In order to solve the problem of difficult treatment of high-concentration chromium-containing wastewater, sulfate-reducing bacteria (SRB) with a high tolerance of hexavalent chromium and a strong ability to reduce the compound were isolated from sludge from a sedimentation tank in a leather industrial park and was identified as Desulfovibrio by morphological observation, routine physiological and biochemical determination, 16S rDNA sequencing and phylogenetic tree construction. After ethanol acclimation, a strain of SRB that could reduce chromium (CR-1) was selected as the research object. The optimum growth conditions for hexavalent chromium removal by the strain were determined by single-factor analysis. The chromium removal mechanism of the strain was analyzed, and a kinetic model of the reduction process was established. The chromium-reducing ability of the strain was 500 mg/L, the optimum pH value was 7, the optimum temperature was 35 °C, the optimum cultivation time was 24 h, and the optimum ratio of bacteria to waste (volume ratio of bacterial solution dosage and chromium-containing wastewater) was 1:5. The mechanism of treatment of Cr(VI) by this strain is mainly based on the reduction of Cr(VI) by H2S accumulated in the cultured bacterial solution and the small amount of H2S generated by bacterial reductase, bacterial growth and SO42− reduction in the waste liquid.
Chromium and its compounds are indispensable raw materials for the metallurgical, metal processing, and electroplating industries, among others. These industrial sectors are widely distributed and discharge a large amount of chromium-containing wastewater every day (Li et al. 2011). The chromium ions in the water mainly exist in the form of Cr(VI) and Cr(III); Cr(VI) can cause genetic defects, may be carcinogenic if inhaled, and poses a persistent risk to the environment (Kotaś & Stasicka 2000; Religa et al. 2014). Research on treatment of chromium-containing wastewater has been conducted for years (Cheung & Gu 2007; Li & Shao 2015). The common treatment methods are adsorption, electrolytic reduction, and chemical and biological methods (Song et al. 1998; Owlad et al. 2009; Bhattacharya et al. 2019). Biological treatment of chromium-containing wastewater has great potential due to its economy, efficiency and safety (Pradhan et al. 2017). In the late 1970s, Romanenko & Koren'Kov (1977) isolated a genus (Pseudomonas) that can reduce Cr(VI) under anaerobic conditions. This was the first report of microbial reduction of Cr(VI). Chai et al. (2005) isolated a highly efficient aerobic bacterium that reduced Cr(VI) from the soil of a Changsha chromium slag yard and found that 300 mg/L Cr(VI) could be reduced by more than 90% within 16 h; Mangaiyarkarasi et al. (2011) isolated an alkalophilic Gram-positive Bacillus subtilis strain from tannery-wastewater-polluted soil, which reduced 50 mg/L Cr(VI) by 100%, and established the reduction kinetics equation; and Banerjee et al. (2019) isolated a Pseudomonas strain with a chromium reduction capacity of 150 mg/L from coal mine wastewater. The growth conditions of the strain and the reduction mechanism of hexavalent chromium were studied in three ways: biological adsorption, biological accumulation and ion carrier adsorption. The degradation ability and reduction mechanism of high-concentration chromium-containing wastewater by the strains isolated to date are insufficiently understood. Alice et al. (2015) isolated a new fungal biosorbent, Penicillium sp. (MSR1), from local tannery wastewater. The optimum conditions for Cr(VI) removal were as follows: biomass dosage of 2 g/L, initial concentration of Cr(VI) of 62.5 mg/L and contact time of 37.5 minutes. The maximum adsorption capacity was approximately 79.9%. Bhattacharya & Gupta (2013) studied the detoxification and resistance mechanism of a newly isolated Acinetobacter strain (B9) to chromium(VI). Strain B9 could tolerate up to 350 mg/L Cr(VI) and reduce the initial concentration of Cr(VI) to 67% within 24 h of culture.
According to the existing researches, the low degradation ability and reduction mechanism of high-concentration chromium-containing wastewater by the strains isolated to date are insufficiently understood. Based on this gap, an anaerobic sulfate-reducing bacterium with strong chromium reduction ability was isolated from the sludge of a sedimentation tank in a leather industrial park. On the basis of strain identification, the growth conditions and optimum reduction conditions for reducing hexavalent chromium were determined, and the mechanism of chromium reduction was studied.
