the A/O system stopped taking in water and kept on aeration.After the third day, the A/O system took in water twice a day.After operation for 15 days, the reactor was operated with continuous influent at half of the design flow.Additionally, the COD of the influent was strengthened during this time.After 25 days, there were large amounts of Vorticella sp. and rotifers in the reactor.The removal of COD and NH 3 -N was relatively high and the quality of the effluent was good.At this time, the influent COD was equal to the design loading of COD.After 30 days, the sludge became brown owing to flocculation, indicating successful reactor setup and good sludge activity.After setup, the reactor was used to treat the wastewater.During the setup period, the aerobic tank must remain aerated, and the dissolved oxygen should be maintained at 3 mg/L, while the pH was kept at about 6.5 to 8. Additionally, the pH of the anaerobic tank was maintained at 6 to 8 and the DO was less than 0.8 mg/L.Finally, KH 2 PO 4 was added to the wastewater to maintain a COD:N:P ratio of 100:5:1.

Microbial diversity analysis
Sample collection.A total of 12 samples were collected from the aeration tank.Samples were collected per half a month.Each sample was dispensed into a 1.5 mL sterile Eppendorf tube and centrifuged at 14,000×g for 10 min.The supernatant was decanted, and the pellets were stored at -20˚C prior to analysis.For all samples, the sample numbers begin with the sample date.DNA extraction and PCR amplification.Microbial DNA was extracted from samples using an E.Z.N.A. 1 activated sludge DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's protocols.The V4-V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR (95˚C for 2 min, followed by 25 cycles at 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s and then final extension at 72˚C for 5 min) using primers 515F 5 0 -barcode-GTGCCAGCMGCCGCGG-3 0 and 907R 5 0 -CCGTCAATTCMTTTRAGTTT-3 0 , which contained an eight-base sequence barcode unique to each sample.PCR reactions were performed in triplicate 20 μL mixtures containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.
Illumina MiSeq sequencing.Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions, then quantified using QuantiFluor™-ST (Promega, Madison, WI, USA).Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols.The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP077353).
Processing of sequencing data.Raw fastq files were demultiplexed, then quality-filtered using QIIME (version 1.17) with the following criteria: (i) 300 bp reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window, discarding truncated reads that were shorter than 50 bp.(ii) Exact barcode matching, any sequences with two nucleotide mismatches upon primer matching or reads containing ambiguous characters were removed.(iii) Only sequences that overlapped by more than 10 bp were assembled according to their overlap sequence.Reads that could not be assembled were discarded.

Statistical analysis
The Shannon-Wiener index was used to assess bacterial diversity.Principal component analysis (PCA) was conducted to examine variations among bacterial communities of these thirty-  As shown in Fig 6, the influent TP concentrations fluctuated between 0.747 and 1.158 mg/ L, while the effluent TP concentrations fluctuated between 0.428 and 0.604 mg/L.Moreover, the average TP removal efficiency was 45.82%.

Effect of combined A/O process on nutrient removal from gibberellin wastewater
These results show that A/O processes had good effects on gibberellin wastewater.Moreover, this method could remove most of the COD, NH 3 -N and TP with removal efficiencies of 68.41%, 93.67% and 45.82%, respectively.

