Abstract

Bioremediation is one of the treatment technologies for the black-odorous water, and obtaining functional bacteria is the key step to its success. In this study, a number of highly efficient pollutant degrading strains were isolated from the sediment of black-odorous river, and were identified by phenotypic and phylogenetic analysis. The composite bacterial agent J1 was mixed by strains A1, A2, A5 and A7, with a volume ratio of 4:4:2:1. And the final compound bacteria injected into black-odorous water were composed of J1 and NS3, with the volume ratio of 1:1. Optimized degradation conditions of compound bacterium agent were as follows: pH 7.5, DO 2.5 mg/L, temperature 30 °C. Under optimized conditions, add 1% by volume to the black-odorous water for a 50-day experimental operation. Finally, the overlying water ammonia nitrogen, chemical oxygen demand (COD), and total phosphorus has been significantly degraded. The research is expected to contribute to the use of bioremediation methods to repair black-odorous water, and the application of isolates can be carried out in-situ for water types similar to pollute waterways.

HIGHLIGHTS

  • Controllable functional microbes for characteristic pollutants were obtained.

  • Physiological, biochemical and degradation characteristics of microbial isolates were explored.

  • A basic framework of in situ bioremediation of black-odorous water was demonstrated.

Graphical Abstract

Graphical Abstract
Graphical Abstract

INTRODUCTION

The black-odorous water is pungent, poisonous, black or gray-black, and loses its ecological function (Ji et al. 2017). Black-odorous water occurs in both developing and developed countries (Le et al. 2010), and has also become a common problem in China in recent decades (Liu et al. 2011; Wang et al. 2016). According to data released in September 2019 from National Urban Black and Smelly Water Management Supervision Platform (China Housing and Urban–Rural Construction Ministry, 2019), the number of black-odorous water in cities across the country has increased to 2,100.

In these water bodies, the sediments generally contain high concentrations of nutrients (N, P), heavy metals (Fe, Mn, Cu, Hg, etc.) and organic pollutants (PAHs, PCBs, etc.) (Wang et al. 2014; He et al. 2018). The concentration of these substances exceeds the self-purification capacity of the water body. The decomposition of organic matter and excessive reproduction of algae leads to insufficient dissolved oxygen (DO) in water, and it kills a lot of algae, decomposition and mineralization, forming oxygen-depleting organic matter and ammonia nitrogen, which leads to water bodies turn black and produce offensive odorous gas (Lu et al. 2012). Studies have shown that when the organic matter concentration reaches the critical load level of 1.0 g/L, the water will begin to blacken, especially the sulfur-containing organic matter, which takes only 7–13 days (Yu & Sun 2016). Volatile organic sulfur compounds (VOSC) are the main causes of odor in water, such as methyl mercaptan (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) (Liang et al. 2018). Nitrogen-containing compounds such as organic amine, cadaverine and putrescine also produce odors, which are mostly produced by proteolytic microorganisms (Pham et al. 2012). In addition, malodor may also be emitted from organic matter that does not include any sulfur or nitrogen components, because some organic matter may release volatile fatty acids (VFA) (Yuan et al. 2018).

The treatment technologies of black-odorous water include four categories: physical, chemical, biological and ecological remediation technologies. The extremely low DO level is the fundamental feature of black-odorous water (Cao et al. 2020). Artificial aeration can increase the DO concentration in water and advance the proliferation of bacteria related to organic matter degradation (He et al. 2013a), affecting the nitrogen transformation pathway. However, this will also lead to sediment resuspension and promote the release of endogenous nitrogen (He et al. 2013b). The technology is becoming obsolete due to high energy consumption and relatively low efficiency (He et al. 2017). Bioremediation is a treatment technology that uses specific organisms (protozoa, microorganisms and plants) to bio-absorb, transform, remove or degrade environmental pollutants under controlled environmental conditions, so as to achieve ecological purification and restoration of ecological effects (Shishir et al. 2019). Because of its low cost, good effect and little interference to the environment, it has become a research hotspot in the field of environmental protection. Microbial agents play a major role in the treatment of water. They can improve black-odorous water in an economical, effective, simple and convenient way, and have been satisfactorily practiced worldwide. However, few microorganisms are able to simultaneously remove phosphorus, nitrogen and organic pollutants.