The sludge containing the strain was taken from the sedimentation tank of the Fuxin Leather Industrial Park. The strain had high tolerance to hexavalent chromium and strong reduction ability.
Preparation of culture medium
Enriched medium 1: KH2PO4 0.5 g/L, NH4Cl 1.0 g/L, MgSO4·7H2O 0.06 g/L, ethanol 3 g/L, yeast extract 1.0 g/L, CaSO4 1.0 g/L, FeSO4·7H2O 0.5 g/L, Na2SO4 3 g/L, CaCl2·6H2O 0.06 g/L, citric acid 0.3 g/L, distilled water 1 L, pH 7, 121 °C, sterilization for 20 minutes.
Enriched medium 2: Identical to enrichment medium 1, but FeSO4·7H2O was not added.
Solid medium: a total of 2% agar was added to the enrichment medium. Sterilization was performed at 121 °C for 20 minutes. (All chemicals and solvents were analytical grade and required no further purification.)
Culture, domestication and purification of strain
The dominant strains were enriched and cultured under anaerobic conditions at 35 °C by using ethanol as a carbon source in enrichment medium 1; an inoculation amount of 5% into sludge was used. Cr(VI) with a concentration of 20–500 mg/L was added to the medium by the increasing gradient method until the strain had been domesticated to treat high-concentration Cr(VI) wastewater with a concentration of 500 mg/L.
All operations were carried out with natural light in an anaerobic operating platform (Bactron II, SHELLAB, USA). Purification and isolation of the strains were performed by dilution coating and sandwich culture on dishes (Wan et al. 2003). When the obtained colony morphologies and the microscopic examination results were the same, this high-efficiency strain was named chromium-reducing strain CR-1.
Molecular biological identification of the chromium-reducing strain
The morphology and movement characteristics of the bacteria were observed by scanning electron microscopy (SEM), and the strain was stained by the conventional staining method.
Colony genomic DNA was extracted by using a Maxwell 16 strain DNA purification kit. Polymerase chain reaction (PCR) amplification was carried out using a universal primer for bacterial 16S rDNA PCR (Chen et al. 2005). After purification and amplification of the product, Beijing Liuhe Huada Gene Technology Service Co., Ltd was commissioned to perform sequencing.
Growth conditions of reducing Cr(VI) by strain CR-1
Through a parallel comparative experiment, 20 mL of bacterial solution (the bacterial count at the logarithmic phase was 3 × 108 cells/mL) in the logarithmic growth period was inoculated into 100 mL of enriched medium 2 containing 500 mg/L Cr(VI), which was then incubated at a constant temperature under anaerobic conditions for 24 h. The growth curve of strain CR-1 is shown in the Supplementary Material. The treated solution was centrifuged for 10 minutes at 4,000 r/min at intervals of 3 h, and the supernatant was collected to measure the concentration of Cr(VI). The single-variable control method was used to investigate different pH values, different culture temperatures, different initial concentrations of Cr(VI), different bacteria:waste ratios (the volume ratio of bacterial solution dosage and chromium-containing wastewater), other heavy metals and different culture times to determine their effect on the chromium reduction characteristics of the high-efficiency chromium-reducing strain.
Analysis of the chromium reduction mechanism of strain CR-1
A bacterial solution of strain CR-1 in the logarithmic phase was obtained aseptically and centrifuged for 20 minutes at 4,000 r/min. The following steps were performed.
Sample pretreatment for the H2S pathway: The supernatant, which contained H2S produced by strain CR-1 during the growth process, was collected. This solution did not contain bacteria but did contain H2S.
