Degradation pathway and microbial mechanism of high-concentration thiocyanate in gold mine tailings wastewater

As one of the inorganic pollutants with the highest concentration in the waste water of gold tailings using biohydrometallurgy, thiocyanate (SCN−) was effectively degraded in this research adopting a two-stage activated sludge biological treatment, and the corresponding degradation pathway and microbial community characteristics in this process were also studied. The results showed that SCN− at 1818.00 mg L−1 in the influent decreased to 0.68 mg L−1 after flowing through the two-stage activated sludge units. Raman spectroscopy was used to study the changes of relevant functional groups, finding that SCN− was degraded in the COS pathway. Based on 16S rRNA high-throughput sequencing technology, the microbial diversity in this system was analyzed, and the results indicated that Thiobacillus played a major role in degrading SCN−, of which the abundance in these two activated sludge units was 32.05% and 20.37%, respectively. The results further revealed the biological removal mechanism of SCN− in gold mine tailings wastewater.


Introduction
China is rich in gold resources, most of which have been developed completely, with the rest hard to mine. In view of this, economical, efficient and environmentally-friendly biohydrometallurgy is explored across the world. However, when minerals are oxidized by microorganisms, suldes or thiosulfates can react with cyanide to form a large number of sulfur cyanates (SCN À ) containing carbon, nitrogen and sulfur, which are moderately toxic and chemically stable. 1 Emission of SCN À will do serious harm to the ecological environment. 2 At present, there are few reports on the treatment of high-concentration SCN À in the wastewater, and according to the existing data, the reducibility and adsorption capacity of SCN À are stronger than that of CN À , and most degradation processes of CN À such as alkaline chlorine oxidation, 3,4 hydrogen peroxide oxidation, 5 ozone oxidation, 6 activated carbon adsorption oxidation, 7 ion exchange, can be also used for the removal of SCN À in wastewater. No matter which chemical or biological treatment is adopted, the conversion of SCN À in aerobic conditions needs a lot of oxidants, which is costly and may produce intermediate product CN À , thus increasing the toxicity of wastewater. 7,8 As an economic and environmental-friendly method, biohydrometallurgy is attracting more and more attention. Considering that SCN À consists of S, C and N, which are all necessary elements for the growth of the living beings, it can provide a carbon source, nitrogen source and sulfur source for microorganisms under aerobic conditions, and generate such metabolites as SO 4 2À , CO 2 , and NH 4 + . 9 Hence, high-load SCN À in the wastewater can be biodegraded efficiently under the condition that dissolved oxygen and hydraulic retention time (HRT) are sufficient. Biological treatment of SCN À can be generally divided into two parts: the carbon removal unit to decompose and release CO 2 , and the nitrication and denitrication unit to remove NH 4 + -N from wastewater. Now, bacteria including Arthrobacter, Escherichia, Methylobacterium, 9,10 Bacillus, 11 Pseudomonas, Acinetobacter, 12 Thiobacillus, 13 Burkholderia, Chryseobacterium, 14 Klebsiella and Ralstonia 15 and others, been isolated and identi-ed from many resources to degrade thiocyanate. Paruchuri et al. found that the mixed culture containing Pseudomonas and Bacillus could degrade SCN À batch culture up to 1400 mg L À1 in 6 days. 16 Chaudhari and Kodam isolated Klebsiella pneumoniae and Ralisto. sp co-cultures from thiocyanate-contaminated sites and found, 15 the SCN À degradation rate was 500 mg (L À1 d À1 ) at the concentration of 2500 mg L À1 thiocyanate at pH 6.0 and 37 C. Different from physical and chemical treatment, biological treatment does not need a large number of oxidants and thus gradually becomes the research hotspot in respect of sulfur-containing cyanide wastewater treatment.
As a mature biological treatment process, activated sludge process plays an irreplaceable role in wastewater treatment. The large surface area and sugar layer of the activated sludge can quickly absorb pollutants in sewage, and effectively biodegrade pollutants relying on the relevant functional microorganisms. Hence, in this paper, a two-stage activated sludge biological treatment process is applied into the effective biodegradation of high-concentration thiocyanate (SCN À ) in the gold mine tailings wastewater. Meanwhile, the functional groups in this system is analyzed to decode the degradation pathway of SCN À , and 16S rRNA high-throughput sequencing technology is also employed to analyze the microbial diversity in the system and further to explore the related biodegradation mechanism of SCN À .