Microbes are a strong driver of most ecosystem processes, but the application standards of bioremediation are often missing, and is still fear-based regulation but risk-based regulation. Therefore, despite the rapid development of omics technology, isolation, screening and identification of excellent microbial resources with environmental remediation potential are still the main ideas and the necessary conditions. To achieve microbial agents for black-odorous water bioremediation, a few efficient degrading bacteria were isolated from natural sediment, included common pollutant degrading bacteria, heterotrophic nitrification–aerobic denitrification bacteria and sulfur autotrophic denitrification bacteria, and their physiological and biochemical characteristics were analyzed.

MATERIALS AND METHODS

Sample collection

A sediment core extractor (UWITEC) was utilized to collect overlying water 0.5 m below the river surface and sediment at a depth of 0–0.20 cm below the river bottom of a typical black-odorous river. The sediment and overlying water were collected from four black-odorous rivers (22°55 N, 112°58 E; 23°17 N, 113°18 E; 29°57 N, 106°45 E; 29°57 N, 106°44 E). The sample was taken to a sterile container and immediately returned to the laboratory. After the sediment samples are homogenized, they are utilized for the determination of physicochemical properties, bacteria screening and experimental simulation of the indoor environment. The overlying water is used for physicochemical properties testing and added to a simulated water tank. The remaining samples are stored at low temperature (4 °C) for a later experiment.

Monitoring of environmental parameter

Environmental parameters of black-odorous water, including temperature (T), pH, and the concentration of DO were measured in situ by using a Water Quality Checker U-10 (HORIBA). The concentrations of chemical oxygen demand (COD), ammonium (NH3-N), total nitrogen (TN), total phosphorus (TP), nitrate nitrogen (NO3), Fe2+, and SO42− were measured according to Chinese NEPA standard methods (Bureau 2002).

Medium

Beef-protein medium (g/L): beef extract of 3, protein peptone of 10, NaCl of 5, and yeast extract of 20 (add when solid culture medium), pH was adjusted to 7.5. Sulfur autotrophic denitrifying bacteria enriched medium (g/L): Na2S2O3·5H2O of 10, KNO3 of 4, KH2PO4 of 4, NaHCO3 of 2, MgCl2·6H2O of 1, and FeSO4·7H2O of 0.02. Sulfur autotrophic denitrifying bacteria primary screening medium (g/L): Na2S2O3·5H2O of 5, KNO3 of 2, KH2PO4 of 2, NaHCO3 of 1, MgCl2·6H2O of 0.5, FeSO4·7H2O of 0.01, and yeast extract of 20. Heterotrophic nitrification bacteria medium (g/L): (NH4)2SO4 of 0.24, succinic sodium of 2.17, yeast extract of 20, and vinyl salt solution of 50 mL, pH was adjusted to 7.0. Vinyl salt solution (g/L): K2HPO4·3H2O of 6.5, MgSO4·7H2O of 2.5, NaCl of 2.5, FeSO4·7H2O of 0.05, MnSO4·H2O of 0.04. BTB medium (g/L): succinic sodium of 4.72, NaNO3 of 0.85, KH2PO4 of 1, FeSO4·7H2O of 0.2, MgSO4·7H2O of 0.1, yeast extract of 20, and bromothymol blue of 1 mL. Test medium for nitrogen and sulfur removal (g/L): Na2S·9H2O of 0.9, KNO3 of 0.1, KH2PO4 of 2, NaHCO3 of 1, MgCl2·6H2O of 0.5, FeSO4·7H2O of 0.01. Medium sterilization conditions were 121 °C, 103.4 kPa, and 20 min. All Na2S2O3·5H2O and Na2S·9H2O need to dissolve, then filter sterilize with 0.22 μm microporous membrane, and mix with the remaining components after sterilization at 121 °C for 20 min.