Cell pretreatment for the bacterial reductase pathway: A sample was centrifuged for 30 minutes and rinsed with an equal volume of 2.5 g/L NaHCO3 buffer solution. At this time, the chromium-reducing strain CR-1 cells were suspended in the buffer solution. The bacterial suspension was placed at 4 °C and the bacterial cells were broken by ultrasonic crusher (950E, Scientz, Ningbo, China). The working parameters of the ultrasonic crusher were: output power 30 W, working for 3 s, resting for 3 s, circulation for 99 times. The breaking effect was then examined by microscope. The supernatant of the acellular extract was obtained at 5,000 r/min, sealed with liquid paraffin and centrifuged for 60 min. The supernatant was the crude enzyme solution.
Preparation of bacterial extracellular polymers for the adsorption route (Xie et al. 2016): The supernatant was discarded, 0.9% NaCl solution was added to the original volume, and the bacteria were resuspended in the solution three times. Then, the pH was adjusted to 11 by adding sodium hydroxide solution, and the mixture was slowly stirred at 100 r/min for 10 minutes, centrifuged at 4,000 r/min for 20 minutes, and filtered through a filter to obtain the extracellular polymers.
Cr(VI) reduction experiments by different pathways: A total of 20 mL of the above-pretreated solution was added to a 100 mL solution of 500 mg/L Cr(VI), incubated in an anaerobic incubator at 35 °C for 24 h, and centrifuged at 4,000 r/min for 10 minutes every 3 h to measure the residual Cr(VI) concentration in the solution.
Cr(VI) was determined by diphenylcarbohydrazide spectrophotometric method (GB7467-87) (UV-2550, Shimadzu, Kyoto, Japan). The total chromium was determined by potassium permanganate oxidation–diphenylcarbazide spectrophotometry (GB7466-87) (UV-2550, Shimadzu, Kyoto, Japan); and the pH value was determined by a pH meter (PHS-3C, LEICI, Shanghai, China).
RESULTS AND DISCUSSION
Molecular biological identification of the chromium-reducing strain
Morphological characteristics of strain CR-1
Strain CR-1 is a Gram-negative spiral bacterium with no spores and polar flagella and exhibits strictly anaerobic growth. CR-1 cannot hydrolyze gelatine and cannot use citrate as a carbon source. The strain can produce a black precipitate and H2S in media containing ferrous ions. An SEM image of strain CR-1 is shown in Figure 1(a). The size of the microorganism is approximately (0.3–0.5) μm × (1–1.5) μm, so CR-1 was preliminarily identified as Vibrio desulfuricans, a member of the bacterial kingdom in the phylum of purple photosynthetic bacteria (Buchanan & Gibbons 1984).
Molecular identification of strain CR-1
The 16S rDNA sequence of strain CR-1 was compared to the registered 16S rDNA sequences in the GenBank database by the Blast search system for homology. As shown in Figure 1(b), strain CR-1 and Desulfovibrio (AY928662.1) belong to the same branch with 99% homology. It can be inferred that strain CR-1 belongs to the genus Desulfovibrio.
Study on the growth conditions of reducing Cr(VI) by strain CR-1
Effect of pH value on the removal rate of Cr(VI)
Figure 2(a) shows that as the reaction proceeded, the removal rate of hexavalent chromium gradually increased. With the same reaction time, the removal efficiency of Cr(VI) was better at approximately pH 7 but was not ideal when the pH was lower than 5 or higher than 9. This result indicated that the optimum pH value for the growth and metabolism of strain CR-1 was approximately 7, at which value the bacterial activity was higher, more metabolites were produced, and the removal efficiency of Cr(VI) was improved.
Effect of temperature on the removal rate of Cr(VI)
As shown in Figure 2(b), the removal rate of Cr(VI) by strain CR-1 increased gradually with an increase in culture temperature from 20 °C to 40 °C, but the removal rate of Cr(VI) decreased sharply when the temperature reached 45 °C. When the temperature is too low, the metabolism of strain CR-1 is weak, the activities of various enzymes in cells are reduced, fewer metabolic products are produced, and the removal rate of Cr(VI) is low. When the temperature is too high, the proteins and nucleic acids of bacterial cells will denature and inactivate, thereby affecting the Cr(VI) removal effect of the bacteria. Zhao et al. (1997) showed that the critical growth temperature of sulfate-reducing bacteria (SRB) under pure culture conditions in wastewater treatment was 45 °C. Li et al. (2018) also showed that the critical growth temperature of the V. desulfuricans G20 strain was 47 °C; at higher temperatures the strain did not grow. It can also be seen in Figure 2(b) that with an increase in culture time and in the temperature range of 30 °C to 40 °C, the removal rate of Cr(VI) by strain CR-1 gradually increased to the same level, and the removal effect was best. This indicates that the chromium-lowering strain CR-1 is a medium-temperature bacterium and that the optimum temperature range is 30–40 °C.
