Lei Lia,
Fanyao Yueb,
Yancheng Li*ac,
Aijiang Yangac,
Jiang Liac,
Yang Lva and
Xiong Zhonga
aCollege of Resources and Environmental Engineering, Guizhou University, Guiyang, Guizhou 550025, China. E-mail: ycli3@gzu.edu.cn
bGuizhou Jinfeng Gold Mine Limited, Qianxinan Buyi and Miao Autonomous Prefecture, Qianxinan, Guizhou 550025, China
cGuizhou Karst Environmental Ecosystems Observation and Research Station, Ministry of Education, Guiyang, Guizhou 550025, China
First published on 7th July 2020
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.
Biological treatment of SCN− can be generally divided into two parts: the carbon removal unit to decompose and release CO2, and the nitrification and denitrification unit to remove NH4+–N from wastewater. Now, bacteria including Arthrobacter, Escherichia, Methylobacterium,9,10 Bacillus,11 Pseudomonas, Acinetobacter,12 Thiobacillus,13 Burkholderia, Chryseobacterium,14 Klebsiella and Ralstonia15 and others, been isolated and identified from many resources to degrade thiocyanate. Paruchuri et al. found that the mixed culture containing Pseudomonas and Bacillus could degrade SCN− batch culture up to 1400 mg L−1 in 6 days.16 Chaudhari and Kodam isolated Klebsiella pneumoniae and Ralisto. sp co-cultures from thiocyanate-contaminated sites and found,15 the SCN− degradation rate was 500 mg (L−1 d−1) at the concentration of 2500 mg L−1 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−) in the gold mine tailings wastewater. Meanwhile, the functional groups in this system is analyzed to decode the degradation pathway of SCN−, 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−.
Index | ① | ② | Removal efficiency 1 | ③ | Removal efficiency 2 | Total removal rate |
---|---|---|---|---|---|---|
pH | 8.82 | 7.13 | — | 6.66 | — | — |
NH4+–N (mg L−1) | 99.21 | 318 | −220.53% | 292 | 8.18% | −194.33% |
NO2−–N (mg L−1) | 4.3 | 0.275 | 93.6% | 0.0174 | 93.67% | 99.60% |
NO3−–N (mg L−1) | 339.44 | 14.28 | 95.79% | 43.83 | −67.42% | 87.09% |
COD (mg L−1) | 2089 | 250.20 | 88.02% | 294.67 | −17.77% | 85.89% |
TOC (mg L−1) | 707.76 | 41.918 | 94.08% | 23.658 | 43.56% | 93.89% |
SCN− (mg L−1) | 1818 | 1.01 | 99.94% | 0.68 | 32.67% | 99.96% |
Hg (ng mL−1) | 47.79 | 46.74 | 2.20% | 1.47 | 96.85% | 96.92% |
After the treatment in the primary unit, the removal rate of NO2−–N and NO3−–N in the wastewater reached 93.6% and 95.79%, respectively and only NH4+–N exhibited an upward trend from 99.21 mg L−1 to 318 mg L−1, suggesting NH4+–N was an intermediate product from SCN− degradation which further led to the accumulation of NH4+–N due to a lack of electron acceptors in the system. Theoretically, 100 mg SCN− can release 24 mg NH4+–N, about 10% of which can be transformed into biomass by microorganism as nitrogen source, and the rest enter into the water body in a form of NH4+, resulting in the increase of NH4+–N concentration in water.12 On the other hand, Huang et al. found that the nitrification of NH4+-N was inhibited due to the existence of SCN−.14
Formula (1): SCN− conversion path.
SCN− + H2O → S2− + NH4+ + CO2 | (1) |
Then, after flowing through the secondary unit, NH4+–N in the wastewater decreased by 26 mg L−1, but NO3−–N increased by 29.55 mg L−1, showing nitrification occurred in this secondary unit. Moreover, it was found that mercury in this unit reduced from 47.79 ng mL−1 to 1.47 ng mL−1 with the removal efficiency high up to 96.92%, suggesting this system had a favorable condition to remove mercury.