Isolation of strains

In order to repair the black-odorous water in situ by bioremediation, characteristic bacterial were isolated from the sediment of the typical black-odorous rivers. After the enrichment, primary screening and re-screening of bacteria, general pollutant degrading bacteria, sulfur autotrophic denitrifying bacteria and heterotrophic nitrification–aerobic denitrification bacteria are finally obtained.

Enrichment: take 10 g of 3 parts of black-odorous water sediment and put them into 250 mL conical flasks containing 100 mL sterilized beef-protein medium, sulfur autotrophic denitrifying bacteria enriched medium and heterotrophic nitrification bacteria medium, respectively. After 3 days of constant temperature culture at 30 °C and 150 r/min in shaking table, 5 mL enrichment media were transferred to fresh liquid enrichment medium for culture, and the operation was repeated until there was obvious bacterial solution (about 4 times).

Primary screening: 1 mL enrichment medium was diluted 10 times, and then coated on beef peptone solid medium, sulfur autotrophic denitrification solid medium and heterotrophic nitrification solid medium, and then cultured in 30 °C constant temperature foster box for 3 days. Single colonies with different morphological characteristics in 10−6 media were selected for three-stage separation and purification in the primary screening solid medium until the morphological characteristics of the purified colonies were different, and then they were stored on the beef extract peptone slope. In addition, the single bacteria obtained from heterotrophic nitrification solid medium were scribed on the BTB medium and cultured at 30 °C for 3 days. Blue colonies around the colonies were aerobic denitrifying strains, and the denitrifying strains were scribed on the beef peptone slope for preservation.

Re-screening: under aseptic conditions, two rings of general pollutant degrading bacteria were selected from the beef peptone slope and inoculated into a conical flask containing 50 mL sterilized black-odorous water for constant temperature cultivation. After 48 h, the COD, NH3-N and TP of black-odorous water were determined. Two rings of sulfur autotrophic denitrifying bacteria were selected from the beef peptone slope and inoculated in the liquid medium for nitrogen and sulfur removal. The contents of nitrate nitrogen and sulfide in the medium were determined after 48 h of constant temperature cultivation at 30 °C. Two rings of heterotrophic nitrification–aerobic denitrification bacteria were selected from the beef peptone slope and inoculated in the liquid medium with ammonia nitrogen as the sole nitrogen source. The ammonia nitrogen content in the medium was determined after 48 h of constant temperature cultivation at 30 °C. Strains with high degradation effect on pollutants were selected and preserved with glycerol.

Identification of strains

For 16S rDNA gene amplification, the genomic DNA of optimum strain was extracted using Andybio DNA kit. The 16S rRNA gene fragments were amplified using universal primers 27F (5′AGAGTTTGATCCTGGCTCA3′) and 1492R (5′GGTTACCTTGTTACGACTT3′). PCR amplification was performed under the following conditions: 5 min at 95 °C, 30 cycles of 3 min at 94 °C, 30 s at 55 °C, and 90 s at 72 °C, plus an additional 5 min cycle at 72 °C. The automatic sequence was carried out by Nanjing Kingsley Biotechnology Co. Ltd. The 16S rDNA sequence was checked in GenBank.