Effect of initial Cr(VI) concentration on the removal rate of Cr(VI)
Figure 2(c) shows that the domesticated strain had different tolerances to different concentrations of Cr(VI). With an increase in the initial Cr(VI) concentration, the removal rate of Cr(VI) by strain CR-1 gradually decreased. When the initial concentration of Cr(VI) was less than 500 mg/L, Cr(VI) was almost completely removed. With an initial concentration of Cr(VI) greater than 500 mg/L, the removal rate of Cr(VI) by strain CR-1 decreased rapidly, which indicated that increased Cr(VI) had an effect on bacterial activity and the toxicity of Cr(VI) caused the inactivation of bacterial cells. Therefore, the bacteria should be domesticated step by step in a high concentration of Cr(VI)-containing wastewater to maintain the bacteria's high activity in wastewater containing high concentrations of Cr(VI).
Effect of bacteria:waste ratio on the removal rate of Cr(VI)
Figure 2(d) shows that the removal rate of Cr(VI) increased with an increased ratio of bacteria to waste at the same concentration of Cr(VI) (10, 15, 20, 25, and 30 mL of bacterial solution was added to 100 mL of 500 mg/L Cr(VI)-enriched medium 2, respectively) before 18 h. This result shows that an increased ratio of bacteria to waste increases the density of bacteria in the solution, so that more Cr(VI) can be reduced. When the reaction time was from 18 h to 24 h, the removal rate of Cr(VI) by the strain tended to be stable; at this time, the strain was also in the stable growth phase. The removal rates of Cr(VI) were almost the same when the ratio of bacteria to waste was 1:5, 1:4 and 1:3.3, but all of them were higher than the ratio of bacteria to waste of 1:10 and 1:1.67. Under the same conditions of effectiveness, the larger the ratio of bacteria to waste, the more bacteria are added, and the higher the cost. Considering the cost and effectiveness simultaneously, the optimal ratio of bacteria to waste was determined to be 1:5.
Effects of Pb2+, Zn2+, and Cd2+ on the treatment of Cr(VI) by the strain
Three metals (Pb2+, Zn2+, and Cd2+) and their mixture were selected as the research object to simulate the metal components typically found in chromium-containing wastewater from the local leather industrial park. Figure 2(e) shows that Pb2+, Zn2+, and Cd2+ have certain inhibitory effects on the removal of Cr(VI) by strain CR-1 and the inhibitory effect increased with increased concentration of heavy metal ions. Moreover, the inhibitory effects of the above three heavy metals on the chromium reduction of strain CR-1, from strong to weak, followed the order Cd2+ > Zn2+ > Pb2+. At Zn2+ > 20 mg/L, obvious inhibition occurred, and the Cr(VI) removal rate decreased significantly. The heavy metal Zn is a trace element necessary for living organisms; it can participate in and promote the biochemical reaction process, but if it exceeds a certain range, the toxicity of Zn2+ ions will harm bacterial cells and inactivate them, decreasing the hexavalent chromium removal rate. Cd2+ is a very toxic heavy metal that cannot be used by bacteria. At Cd2+ > 20 mg/L, strong inhibition of Cr(VI) reduction by bacteria is apparent. This inhibitory effect may be because, at an excessive concentration of Cd2+, the toxicity of Cd2+ ions caused the inactivation of various enzymes in the cells of chromium-reducing strain CR-1 in solution. Pb2+ had little effect on chromium reduction by strain CR-1, which may be because Pb2+ had no effect on reductase in strain CR-1. Song et al. (2016) also showed that the activity of adenosine 5′-phosphosulfate reductase and sulfite reductase decreased significantly when the initial concentration of Pb2+ reached 1100 mg/L. These three heavy metal ions are liable to form sulfide precipitates with S2− in water, but Cr2S3 is extremely easy to hydrolyze. Under the pH condition of this reaction, Cr3+ exists as Cr(OH)2+. Mothe et al.(2016) believed that, at a low initial metal concentration, precipitation of the insoluble metal due to sulfide produced by SRB avoids any toxic effect of the metal on SRB. On the other hand, at an elevated concentration, these metals tend to be toxic to microorganisms due to their enhanced bioavailability, thus resulting in denaturation and deactivation of enzymes, rupture of cell organelles and membrane integrity, etc.
