3.1 C3H8-MBfR processing Cr (VI) performance overview
Figure 2 shows the concentration of influent Cr(VI), effluent Cr(VI) and effluent soluble total chromium in the three stages of the C3H8-MBfR reaction system. Figure 3 shows the Cr(VI) removal rate, Cr(VI) flux and surface load in the three stages of the C3H8-MBfR reaction system. According to the concentration of Cr(VI) in the effluent and total soluble chromium in the effluent, the content of soluble Cr(III) in the effluent can be neglected, which can be regarded as almost all the Cr(VI) removed in the reaction system is converted into Cr (III) Proof of precipitation.
According to Fig. 3, as the load of Cr(VI) increases, the removal rate of Cr(VI) gradually decreases. When the concentration of Cr(VI) in the first stage of influent is 0.5 mg·L− 1, Cr(VI) is removed The rate is stable at about 95%. When the influent Cr(VI) concentration is increased to 1.0 mg·L− 1, the removal rate is reduced to 80%, and in the third stage, the influent Cr(VI) concentration is 2.0 mg·L− 1 At this time, the Cr(VI) removal rate continued to decrease, and finally the removal rate stabilized at 75%.
3.2 The effect of Cr(VI) on EPS
We measured the amount of EPS produced by microorganisms at different stages of the inoculation sludge and the experiment, and the results are shown in Table 2.
Table 2
Contents of EPS in inoculated source and biofilm samples at different stages
|
Protein
(mg/g·vss)
|
Polysaccharides(mg/g·vss)
|
Inoculated sludge
|
1.13
|
1.35
|
Stage 1
|
1.51
|
2.24
|
Stage 2
|
7.49
|
9.43
|
Stage 3
|
2.88
|
3.48
|
According to Table 2, when Cr(VI) is not added, the amounts of PN and PS in EPS secreted by microorganisms are 1.13 mg/g·vss and 1.35 mg/g·vss, respectively. After adding Cr(VI), at the end of the first phase of the experiment, the production of PN and PS were 1.51 mg/g·vss and 2.24 mg/g·vss, respectively. PN and PS were increased compared to inoculated sludge. At the end of the second phase, the amount of PN increased to 7.49 mg/g·vss, the amount of PS increased to 9.43 mg/g·vss, and the increase in PN and PS increased. At the end of the third stage, the amount of PN was reduced to 2.88 mg/g·vss, and the amount of PS was reduced to 3.48 mg/g·vss. The amount of EPS secreted by microorganisms increases with the increase of Cr(VI) load in stage 1 and stage 2. In stage 3, when the Cr(VI) inflow is 0.5 mg·L− 1, the amount of EPS decreases compared with stage 2. Up to 60%. We speculate that the EPS secreted by microorganisms has a certain effect on the reduction of Cr(VI), but the high load of Cr(VI) will also inhibit the secretion of EPS by microorganisms.
3.3 Characterization of cell morphology and sedimentation of chromate reducing bacteria
Figure 4 shows the changes in the cell morphology of chromate-reducing bacteria at each stage in the reaction system and the signals of each element contained in the biofilm sediment at each stage.
Scanning electron microscopy (SEM) analysis results show that when Cr(VI) is not added, most of the microorganisms from the inoculation source have a smooth surface and mostly rod-shaped. In the first and second stages after adding Cr(VI), obvious irregular wrinkles appeared on the cell surface, and the first stage was particularly obvious. According to EDS analysis, it is found that Cr signals have appeared in the biofilm deposits in the first stage, and obvious Cr signals were detected in the second and third stages. It is speculated that the irregular folds on the cell surface are Cr deposits; in the third stage of the experiment, The cell surface returned to smoothness, indicating that the chromate reducing bacteria gradually adapted to the Cr(VI) environment as the experiment progressed. Studies have shown that chromate has strong oxidizing ability and can change cell morphology directly or through the production of reactive oxygen species (ROS). In many studies, it has been reported that the cell surface of Cr(VI) reducing bacteria appears deformed and wrinkled(Lai, C. Y. er al.,2016; Shi, L. D. et al.,2019; Sheng, G. P. et al.,2008; Kantar, C., Demiray, H.& Dogan, N. M. J. C.,2011; Liu, Y.; Lam, M. C., Fang, H. H. J. W. S.& Research, T. A. J. o. t. I. A. o. W. P.,2001; Guibaud, G. et al.,2005; Wu, C. Y., Peng, Y. Z., Wang, S. Y.& Ma, Y. J. W. R.,2010; Pei, Q. H.; et al., 2009)
After the third stage, we analyzed the precipitate in the reaction system by XPS. The result is shown in Fig. 5. We used chromium chloride hexahydrate (CrCl3·6H2O) and potassium dichromate (K2Cr2O7) as Cr( III) and Cr(VI) standard materials, XPS results show that the sample pattern is consistent with the Cr(III) standard material pattern, which can prove that the Cr in the biofilm sediment in the reaction system is trivalent. At the same time, we also analyzed the precipitate by XRD, and the results are shown in Fig. 6. XRD results also showed the presence of Cr(OH)3·3H2O in the precipitate, which is consistent with the results of Min Long et al. (2017) 29. Therefore, through XPS and XRD analysis, we can conclude that there is Cr(III) precipitation on the biofilm in the reaction system, and Cr(III) exists in the form of Cr(OH)3·3H2O.