Treatment of actual wastewater by mixed bacteria

There is no standard for the evaluation of black-odorous water, so the removal of ammonia nitrogen and COD is taken as the degradation index in this research, and TP is taken as the auxiliary parameter to carry out the degradation experiment. The degradation experiment was carried out for 50 days, in which aeration was carried out in the first 20 days, and the experimental device was made of plexiglass. The reactor has a height of 1 m, an outer diameter of 100 mm and a wall thickness of 3 mm. On the right side of the reactor, the bottom sludge aeration port is set at 10 cm from the bottom, and the overlying water sampling port is set at 50 cm from the bottom. The top of the reactor is equipped with aeration port and water supply port. The sand core aeration head is used for aeration, and the aeration rate is 10 L/min. The height of sediment is 20 cm and that of overlying water is 50 cm. Intermittent aeration, 8 h a day, 4 h in the morning and 4 h in the afternoon, controlled by time control switch. Sediment is wrapped with lightproof material, and the specific experimental device is shown in Figure 1. Three groups of reactors were set, the control group without any operation. The aeration group without bacteria, which only carries out deep water aeration to control the aeration depth to avoid sediment disturbance. The aeration and adding bacteria agent group is the same as the aeration group except adding bacteria agent. The sediment sampler was used to take the sediment into the column reactor, and the overlying water was slowly injected by siphon method to avoid disturbing the sediment. In the aeration stage, compound microbial agent was added to aeration and adding bacteria agent group every 10 days, with the addition ratio of 1% of the reactor volume. After the dissolved oxygen increased to 2.5 mg/L, aerobic denitrifying bacterial agent was added to enhance nitrogen removal. Because the nature of the raw water in the black-odorous river is easy to change, chemicals are added to the original water to control the concentration of pollutants in the overlying water in the range of COD 68–72 mg/L, NH3-N 20–25 mg/L and TP 2.0–2.5 mg/L. The contents of COD, NH3-N, TP and DO in overlying water were detected every 2 days, and the contents of organic carbon, TN, TP, Fe2+, SO42− in sediment samples were detected every 10 days.

Figure 1

Experimental device. ①&③ aeration outlet, ② sampling port, ④ water supply outlet, ⑤ aerator, ⑥ time switch.

Figure 1

Experimental device. ①&③ aeration outlet, ② sampling port, ④ water supply outlet, ⑤ aerator, ⑥ time switch.

Statistical analysis

All the above experiments were repeated in triplicate, and the average data are reported. A one-way analysis of variance with Tukey's test was used to determine any significant differences between treatments (p < 0.05).

RESULTS AND DISCUSION

Isolation of highly efficient malodorous black-odorous degrading bacteria

The colony morphology of the isolated strains was observed by cell staining to ensure the diversity of the selected strains.

After primary screening, 45 strains were screened out from four kinds of sediment by using three different types of media. After re-screening, four strains with good degradation effect on general pollutants, one strain with good degradation effect on ammonia nitrogen and one strain with good desulfurization and denitrification ability were screened. The strain numbers are A1, A2, A5, A7, HF2 and NS3, respectively. The degradation ability of each strain of pollutants is given in Table 1. The degradation rates of COD by A1, A2, A5 and A7 were above 70%. A1 and A7 had the best degradation effect on COD, 82.4% and 79.6%, respectively. A2 had better degradation effect on COD and TP. The NH3-N degradation rate of HF2 reached 89.7%, and the denitrification effect was evident. The denitrification rate of NS3 was 48.2%, and the desulfurization efficiency was 35.3%.

Table 1

Removal rate of pollutants by strains

ItemRemovel rate (%)
CODNH3-NNO3TPS2−
A1 82.4 21.2 — 41.6 — 
A2 72.8 38.4 — 35.7 — 
A5 76.3 10.8 — 9.8 — 
A7 79.6 16.2 — 7.1 — 
HF2 — 89.7 — — — 
NS3 — — 48.2 — 35.3 
ItemRemovel rate (%)
CODNH3-NNO3TPS2−
A1 82.4 21.2 — 41.6 — 
A2 72.8 38.4 — 35.7 — 
A5 76.3 10.8 — 9.8 — 
A7 79.6 16.2 — 7.1 — 
HF2 — 89.7 — — — 
NS3 — — 48.2 — 35.3 

The colony of strain HF2 was white, regular edge, convex surface, moist and glossy. Gram staining results under the microscope showed that the strain HF2 was gram-positive, rod-shaped. Colony morphology and Gram staining results of other strains are shown in Figure 2. 16S rDNA identification results of each strain are compared with blast in gene bank to determine the species of the strain. A1 is similar to Klebsiella sp., A2 is similar to Pseudomonas sp., A5 is similar to Comamonas sp., A7 is similar to Chryseobacterium sp., HF2 is similar to Bacillus sp., and NS3 is similar to Roseateles sp. The sequence homology of each strain and similar strains is greater than 99%.