Water quality and method of the test
The tailing wastewater was selected from a gold mine in Guizhou Province, with the water quality shown as below: COD, 2089.00 mg L À1 ; SCN À , 1818.00 mg L À1 ; NH 4 + -N, 1.98 mg L À1 ; NO 2 À -N, 4.30 mg L À1 ; NO 3 À -N, 339.44 mg L À1 ; and TOC, 707.76 mg L À1 , in a mode of "regulating tank + two-stage activated sludge units + radial ow sedimentation tank". Inuent ow reached 1000 m 3 d À1 and the HRT of primary and secondary activated sludge units lasted 35.5 h and 10.5 h, respectively, with the MLSS being 4000 mg L À1 , and the dissolved oxygen 2.5-3.5 mg L À1 . Then, the mixture is separated in the radial ow sedimentation tank, and the separated sludge owed back to the primary unit, with the reux ratio reaching 200%. Sodium hydroxide solution was added to adjust pH value to 6-9 during operation. To ensure that a certain amount of organic matter can be provided for the growth and reproduction of microorganisms in the tanks, nutrition ratio of C : N : P should be maintained 100 : 5 : 1. A nutrition dosing system for phosphorus solution was established to keep the balance of C, N and P in the tanks. While the system became stable, wastewater quality in the regulating tank, the primary and the secondary activated sludge units was assayed, and the microorganism in these two units were also analyzed by 16S rRNA high-throughput sequencing.

Sample collection and pretreatment
Wastewater samples in the regulating tank was numbered ①, the primary activated sludge unit ②, and the secondary activated sludge unit ③. The sludge water mixture of the primary unit was marked A and the sludge water mixture of the secondary unit was marked B. Sample ①②③ of 1 L was refrigerated in a cleaned and cold plastic bucket, and stored at À4 C in the laboratory, and Sample A and B of 1 L was collected and stored at À20 C aer centrifugation in the laboratory.

Raman spectrum analysis
The water samples were frozen at À20 C and then dried to powder. Then, a proper amount of powder was taken to the center of quartz slide for spectral integration using confocal micro Raman spectrometer (LRS-8, Shanghai, China), with excitation light source being 632.8 nm He-Ne laser, excitation wavelength 532.08 nm, and grating 1800 L mm À1 ; and this process lasted 10 s and was repeated 4 times, in order to obtain a relatively smooth spectrum ranging form 100 cm À1 to 4000 cm À1 . In addition, every spectrum sample was repeatedly collected three times to improve the accuracy. All the spectra were collected in a dark room at the laboratory temperature (23 C) and pressure (0.1 MPa). Original 8.0 soware was used for data analysis.

16S rRNA high throughput sequencing analysis
Microorganisms in the two units were collected and frozen at À20 C, and then sent to Shanghai Meiji for 16S rRNA highthroughput sequencing. An universal bacterial primer: 338F (5 0 -ACTCCTACGGGAGGCAGCAG-3 0 ), was used to amplify V3-V5. Microbial DNA was extracted for PCR amplication and product purication pre-experiment. Passing the preexperiment, the formal experiment could be initiated, using TransGen AP221-02: TransStart Fastpfu DNA Polymerase, 20 mL reaction system: 5 Â FastPfu buffer 4 mL, 2.5 mmol L À1 dNTPs 2 mL, forward primer (5 mM) and reverse primer (5 mM) 0.8 mL, FastPfu polymer 0.4 mL, BSA 0.2 mL, and template DNA 10 ng, then, adding ddH2O to 20 mL, as well as ABI GeneAmp® 9700 PCR amplication instrument, with the reaction parameters: pre denaturation at 95 C for 3 min, denaturation temperature at 95 C for 30 s, annealing temperature at 55 C for 30 s, extension temperature at 72 C for 45 s, in 72 cycles, and extension at 72 C for 10 min. Aer amplication, PCR product sample of 3 L were loaded and assayed by 2% agarose gel electrophoresis. Aer that, an Illumina platform library was constructed, and then the data obtained were analyzed for biological information, including OTU clustering, species annotation and classication, community composition and others.