The FTIR results in Fig. 7 also further prove that Cr(III) is adsorbed on the biofilm. Figure 7 compares the FTIR spectra of the biofilm before and after adding Cr(VI). The results show that the addition of Cr changes the absorption peak greatly. It can be inferred that the hydroxyl group, carboxyl group, nitro group and sulfonic acid group of the biofilm are related to the combination of Cr by the change of the absorption peak. The biofilm sample showed a broad, stretched and strong peak at 3413.9 cm-1, which is characteristic of -OH stretch. The adsorption band of the sample at 2925.9 cm-1 is characteristic of -CH tensile vibration. The adsorption peaks at 1661.5 cm-1 and 1453.2 cm-1 reflect the stretching vibration of C = C and the asymmetric stretching vibration of C-O-C, which are related to the obvious deviation in the adsorption process. After Cr was deposited on the biofilm, a new adsorption band appeared at 1738.8 cm-1, which is a characteristic of -COOH stretching. Studies have shown that negatively charged chemical reaction functional groups such as -OH and -COOH can bind to high-valence metals, and can form a barrier that hinders the penetration of heavy metals into cells, thereby reducing the toxicity of heavy metals to cells(Joshi, P. M. et al., 2009; Kang, F. et al., 2014; Wang, Z. et al., 2014; Zhu, L. et al., 2012).
3.4 Microbial community structure analysis
We performed high-throughput sequencing analysis on the inoculated sludge and the sludge samples of the various stages of the experiment, and obtained the original sequences of the four samples. The original sequences were subjected to quality control according to 97% similarity and then subjected to OTU cluster analysis. The number of effective sequences was summed up. The number of OTUs is shown in Table 3.
Table 3
Effective sequences and OTU of different samples
|
Inocula
|
Stage1
|
Stage2
|
Stage3
|
Valid sequence
|
64380
|
65192
|
69218
|
53600
|
OTU
|
948
|
650
|
640
|
601
|
As shown in Table 3, at a certain depth of sequencing, the effective sequence number and OTU number of different samples are different. In general, the number of OTUs in the inoculated sample is the largest, and the effective sequence number is larger. ) As the concentration increases, the OTU number gradually decreases, indicating that high concentrations of Cr(VI) will reduce the abundance of the flora.
Through the Alpha diversity analysis of the samples, the Alpha diversity indicators of different samples in Table 4 are obtained. Among them, the Sobs, chao, and ace indexes reflect the abundance of the community and are related to the actual number of species in the microbial community. The larger the value, the larger the community. Rich. The shannon and simpson indexes reflect community diversity, and the coverage index reflects community coverage. According to Table 4, the Sobs, chao, and ace index values of the inoculum were significantly higher than those of each stage of the experiment, indicating that the addition of Cr(VI) had a great impact on the abundance of the flora. The shannon and simpson indexes fluctuate to varying degrees throughout the reactor operation stage, indicating that different Cr(VI) concentrations have a significant impact on the composition of the microbial community. This result is consistent with the research conclusion of Aquino(Aquino, S. F.& Stuckey, D. C.,2004).