Illumina MiSeq sequencing results and microbial community structure
As shown in Table 2, there were 18,563 effective reads for 12 activated sludge samples.On average, the samples had 955.9 OTUs.The diversity of the bacterial community was determined based on the Shannon-Wiener index, which ranged from 4.81 to 5.43 (average = 5.18) [9].These values are typical for diverse microbial populations without a few strongly dominant taxa [10].
As show in Fig 7, Proteobacteria was the dominant phylum in all samples, accounting for 27.19%-61.33% of the total effective bacterial sequences.These findings are concordant with those of our previous study, which showed that Proteobacteria was the dominant community member [11][12][13][14][15]. Evidence by Miura et al. [16] has shown that Proteobacteria was considered to be the most dominant phylum in MBR that played an important role in the removal of organic matter.The abundance of Proteobacteria obviously increased with operation of the aeration tank.Other dominant phyla included Chloroflexi (4.09%-34.25%),Bacteroidetes (1.56%-21.35%)and Actinobacteria (1.90%-19.37%).Chloroflexi has been detected as the predominant bacteria in anaerobic bioreactors and found to be responsible for the degradation of carbohydrates [17].Chloroflexi also showed a great decrease with increased running time.Similar results were observed in previous studies that showed Bacteroidetes was the dominant community [18][19][20][21].The anaerobes Bacteroidetes play an important role in the fermentation system to break down macromolecules such as protein, starch, cellulose and fiber [22].Bacteroidetes also had been found to be responsible for the degradation of complex organic matters [23].The relative abundance of Bacteroidetes decreased greatly at first, then increased to a relatively high abundance in sample collected on August 13, 2015, then finally stabilized.Actinobacteria play an important ecological role in recycling substances in natural ecosystems and are able to degrade a variety of environmental chemicals [24][25][26].Actinobacteria was also the main phyla in a study conducted by Chen et al. [27].The abundances of the dominant phyla were also different between samples.For example the abundance of Chloroflexi in sample collected on May 25 was significantly higher than that of other samples.Planctomycetes was the dominant route for ammonia removal in UASB reactor [28].The relative abundance of anammox bacteria Planctomycetes increased with operation of the aeration tank and became one of the major phyla.Huang et al. [29] found that Planctomycetes was the major phylum in soil samples.Across different activated sludge samples, the relative abundances of some bacterial phyla, such as Nitrospirae and Chlamydiae, varied significantly.For instance, the relative abundance of the phylum Nitrospirae in sample collected on November 17, 2015 was significantly higher than in other samples.In contrast, the relative abundance of Firmicutes did not change  significantly throughout the experimental period.Moreover, Firmicutes and Gemmatimonadetes have lower abundance in all activated sludge samples.This finding differed from those of previous studies, which showed Firmicutes and Gemmatimonadetes were the dominant phyla [30][31][32].Cyanobacteria was a rare group in this study.Most Cyanobacteria can produce extracellular polymeric substances, mainly polysaccharide, which could adsorb heavy metals dispersed in the environment [33,34].during the latter portion of the experiment.These findings are consistent with those of previous studies that showed Nitrospira was the major phylum in the recirculating aquaculture system [35].Nitrosomonas could be a minor component in activated sludge samples.Nitrosomonas are regarded as the dominant ammonia-oxidizing bacteria, while Nitrospira are the main nitrite-oxidizing bacteria in wastewater treatment plants [36].The bacterial community composition showed little difference as the operating time increased in this study.Anaerolineaceae_uncultured and Ottowia decreased significantly as operating time increased.Coxiellaceae_uncultured was undetected in most samples.Mycobacterium was a minor component in most activated sludge samples, which was much different from the results of a previous study [37].Some species of Blastocatella are aerobic, chemoorganotrophic organisms with a strictly respiratory type of metabolism [38].The sulphur oxidizing bacteria genus Thiothrix, which increased significantly during the operation period, has also been found in aerobic granules incubated in brewery wastewater [39].The relative abundance of Caldilineaceae_uncultured, Saprospiraceae_uncultured and Anaerolineaceae_uncultured decreased significantly as  Multiple samples similarity tree analysis was used to identify similarities and differences among the 12 bacterial community structures ( Fig 10).Overall, samples collected from May, June and July formed one cluster, while those from August, September, October, November and December formed another cluster.The samples which were clustered together also  ,pH and DO.The correlation of genus OM190_norank (G8) and Saprospiraceae_uncultured (G9) was similar, with positive correlation with pH, and negative correlation with temperature.Besides, Saprospiraceae_uncultured (G9) and Anaerolinea-ceae_unclassified (G41) also negatively with SO 4 2-and DO.

Conclusion
The combined processes of multi-stage anaerobic bioreactor + micro-aerobic + anoxic/aeration + biological contact oxidation were used to treat gibberellin wastewater.The A/O process led to good removal of COD, NH 3 -N and TP.Proteobacteria was the dominant phylum in all samples, followed by Chloroflexi, Bacteroidetes and Actinobacteria.Novosphingobium and Nitrospira were dominant genus in most samples.Illumina MiSeq sequencing showed that bacterial community structure and diversity changed with DO, temperature and SO 4 2-.
Fig 2 shows a schematic diagram of the treatment system.

2 -,
As shown inFig 3, the influent COD concentrations fluctuated between 346 and 772 mg/L, with the lowest concentration (346 mg/L) being observed at 29 days and 121 days.Moreover, the highest influent COD concentration of 772 mg/L was observed at 53 days.The effluent COD concentrations fluctuated between 50.29 and 76.45.Taken together, these results indicate that removal efficiency changed with influent COD concentration.Moreover, the average COD removal efficiency was 68.41%.As shown in Fig 4, the influent NH 3 -N concentrations fluctuated between 17.87 and 72.50 mg/L, with 17.87 being observed on days 33 and 81 and 72.50 being observed at 149 days.However, the effluent NH 3 -N concentration remained relatively stable, and the NH 3 -N removal efficiency was around 93.67%.As shown in Fig 5, the concentration of effluent SO 4 2-was higher than that of influent SO 4 possibly because some of the SO 4 2-had transformed into H 2 S, and some of the H 2 S had transformed into SO 4 2-in the A/O system.

Fig 6 .
Fig 6.Removal of TP by A/O processes.https://doi.org/10.1371/journal.pone.0186743.g006 Planctomycetes and Nitrospirae increased greatly with operation time.Proteobacteria, Chloroflexi, Bacteroidetes and Actinobacteria are predominant phyla in the activated sludge.The distribution of sequences at the genus level in each sample is shown in Fig 8.The top 10 genera of each sample were selected, while all remaining genera were categorized as "other."Anaerolineaceae_uncultured was the major genus in sample collected on May 25, 2015, while Novosphingobium and Nitrospira were dominant in most samples, especially

Fig 7 .Fig 8 .
Fig 7. Abundances of different phyla in 12 activated sludge samples.The abundance is presented in terms of the percentage of total effective bacterial sequences in a sample.https://doi.org/10.1371/journal.pone.0186743.g007

Fig 9 .
Fig 9. Heat map of top 10 genera in each sample.A total of 47 genera were selected from 12 samples.The color intensity in each panel shows the percentage of a genus in a sample based on the color key at the bottom of the figure.https://doi.org/10.1371/journal.pone.0186743.g009