The results of the experiments with the metal mixture addition at different concentrations (the composition of the metal mixture was the same and the total mixed concentration was 10, 20, 30, 40, and 50 mg/L) are also shown in Figure 2(e). Compared with individual metal addition, there was no obvious effect on the reduction effect of SRB with the metal mixture addition, and it also did not show significant synergistic inhibitory effects. However, the SRB strain cultured by Hao et al. (1994) showed a synergistic inhibitory effect on a mixture of six heavy metals including Pb2+, Zn2+, and Cd2+. In comparison, our strain CR-1 is undoubtedly beneficial to the actual industrial wastewater treatment.
Effect of reaction time on the removal rate of Cr(VI)
Figure 2(f) shows that the concentration of Cr(VI) in the chromium-containing waste liquor decreased rapidly in the first 3 h, which was due to the production of the metabolite H2S by the growth and metabolism of a certain amount of strain CR-1 in the original bacterial liquor. At the beginning of the reaction, the chemical reduction of Cr(VI) by H2S should play the most important role in the removal of Cr(VI). Therefore, in the initial stage of the reaction, the concentration of Cr(VI) decreased significantly. Then, the bacteria entered the logarithmic growth phase; during this period, the Cr(VI) concentration rapidly decreased. This decrease was because the growth activity of bacteria during the logarithmic growth period was strong, reductase metabolism was active, and the amount of metabolites produced was relatively high, thus accelerating the removal of Cr(VI). After this period, the removal rate of Cr(VI) tended to be stable, indicating that the bacteria had reached a stable stage. However, the change trend of total chromium in solution was not significant, which indicated that the total chromium is not changing, meaning all hexavalent chromium is being reduced to trivalent chromium. Finally, the removal rate of hexavalent chromium reached the integrated wastewater discharge standard (NEPA 1996).
Mechanism analysis of chromium reduction in strain CR-1
Currently, most researchers believe that there are three mechanisms for the removal of Cr(VI) by microorganisms. The first mechanism is that microbial metabolites can reduce Cr(VI). For example, SRB can reduce SO42− to H2S and S2−, and H2S and S2− can reduce hexavalent chromium to a low valence state for removal purposes. The second mechanism is that microorganisms can rely on the reductase itself or some reducing substances to reduce Cr(VI). The third mechanism is that a high polymer content on the surface of microbial cells can directly adsorb and therefore remove Cr(VI) (Chen 2006; Srivastava & Thakur 2007).
H2S is an important metabolite in the process of dissimilation and reduction of sulfate, which poses a direct threat to the survival of most organisms, especially aerobic organisms. However, when there are heavy metal ions in the environment, insoluble precipitate in water can be generated by direct reaction with heavy metal ions to restore and detoxify the environment. Figure 3 shows that the H2S pathway has a much better effect on the reduction of Cr(VI) than other pathways because, after a period of cultivation, a large amount of H2S is produced in the solution, which will reduce Cr(VI). In the first 3 hours of the reaction, the centrifugal solution containing only H2S was added to the waste liquid containing Cr(VI), the H2S in the solution immediately reduced most of the Cr(VI), and the removal rate of Cr(VI) increased rapidly. Then, the reduction rate of Cr(VI) by H2S remained almost unchanged. The reaction rate is very similar to that of a chemical reaction, and it is highly likely that it is the result of a direct reaction. The removal rates of Cr(VI) by the H2S pathway were 40.3% and 47.2% when the reaction lasted for 3 h and 24 h, respectively. However, the removal rate of Cr(VI) by the original bacterial solution was 99.6%, which was quite different. The reasons for this result may be as follows. First, strain CR-1 is still growing and metabolizing in the original bacterial solution. The metabolic result is that H2S will continue to be produced. SRB can reduce SO42− and produce H2S and S2−, which can reduce hexavalent chromium to a low-valent state for removal. The H2S in the H2S pathway is collected by centrifugation, and its content will only be consumed and thus will not increase during the reaction. Therefore, as the reaction time is extended, the removal rate of Cr(VI) varies. There is no CR-1 strain in the H2S pathway. After 3 h, the residual concentration of Cr(VI) in the original bacterial solution continued to decrease, and the removal rate continued to increase, but with the consumption of H2S, the removal rate gradually became slower. At 24 h, the removal rate of Cr(VI) was stable at 99.6%.