Figure 2

The result of colonial morphology and gram staining.

Figure 2

The result of colonial morphology and gram staining.

Preparation of compound bacterium agent

Depending on the different functions of microbial agents, four general pollution degrading bacteria A1, A2, A5 and A7 were recombined. L9 (34) orthogonal experiment was carried out at three inoculation levels of 1 (0.5%), 2 (1%) and 3 (2%), temperature of 30 °C and rotational speed of 150 r/min. The experimental data were calculated after 2 days. The range analysis results demonstrate that the order of influence of the four bacteria on COD degradation rate is A1 > A7 > A5 > A2. K value analysis showed that the optimal combination of COD degradation was A1: A2: A5: A7 = 3:1:3:2; range analysis showed that the order of NH3-H degradation rate was A2 > A1 > A7 > A5. K value analysis showed that the optimal combination of NH3-H degradation was A1: A2: A5: A7 = 3:3:2:1. The order of influence of the four strains on the degradation rate of TP was A1 > A2 > A5 > A7, and the optimal combination of TP degradation was A1: A2: A5: A7 = 3:3:2:1. Comprehensive analysis showed that when the optimal ratio of compound bacterium agent J1 was A1: A2: A5: A7 = 4:4:2:1, the degradation rate of each pollutant was the best.

In order to determine the optimal addition proportion, different additional proportion experiments were carried out for J1 and NS3. Because NS3 is sulfur autotrophic denitrifying bacteria, the degradation effect of organic matter and TP are general. In order to ensure the degradation effect of conventional pollutants and reduced sulfide at the same time, J1 and NS3 were inoculated in black-odorous wastewater at 1% total inoculation ratio with volume ratios of 1:1, 2:1 and 3:1, respectively. By measuring the degradation rate of pollutants in different volume ratios, the optimal ratio of the two bacteria was determined. The S2− removal rates of 1:1, 2:1 and 3:1 were 38.4%, 30.7% and 26.5%, respectively. The volume ratio of 1:1 was used.

Condition optimization and analysis

The temperature (20, 25, 30, 35 and 40 °C), pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9) and rotational speed (130, 140, 150, 160, 170, 180, 190 and 200 r/min) were investigated on the growth of the compound microbial agent.

Effect of rotating speed on degradation. The effect of rotation speed on the growth of bacteria lies in the effect of rotation speed on the content of DO. With the increase of rotation speed, DO content also increases, but when the rotation speed exceeds a certain value, the strain cells will be destroyed due to the increasing shear force, thus affecting the biomass. In this experiment, J1, HF2 and NS3 were inoculated into LB medium with pH 7 at 1% inoculum ratio, respectively. They were incubated at 30 °C for 24 h in a shaker at 130, 140, 150, 160, 170, 180, 190 and 200 r/min, and the biomass was determined. It can be seen from Figure 3(a) that the biomass of the inoculant increases with the increase of the rotation speed of the shaker, and gradually decreases after exceeding a certain rotation speed. The results showed that the optimal rotation speed of J1 was 160–180 r/min. Optimal rotation speed of HF2 was 170–200 r/min, and the optimal rotation speed of NS3 was 150–180 r/min.

Figure 3

Effects of environmental factors on the growth of bacteria. (a) rotation speed, (b) pH, (c) temperature.

Figure 3

Effects of environmental factors on the growth of bacteria. (a) rotation speed, (b) pH, (c) temperature.

Effect of pH on degradation: Inoculate J1, HF2 and NS3 into LB medium with a pH of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9. Biomass was determined after 24 h incubation at 30 °C and 150 r/min. It can be observed in Figure 3(b) that the growth amount of J1 is very little under the acidic condition of pH 4. With the increase of pH, the biomass gradually increases, and the optimal growth pH of J1 is 6.5–7.5. When pH is 7.5, the biomass of HF2 was the largest, and the optimum pH of HF2 was about 7.5–8. The variation trend of biomass of NS3 with pH was similar to J1 and HF2. The optimum pH for growth of NS3 was about 7–7.5.