Table 4
Indices of Alpha diversity of different samples
|
sobs
|
shannon
|
simpson
|
ace
|
chao
|
coverage
|
Inocula
|
948
|
5.439196
|
0.009839
|
1008.521432
|
1023.795455
|
0.996954
|
Stage1
|
630
|
4.047472
|
0.058822
|
809.12519
|
799.036585
|
0.995615
|
Stage2
|
575
|
4.105986
|
0.045749
|
706.905918
|
731.935484
|
0.996324
|
Stage3
|
593
|
3.791467
|
0.089824
|
740.746065
|
727.463415
|
0.996087
|
Figure 8 shows the inoculation source and the composition of the microbial community in each stage at the class level. At the class classification level, Actinobacteria, Alphaproteobacteria and Gammaproteobacteria dominate. Among them, the abundances of Actinobacteria and Alphaproteobacteria in the inoculated sludge are 10.84%, respectively. 5.67%. With the addition of Cr(VI), the abundances of Actinobacteria and Alphaproteobacteria in the first stage increased to 45.97% and 16.97% respectively, occupying a dominant position. With the increase of the influent Cr(VI) concentration, Alphaproteobacteria The abundance gradually increased, occupying a dominant position in both Stage2 and Stage3, while the abundance of Actinobacteria gradually decreased, and the abundance in Stage3 decreased to 15.68%, which is consistent with the gradual decrease of the Cr(VI) removal rate.
Figure 9 shows the inoculation source and the composition of the microbial community in each stage at the genus level. At the genus level, the abundance of Nocardia, Rhodococcus and Mycobacterium belonging to Actinobacteria in the inoculated sludge is zero. The abundance of both increases. The genera Nocardia, Rhodococcus and Mycobacterium have been shown to have the ability to use gaseous normal alkanes as the sole carbon source(Hamamura et al., 2001; William, A.; Alaa, M.& Letters, C. M. J. J. F. M., 1994 ). N.R.WOODS (1989) found that a strain of Rhodococcus rhodochrous isolated in the soil can use propane as the sole carbon source(Woods, N. R.& Murrell, J. C. J. M., 1989 ). Some studies have found many strains of propane oxidizing bacteria in Rhodococcus and Mycobacterium, which can use propane as a substrate to biodegrade trichloroethylene, methyl tert-butyl ether, dioxane and other organic pollutants(Wackett, L. P. J. A.& Microbiology, E., 1989; Tupa, P. R.& Masuda, H. J. J. o. G., 2018; Deng et al., 2018). Studies have reported that Nocardia has the ability to remove Cr(VI)(Zhang, X et al., 2019). Dimitroula et al (2015) reported that a strain of acidophilus can completely reduce 20 mg·L-1 Cr(VI) within 50 h(Dimitroula, H. et al., 2015), Previous studies have also reported that strains belonging to the genus Rhodococcus can reduce Cr(VI)(Sun, J. Q. et al., 2011; Revelo Romo, D. M. et al., 2019; Kuyukina, M. S. et al., 2017; Patra, R. C. et al., 2010). Soumya Banerjee (2017) reported that R. erythropolis removes Cr(VI) through bioconcentration(Banerjee, S. et al., 2017). Nocardia and Rhodococcus had the highest abundance in Stage1, and then the abundance decreased with the increase of the influent Cr(VI) concentration, which was consistent with the decreasing trend of the Cr(VI) removal rate.
3.5 Functional gene analysis
Table 5 shows the predicted abundance of some functional genes of metagenomics on the biofilm in the inoculation source and the various stages of the experiment based on the PICRUST 2 function prediction analysis. Among them, the abundance of ABC transport system at all stages of the experiment was higher than that of the inoculation source, and its abundance was the highest in Stage 1. The abundance of ABC transporters at all stages of the experiment was higher than that of the inoculation source. ABC transporters can transport heavy metals through the cell membrane and excrete them from the cell, and are part of the cell defense system(Torre, C. D. et al., 2012). The abundance of chromate reductase and chromate transporter gradually increased with the increase of the influent Cr(VI) concentration, and reached the highest in Stage 3. Cytochrome c belongs to chromate reductase and has Cr(VI) reduction ability, which reduces Cr(VI) to Cr(III) through Cr(V) intermediates. Its abundance increases with the concentration of Cr(VI) influent. High and increase(Shi, X. L. et al., 1990; Ackerley, D. F. et al., 2004).NADH, as an electron donor for reduction of intracellular Cr(VI), has an abundance of 2 times that of the inoculation source at each stage, and its abundance in Stage 1 with the highest Cr(VI) removal rate is 3 times that of the inoculation source(Thatoi, H. et al., 2014). Flavin reductase and nitrate reductase are both soluble chromate reductase and extracellular enzymes, and their abundance gradually increases with the progress of the experiment(Cheung, K. H. et al., 2017). The abundances of cytochrome c551 and thioredoxin reductase, which are also related to chromate reduction, increase in Stage 1, and then decrease with the increase of Cr(VI) influent concentration(Li, L. Z. P. L. X.,2013; Zhong, L. et al., 2017). Cysteine is a non-enzymatic chromate reducing agent, and its abundance in the reaction system is negligible(Poljsak, B. et al., 2010).