In the bacterial reductase pathway, since SO42− is not contained in the solution, this pathway serves only as a reductase in the bacterial cell to reduce Cr(VI). Through experiments, it was found that for strain CR-1, the reduction of Cr(VI) by the bacterial reductase pathway was not as effective as that by the H2S pathway and did not dominate the reduction of Cr(VI), but the bacterial reductase pathway did reduce Cr(VI). After 3 and 24 h of reaction, the removal rate of Cr(VI) was only 5.5% and 35%, respectively. Notably, the removal rate 3 h before the reaction was particularly low, which may be because the concentration of 500 mg/L Cr(VI) is high at the initial stage of the reaction, which has a significant inhibitory effect on the reductase.
Microbially derived extracellular polymeric substances (EPSs) are complex mixtures of polymers secreted by microorganisms, including polysaccharides, proteins, nucleic acids, fats, and humic acids. Therefore, SRB often contain hydroxyl, carboxyl, amino, sulfonic, mercapto, and carbonyl groups in the shell, which cause the shell to be negatively charged. By electrostatic attraction, the shell adsorbs heavy metal cations and forms complexes with metal ions, thus fixing metal in a polymer matrix (Pal & Paul 2008). Figure 3 shows that the effect of the microbial EPS adsorption pathway on the treatment of chromium(VI) is unsatisfactory and that the removal rate of chromium(VI) is almost unchanged because chromium(VI) exists in solution as a CrO42− anionic group (Zhao et al. 2006). EPSs of SRB, acting as adsorbents, can better adsorb metal cations but cannot adsorb heavy metals in anionic groups.
After analysing the reduction of Cr(VI) by the adsorption pathway, bacterial reductase pathway and H2S pathway, a hypothesis of the Cr(VI) removal mechanism of SRB (CR-1) was proposed. In the first 3 h of removal of Cr(VI) by strain CR-1, the H2S pathway was dominant; between 3 h and 24 h, due to the large consumption of H2S in the first 3 h by the H2S pathway, the reductase in the reductase pathway gradually adapted to Cr(VI), and with the consumption of nutrients, the removal rate of Cr(VI) by strain CR-1 gradually plateaued. The adsorption pathway hardly worked during the removal process.
In summary, the reductase pathway and H2S pathway alone cannot achieve the removal effect of the original bacteria–solution pair including SRB (CR-1). These two mechanisms do not overlap but show different dominant changes in different reaction stages. At the initial stage of the reaction, the sulfate in the original bacterial solution is relatively abundant, and Cr(VI) is toxic to strain CR-1, so from the point of avoiding toxicity, strain CR-1 may preferentially choose to reduce sulfate. Based on this, the H2S pathway dominated the first 3 h of the reaction. With the reduction of sulfate and the consumption of nutrients, H2S alone is not enough to complete the reduction of residual Cr(VI), which must be removed by the bacterial reductase pathway.