The COS pathway:15
NC–S− + 2H2O → OCS + NH3 + OH− | (2) |
OCS + H2O → H2S + CO2 | (3) |
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−1 in the regulating tank, specifically speaking, 634 cm−1 and 757 cm−1, respectively. In the primary unit, there was only one vibration at 636 cm−1, and in the secondary unit, there was only one vibration at 627 cm−1, with the vibration frequency less than that in the regulating tank and the primary unit. At the same time, the CN stretching vibration frequency of M–S–CN, a metal thiocyanate salt located at 2160–2040 cm−1, 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−1, that is to say, this peak belongs to CN stretching vibration, and the absorption band in the secondary activated sludge unit is the weakest, indicating that the C–S bond and CN bond fracture of SCN− (electronic formula is NC–S−) mainly occur in the primary activated sludge unit. The CS stretching vibration peak at 1200–1020 cm−1 only appeared in the regulating tank and the primary effluent, and disappeared in the secondary effluent, indicating that the CS bond fracture of OCS mainly occurred in the secondary unit.22 In Minogue's report, the Raman spectral frequencies of 981 cm−1 and 451 cm−1 belong to the vibration of SO42−. The vibration frequency (about 460 cm−1) caused by the symmetric angle change of SO42− from the regulating tank to the primary effluent changed slightly, but then weakened to the lowest extent in the secondary effluent; however, the S–O symmetric stretching vibration peak (about 980 cm−1) of SO42− was very strong at the beginning, and then weakened gradually from the regulating tank to the primary effluent and then to the secondary effluent, indicating that H2S can be further oxidized into SO42− both in the primary and secondary units. Accordingly, Raman spectrum analysis indicated that biodegradation of SCN− belonged to COS pathway, where the N–C bond and S–C bond in SCN− was hydrolyzed into COS and NH3 in the primary unit; then the C–S bond in COS was broken into CO2 and H2S mainly in the secondary unit; and finally, H2S was further oxidized into SO42−.
Fig. 1 Raman spectrum analysis of water quality—①: regulating tank; ②: primary activated sludge unit; ③: secondary activated sludge unit. |
Sample | Sequences | OTUs | Shannon | ACE richness | Chao richness | Courage |
---|---|---|---|---|---|---|
A | 38146 | 154 | 2.65 | 163.38 | 159.22 | 0.9996 |
B | 35886 | 151 | 2.81 | 160.50 | 158.16 | 0.9995 |
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.
Fig. 2 Venn diagram of microbial community—A: primary activated sludge unit; B: secondary activated sludge unit. |
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 influent of sewage treatment plant.24
Fig. 3 Microbial classification at a Phylum level—A: primary activated sludge unit; B: secondary activated sludge unit. |
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−, belongs to this family, and it is a special autotrophic bacterium which can utilize oxygen, nitrate and nitrite as electron acceptors to oxidize sulfide, 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−, 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− of about 500 mg L−1 in 90 h.14 It is reported that Burkholderiaceae can also degrade hydrocarbons, phenol and PAHs (pyrene) in coking wastewater.27,28
Fig. 4 Microbial classification at a Family level—A: primary activated sludge unit; B: secondary activated sludge unit. |
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 sulfide or elemental sulfur of Thiobacillus was sulfuric acid, and as a specific autotrophic bacterum using chemical energy, Thiobacillus could effectively degrade SCN−, during which SCN− would generate metabolites such as NH3, NO2− and S2−.29
Fig. 5 Heatmap of microbial community (A: primary activated sludge unit; B: secondary activated sludge unit). |
The results showed that thiocyanate can be degraded in two pathways: Thiobacillus, a SCN− 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 polluted soil, belonged to CNO degradation pathway.31 Thiobacillus was the dominant strain in this experiment, suggesting the degradation of SCN− belonged to COS degradation pathway,30 consistent with the afore results of Raman spectrum analysis.
Moreover, results of this experiment can reaffirm some of other researches. For example, Kelly et al. put forward that COS can generate CO2 and H2S through C–S bond fracture,32 and H2S can further oxidize to generate SO42−, where the resulting energy can also facilitate the growth of microorganisms. Stratford et al. and Arakawa et al. extracted thiocyanate hydrolase from the isolated strain and identified that thiocyanate hydrolase was an inducible enzyme.33,34 Huddy et al. proved that Thiobacillus was the dominant bacteria to degrade SCN− 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 NH4+–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 verified 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 |