Compared with the vaccination source, some genes related to the oxidation of propane are enriched (aldehyde dehydrogenase, propanal dehydrogenase, acetone monooxygenase, propane monooxygenase coupling protein, NAD+-dependent secondary alcohol dehydrogenase Adh1), and there are four main pathways for propane oxidation, namely Monooxygenase-mediated terminal oxidation of propan-1-ol, monooxygenase-mediated sub-terminal oxidation of propan-2-ol, oxidized by the oxygenase mechanism to produce prop-1-ol and prop- The 2-alcohol mixture produces propane-1,2-diol by oxidizing terminal and sub-terminal carbon atoms by dioxygenase(Hamamura, 2001). In this study, based on the measured abundance of functional genes, it can be inferred that during the experiment, under the action of microorganisms, propane passed through the propane-1-ol or prop-2-ol end or sub-end through a monooxygenase-mediated pathway. Oxidation and production of metabolic intermediates act as electron donors to drive the reduction of Cr(VI).
Table 5
The predicted abundance of some functional genes of metagenomics on the biofilms of the inoculation source and each reaction stage
KO
|
Description
|
Inocula
|
Stage 1
|
Stage 2
|
Srtage 3
|
K07240
|
chromate transporter
|
27486.79
|
28689.02
|
31744.32
|
60895.3
|
K19784
|
chromate reductase
|
4723.78
|
6452.6
|
5798.52
|
17870.57
|
K07058
|
membrane protein
|
42219.8
|
72819.57
|
50357.21
|
59327
|
K01990
|
ABC-2 type transport system ATP-binding protein
|
205351.2
|
271377.7
|
197511.1
|
157927.9
|
K15738
|
ABC transport system
|
25672.89
|
30194.44
|
26576.74
|
31522.89
|
K08738
|
cytochrome c
|
23504.55
|
27133.8
|
46593.99
|
67500.57
|
K00384
|
thioredoxin reductase [EC:1.8.1.9]
|
66375.32
|
83128.9
|
66328.38
|
73029.88
|
K12263
|
cytochrome c551
|
9
|
326
|
93
|
19
|
K03885
|
NADH dehydrogenase [EC:1.6.99.3]
|
27129.77
|
66895.89
|
51875.45
|
45100.63
|
K09024
|
flavin reductase [EC:1.5.1.-]
|
1507.08
|
1417.5
|
3764.72
|
2944.37
|
K00370
|
nitrate reductase
|
2559.66
|
9411.5
|
6836.5
|
15638.67
|
K11249
|
cysteine
|
1
|
0.017
|
0.017
|
0.017
|
K00138
|
aldehyde dehydrogenase [EC:1.2.1.-]
|
7058.5
|
26054.63
|
15886.95
|
23613.48
|
K18366
|
propanal dehydrogenase
|
324.66
|
74
|
134.67
|
688.34
|
K18371
|
acetone monooxygenase
|
295
|
778
|
2742
|
1945.67
|
K18226
|
propane monooxygenase coupling protein
|
123
|
864
|
4163.5
|
1583.5
|
K18382
|
NAD+-dependent secondary alcohol dehydrogenase Adh1 [EC:1.1.1.-]
|
610
|
1003
|
4199.5
|
1600.5
|
3.6 Research on Cr(VI) Reduction Mechanism
According to the analysis of microbial community diversity and PICRUST 2 function prediction analysis, the reduction mechanism of Cr(VI) in the reaction system is speculated.
The reduction of Cr(VI) includes enzymatic reduction and non-enzymatic reduction(Derek et al., 2009; Losi, M. E. et al., 1994; Fendorf, S et al., 1996). Cysteine is a non-enzymatic chromate reducing agent. According to the prediction of the abundance of functional genes, the abundance of this gene in the reflection system is negligible. Therefore, in this study, the reduction of Cr(VI) is mainly through an enzymatic process.
Enzyme-mediated reduction of Cr(VI) can be divided into two parts, the reduction of Cr(VI) in the cell membrane and the reduction of Cr(VI) outside the cell membrane. Cr(VI) in the cell is reduced to Cr(III) under the action of chromate reductase and cytochrome c. This process may release reactive oxygen species (ROS), and the generation of ROS will affect cell viability and Cr(VI) reduction(Thatoi, H. et al., 2014). The extracellular Cr(VI) is reduced to Cr(III) under the action of flavin reductase and nitrate reductase, the reduction product Cr(III) combines with the functional groups on the cell surface(Ngwenya, N. et al., 2011).