Degradation of SCN À by activated sludge process
High-concentration SCN À wastewater was treated by two-stage activated sludge biological treatment process, in the stable period, water quality of the inuent and effluent was shown in Table 1. Among them, SCN À decreased from 1818.00 mg L À1 to 1.01 mg L À1 and the removal rate reached 99.94% aer the owing through the primary unit, and then, the total removal rate of SCN À increased to 99.96% aer the secondary unit, which was consistent with the removal rate of COD (85.89%) and the reduction rate of TOC in water (93.89%). As a main source of COD in gold wastewater, decreases of SCN À concentration can lead to COD decrease.
Aer the treatment in the primary unit, the removal rate of NO Then, aer owing through the secondary unit, NH 4 + -N in the wastewater decreased by 26 mg L À1 , but NO 3 À -N increased by 29.55 mg L À1 , showing nitrication occurred in this secondary unit. Moreover, it was found that mercury in this unit reduced from 47.79 ng mL À1 to 1.47 ng mL À1 with the removal efficiency high up to 96.92%, suggesting this system had a favorable condition to remove mercury.

Raman analysis of SCN À degradation pathway
The results showed that there were two pathways to degrade SCN À . One was COS pathway. Under the action of thiocyanate hydrolase, the nitrogen carbon bond in SCN À hydrolyzed to COS and NH 3 , where the C-S bond was broken into CO 2 and H 2 S, and H 2 S oxidized to SO 4 2À . The other was CNO pathway. The sulfur carbon bond in SCN À was broken and hydrolyzed to cyanate (CNO À ) and HS À , where CNO À was hydrolyzed into NH 3 and CO 2 by cyanate hydrolysis enzyme, and S 2À was further oxidized to sulde and SO 4 2À . 9 The COS pathway: 15 It can be seen from the analysis of three groups of data by comparing the Raman spectrogram (Fig. 1) and Raman spectrogram analysis ( Table 2) of water quality, there were two vibrations in the absorption band of sulfur carbon C-S located at 730-600 cm À1 in the regulating tank, specically speaking, 634 cm À1 and 757 cm À1 , respectively. In the primary unit, there was only one vibration at 636 cm À1 , and in the secondary unit, there was only one vibration at 627 cm À1 , with the vibration frequency less than that in the regulating tank and the primary unit. At the same time, the C^N stretching vibration frequency of M-S-C^N, a metal thiocyanate salt located at 2160-2040 cm À1 , also appears in the water of the regulating tank. 20, 21 Tian et al. reported that the S-and N-combined thiocyanate showed C-N stretching vibration in the range of 2050-2165 cm À1 , that is to say, this peak belongs to C^N stretching vibration, and the absorption band in the secondary activated sludge unit is the weakest, indicating that the C-S bond and C^N bond fracture of SCN À (electronic formula is N^C-S À ) mainly occur in the primary activated sludge unit. The C]S stretching vibration peak at 1200-1020 cm À1 only appeared in the regulating tank and the primary effluent, and disappeared in the secondary effluent, indicating that the C]S bond fracture of O]C]S mainly occurred in the secondary unit. 22 In Minogue's report, the Raman spectral frequencies of 981 cm À1 and 451 cm À1 belong to the vibration of SO 4 2À . The vibration