Effect of temperature on degradation: In this experiment, J1, HF2 and NS3 were added into LB medium with pH 7 by the volume ratio of 1%. Put separately in the incubation shaker at the temperature of 20, 25, 30, 35, 40 °C and rotating speed of 150 r/min; the biomass was determined after 24 h. It can be seen from Figure 3(c) that the influence trend of temperature on the three bacteria agents are similar. The biomass increased with the increase of temperature, which was caused by the increase of enzymatic activity, membrane fluidity, microbial metabolism and growth rate. When the temperature reached 30 °C, the biomass reached the maximum, and then decreased with the increase of temperature. Because excessively high temperature will lead to denaturation and inactivation of proteins, enzymes and nucleic acids in cells, and even the death of microorganisms.

Under optimal growth conditions, the growth curve of J1, HF2 and NS3 were determined, and the results are shown in Figure 4.

Figure 4

Growth curve of compound agent. (a) compound bacterium agent J1, (b) strain HF2, (c) strain NS3.

Figure 4

Growth curve of compound agent. (a) compound bacterium agent J1, (b) strain HF2, (c) strain NS3.

Treatment of black-odorous water by mixed bacteria

Concentration of COD in overlying water. The change of COD concentration in overlying water is shown in Figure 5(a). At the beginning of the experiment, the COD concentration in the overlying water of control group increased due to the release of sediment pollution, reached a high value of 92 mg/L on the 8th day, then stabilized, and began to decline, which may be due to the migration of water pollutants into the sediment. After that, the COD concentration of overlying water fluctuated, and then slowly rose to 71 mg/L, which was close to the level at the beginning of the experiment. After the experiment of aeration group, the COD concentration of overlying water showed a downward trend, and then fluctuated slightly. After 30 days, the COD concentration began to rise, and at the end of the experiment, the COD concentration was 45 mg/L. The change of COD concentration of overlying water in the aeration and adding bacteria agent group was similar to that in the aeration group, showing a trend of first decreasing and maintaining stability, and then slowly rising. However, the COD concentration was lower than that in the aeration group, which may be caused by the addition of bacteria agent.

Figure 5

Fluctuations of monitoring indicators in overlying water. (a) COD, (b) NH3-N, (c) TP, (d) DO.

Figure 5

Fluctuations of monitoring indicators in overlying water. (a) COD, (b) NH3-N, (c) TP, (d) DO.

Concentration of NH3-N in overlying water. According to Figure 5(b), in the early stage of the experiment, the NH3-N concentration in the overlying water of the control group showed an upward trend due to the release of sediment pollution, reaching a peak value of 30.64 mg/L on the 10th day, then remained stable, decreased slightly during the period, and rose slowly in the later stage. At the end of the experiment, the NH3-N concentration in the overlying water was 26.75 mg/L, NH3-N concentration in aeration group showed a downward trend in the early stage, which may be due to the increase of DO concentration, the enhancement of nitrification in surface sediment and water body, and the decrease of NH3-N concentration in water body. It reached the lowest value of 6.55 mg/L on the 20th day, and then increased steadily. At the end of the experiment, NH3-N concentration in the overlying water body was 9.11 mg/L. NH3-N concentration in aeration and adding bacteria group decreased faster than that in the aeration group at the initial stage because of the increase of DO in water body and the effect of ammonia nitrogen degrading bacteria. The fluctuation trend of DO is shown in Figure 5(d). The lowest value was 1.47 mg/L on the 28th day, and then increased slightly. At the end of the experiment, NH3-N concentration in the overlying water was 3.67 mg/L.