Figure 3 shows that the effect of the microbial extracellular polymer adsorption pathway on the treatment of Cr(VI) is unsatisfactory and the concentration of Cr(VI) has almost no change because Cr(VI) exists in solution as a CrO42− anionic group. In the bacterial reductase pathway, because there is no SO42−, this pathway includes only the reduction of Cr(VI) by reductase in bacterial cells. Although a small amount of Cr(VI) can be reduced, this pathway does not dominate the reduction of Cr(VI). The effect of the H2S pathway on the reduction of Cr(VI) is much better than that of other pathways because after a period of culture, the large amount of H2S in solution will reduce a large amount of Cr(VI). After centrifugation, the solution containing only H2S was added to waste liquid containing Cr(VI), and the H2S in the solution immediately reduced most of the Cr(VI), indicating that the reduction of Cr(VI) by H2S is a chemical reaction; therefore, the reduction rate of Cr(VI) by H2S was almost unchanged thereafter. It can be concluded that the mechanism of reduction of Cr(VI) by strain CR-1 primarily depends on Cr(VI) removal by the accumulated H2S in the culture medium, while the removal of the remaining part depends on bacterial reductase and the small amount of H2S produced by bacteria in the waste liquid by the reduction of SO42−, while the adsorption of Cr(VI) by extracellular polymers is almost completely ineffective.
Reduction kinetics of chromium reduction by strain CR-1
km: hexavalent chromium reduction rate constant (mg Cr(VI)/(h·bacteria))
C: hexavalent chromium concentration at time t (mg/L)
Kc: half-velocity constant (mg Cr(VI)/L)
X: cell concentration at time t (number of cells/L)
C0: initial hexavalent chromium concentration (mg/L)
X0: initial bacterial concentration (number of cells/L)
Rc: maximum hexavalent chromium reduction in a single cell (mg/L)
According to the bacterial count, the initial bacterial density in the suspension was X0 = 6 × 1010/L, and the known initial concentration of Cr(VI) was C0 = 500 mg/L because the bacterial density in the suspension was 3 × 1011/L and the bacterial waste ratio was 1:5. The fitting curve of the reduction kinetics equation is shown in Figure 4.
The maximum reduction amount of Cr(VI) by the chromium-reducing strain CR-1 was estimated to be RcX0 = 201.98 mg/L (X0: initial bacterial concentration (cell number/L)). The actual reduction amount of Cr(VI) after 24 h was 187.4 mg/L. The maximum reduction amount estimated by the kinetic equation was only 14.58 mg/L, which was different from the actual reduction amount. The obtained parameters indicate that the strain (CR-1) used in this experiment has a good ability to reduce chromium.
In summary,we obtained an anaerobic sulfate-reducing bacterium, V. desulfuricans, which can reduce hexavalent chromium. It has high tolerance and strong reducing power with ethanol as a carbon source, and can reduce chromium-containing wastewater with concentration of 500 mg/L Cr(VI) to meet the integrated wastewater discharge standard. The optimum conditions are a pH of approximately 7, a temperature of 35 °C, a bacteria to waste ratio of 1:5. The inhibitory effects of the three heavy metals on the chromium reduction of strain CR-1, from strong to weak, followed the order Cd2+ > Zn2+ > Pb2+. When three heavy metal cations coexisted and existed alone, they had little effect on the reduction effect of the SRB strain, and did not show obvious synergistic inhibition. The mechanism of treating Cr(VI) by the dominant chromium-reducing strain CR-1 is mainly based on the reduction of Cr(VI) by H2S accumulated in the cultured bacterial liquor and the small amount of H2S produced by bacterial reductase, bacterial growth and the reduction of SO42− in waste liquor. Based on the reduction kinetics experiment of the chromium-reducing strain, a reduction kinetics equation was established, which can predict the maximum reduction amount of hexavalent chromium by the strain under anaerobic conditions, and evaluate the strain's reducing ability.
The study shows that the strain CR-1 could be effectively used as a bioremediation tool for alleviation of treating high-chromium-concentration industrial wastewater.
The authors acknowledge the support of National Key R&D Program of China (No. 2017YFC1503106), Liaoning BaiQianWan Talents Program of China (No. 2018C01) and Liaoning Provincial Natural Science Foundation of China (No. 2019-ZD-0037).
The Supplementary Material for this paper is available at https://dx.doi.org/10.2166/wst.2020.057.