Analysis of microbial community structure
Biolms in the primary and secondary units were sampled for 16S rRNA high-throughput sequencing to analyze the bacteria community structure that degraded SCN À , and OTU clustering was carried out for non-repetitive sequences (excluding single sequences) according to 97% similarity by calculating common diversity indexes (Table 3) including richness index (Chao/ACE index), coverage index, Shannon index and Simpson index. Couerage index refers to the sequencing depth and coverage rate of the samples. Coverage rates of sample A and B was 0.9996 and 0.9995, respectively, indicating that most of the bacterial populations were detected. The small difference among Shannon index, Simpson index, ACE index and Chao index of sample A and B, as well as the Venn diagram of sample A and B microbial communities (Fig. 2) where the two sludge samples shared 90.63% of the total OTUs, proved similar microbial community diversity between them.
Further taxonomic analysis of OTU representative sequences found that over 95% of the microbial communities of A and B in the entire reaction were composed of three phylum-level bacterial communities (Fig. 3), referring to Proteobacteria, Bacteroidetes and Deinococcus Thermus, respectively, of which Proteobacteria was the dominant with the respective abundance reaching 60.6% and 54.9%. Bacteroidetes was the second dominant, accounting for 21.7% and 22.5%, respectively. Manz et al. found that Proteobacteria possessed the capacity of water treatment. 23 McLellan et al. also found that Bacteroidetes is the dominant bacteria in the inuent of sewage treatment plant. 24 The dominant family-level bacteria of the two samples were Hydrophillaceae, Chitinophagaceae and Trueperaceae, with the similar relative abundance (Fig. 4). Relative abundances of Hydrogenopholaceae in the sample A and B were 32.05% and 20.37%, respectively. Thiobacillus, which has been reported to be capable of degrading SCN À , belongs to this family, and it is a special autotrophic bacterium which can utilize oxygen, nitrate and nitrite as electron acceptors to oxidize sulde, thiosulfate and sulfur, so as to obtain energy. 25,26 The respective relative abundance of Chitinophagaceae was 18.5% and 19.34%, and Trueperaceae was 15.58% and 19.96%. In addition, Burkholderiaceae, another one that can degrade SCN À , accounted for 2.65% and 2.9%, respectively, in these two samples. As a degrading microorganism that can grow and metabolize with thiocyanate as the only carbon source. Burkholderia sp. can completely degrade SCN À of about 500 mg L À1 in 90 h. 14 It is reported that Burkholderiaceae can also degrade hydrocarbons, phenol and PAHs (pyrene) in coking wastewater. 27,28 As shown in Fig. 5, the dominant genera-level bacteria of the two samples were Thiobacillus (32.05% and 20.37%), Truepera (15.58% and 19.96%), Chitinophagaceae (13.45% and 14.02%), and Dokdonella (7.95% and 10.94%). Oxidation-reduction sulde or elemental sulfur of Thiobacillus was sulfuric acid, and as a specic autotrophic bacterum using chemical energy, Thiobacillus could effectively degrade SCN À , during which SCN À would generate metabolites such as NH 3 , NO 2 À and S 2 À . 29 The results showed that thiocyanate can be degraded in two pathways: Thiobacillus, a SCN À degrading bacterium isolated from coking wastewater treatment plant, belonged to COS degradation pathway, 30 while Pseudomonas Putida and Pseudomonas stutzeri, separated from gold smelting wastewater Thiobacillus was the dominant bacteria to degrade SCN À in ASTER™ biological treatment system using gene clone library analysis. 35 In addition, the anammox bacteria Candidatus Campbell bacteria was detected, but its abundance was very small, about 0.17%, so it exerted a poor effect on the removal of NH 4 + -N.
The sequencing results showed that the nitrifying bacteria and nitrobacteria were not the dominant, and were excluded in the community Heatmap diagram (Fig. 5). What's more, results in Table 1 also veried that the ammonia nitrogen in this system did not decline. The reason might be that Thiobacillus became the dominant bacteria, and oxidized sulfur to sulfate, causing the pH value of the system decreased. However, nitrifying bacteria prefer a relatively weak alkaline environment. Kim et al. also studied that nitrifying bacteria in aerobic sludge cannot be detected due to the inhibition of toxic compounds. 36 Hence, nitrifying bacteria were not dominant in this experimental condition.    This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 25679-25684 | 25683

Conclusions
In this research, high-concentration SCN À gold tailing wastewater was treated in a two-stage serial activated sludge process, where SCN À of 1818 mg L À1 was reduced to 0.68 mg L À1 , with the degradation efficiency high up to 99.96%. In addition, the removal efficiency of COD, TN and Hg reached 85.89%, 85.51% and 96.92%, respectively. Meanwhile, according to Raman spectrum analysis of the functional groups' changes, SCN À was transformed and degraded in the same COS pathway as thiocyanate was biodegraded. Analysis of 16S rRNA highthroughput sequencing identied. Thiobacillus was the dominant bacteria to degrade SCN À , with the abundance of 32.05% and 20.37% respectively in the two-stage activated sludge units.

Conflicts of interest
The authors declare that there are no conicts to declare.