Concentration of TP in overlying water. Phosphorus is an indispensable nutrient element in the process of microbial growth. The phosphorus in water is mainly converted into soluble phosphorus from various organic phosphorus or insoluble inorganic phosphorus in domestic sewage and farmland wastewater, and then into the river through pipeline or surface runoff, and the sediment will also release some soluble phosphorus (Liang et al. 2018). As can be seen from Figure 5(c), due to the release of sediment pollution, TP concentration in overlying water of the control group showed a slow upward trend at the initial stage, reaching a peak of 3.12 mg/L on day 18, and then remained stable. At the end of the experiment, the TP concentration in the overlying water was 3.06 mg/L. Except for the control group, the change trend of TP concentration in the overlying water was basically the same in the other experimental groups. After aeration, the concentration of TP in overlying water decreased significantly and remained stable. The TP concentration of the two groups of aeration group increased significantly in the later stage. At the end of the experiment, TP concentrations in the overlying water of the aeration group and aeration and adding bacteria agent group were 0.92 and 0.65 mg/L, respectively.

Effect of compound bacteria on sediment of black-odorous water

As the sediment will have a certain amount of natural settlement after standing, the thickness will be reduced, so the control group is taken as the benchmark to eliminate the influence of natural settlement and check the sediment reduction rate. As showed in Table 2, the natural sedimentation rate of sediment is about 1.99%, and the sediment reduction rate of aeration group and aeration and adding bacteria agent group is 2.47% and 3.81%, respectively. Comparing the experimental group with and without microbial agent, it can be found that the sediment thickness of the experimental group with a microbial agent is lower than that of the control group, which indicates that the microbial agent has a certain effect on sediment reduction.

Table 2

Thickness of sediment before and after the experiment

ItemThickness before experiment (cm)Thickness after experiment (cm)Corrected thickness (cm)Revised reduction (cm)Revised reduction rate (%)
20.1 19.7 19.7 0.40 1.99 
20.2 19.3 19.7 0.49 2.47 
20.7 19.5 19.9 0.78 3.81 
ItemThickness before experiment (cm)Thickness after experiment (cm)Corrected thickness (cm)Revised reduction (cm)Revised reduction rate (%)
20.1 19.7 19.7 0.40 1.99 
20.2 19.3 19.7 0.49 2.47 
20.7 19.5 19.9 0.78 3.81 

It can be seen from Figure 6 that the OM content in the sediment of the control group remained basically unchanged, while that of the other treatment groups decreased, and aeration and adding bacteria agent group was the best, with the OM reduction rate of 55.96%. For total nitrogen in sediment, the change trend is the same as OM, and the reduction of aeration and adding bacteria agent group is the largest, reaching 61.63%. For the TP content in the sediment, the TP content in the sediment of each treatment group increased, and the aeration group increased the most, which also verified that aeration can promote the phosphorus absorption of the sediment to the overlying water. It is widely reported that the blackening of water body is due to the presence of metal sulfides in covering water, such as ferrous sulfide (FeS) and manganese sulfide (MnS), which are formed by the combination of Fe2+ and Mn2+ with S2− (Wobus et al. 2003). The content of Fe2+ in the sediment reflects the oxidation-reduction potential of the sediment. When the content of Fe2+ is greater, the sediment is in the reducing state, that is, the sediment is in the anaerobic state. When the content of Fe2+ is lower, the reducing state of the sediment is changed. Among the three treatments, the Fe2+ content in the sediment decreased, and aeration and adding bacteria agent group decreased the most, with a reduction rate of 60.14%, indicating that the anaerobic state of the sediment was significantly improved. Sulfur mainly enters water in the form of SO42− and organic sulfur. Under the hypoxic environment, microorganisms utilize SO42−, NO3, NO2, and carbon as electron acceptor, oxidize organic sulfur to produce H2S, and S2− and HS are also produced. SO42− can also be further reduced to sulfide by using organic matter in water. The sulfide can be divided into AVS and pyrite (FeS2). When H2S diffuses upward to the aerobic belt of water body, it will be oxidized to SO42− or other sulfur with intermediate valence. It can also react with active iron oxides in the sediment to form FeS under the action of microorganisms, and further react with soluble sulfur, elemental sulfur and polysulfide to form pyrite. The concentration of SO42− in the sediment can aspect reflect the concentration of S2− in the sediment. After the end of the experiment, the concentration of SO42− in the sediment of aeration and adding bacteria agent group increased significantly, and the aspect reflected the decrease of S2− concentration, the S2− in the sediment was reflected, the content of FeS and MnS decreased, and the blackening phenomenon of the sediment was improved.

Figure 6

Fluctuations of monitoring indicators in sediment: (a) organic matter, (b) TN, (c) TP, (d) SO42−, (e) Fe2+.

Figure 6

Fluctuations of monitoring indicators in sediment: (a) organic matter, (b) TN, (c) TP, (d) SO42−, (e) Fe2+.

CONCLUSION

Microorganisms are the main drivers of biogeographic cycle, involved in the transformation of matter, so they can naturally be used as decomposers to repair black and odorous water bodies. The effectiveness and reliability of functional bacteria are an important basic work and a key step for the success of river in situ remediation. In this study, four trad-itional pollutant degrading bacteria and two characteristic pollutant degrading bacteria, were isolated from the sediment of black-odorous river, and their physiological characteristics and 16S rDNA sequence were determined. Mixed bacteria to treat black-odorous water was added in the in-situ simulation experiment. The results showed that the addition of mixed bacteria had a better degradation effect on the main pollutants of black-odorous water. In this study, an economic, feasible and environment-friendly treatment method for black-odorous water was proposed, which laid a foundation for the use of microorganisms to treat black-odorous water in-situ. However, it is difficult to maintain long-term stability and high efficiency by using bacteria to treat black-odorous water. In the future, how to establish long-term stable and coexisting functional zones in an aquatic ecosystem to improve water quality will be a direction of bioremediation.

ACKNOWLEDGEMENTS

This work was supported by the Science and Technology Planning Project of Chongqing Ecology and Environment Bureau, China under grant [number: 2019-125].

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

REFERENCES

REFERENCES
SEPA
2002
Methods for Monitoring and Analysis of Water and Wastewater
, 4th edn.
China Environmental Science Press, Beijing
,
China
.
Cao
J. X.
,
Sun
Q.
,
Zhao
D. H.
,
Xu
M. Y.
,
Shen
Q. S.
,
Wang
D.
,
Wang
Y.
&
Ding
S. M.
2020
A critical review of the appearance of black-odorous waterbodies in China and treatment methods
.
Journal of Hazardous Materials
385
.
https://doi.org/10.1016/j.jhazmat.2019.121511
.
He
W.
,
Shang
J. G.
,
Lu
X.
&
Fan
C. X.
2013a
Effects of sludge dredging on the prevention and control of algae-caused black bloom in Taihu Lake, China
.
Journal of Environmental Sciences
25
(
3
),
430
440
.
https://doi.org/10.1016/S1001-0742(12) 60098-9
.
He
Y.
,
Chen
Y. X.
,
Zhang
Y. F.
&
Huang
M. S.
2013b
Role of aerated turbulence in the fate of endogenous nitrogen from malodorous river sediments
.
Environmental Engineering Science
30
(
1
),
11
16
.
https://doi.org/10.1089/ees.2011.0538
.
He
Z. H.
,
Long
X. X.
,
Li
L. Y.
,
Yu
G. W.
,
Chong
Y. X.
,
Xing
W.
&
Zhu
Z.
2017
Temperature response of sulfide/ferrous oxidation and microbial community in anoxic sediments treated with calcium nitrate addition
.
Journal of Environmental Management
191
,
209
218
.
https://doi.org/10.1016/j.jenvman.2017.01.008
.
He
Z. H.
,
Huang
R.
,
Liang
Y. H.
,
Yu
G. W.
,
Chong
Y. X.
&
Wang
L.
2018
Index for nitrate dosage calculation on sediment odor control using nitrate-dependent ferrous and sulfide oxidation interactions
.
Journal of Environmental Management
226
,
289
297
.
https://doi.org/10.1016/j.jenvman.2018.08.037
.
Ji
X.
,
Zhang
W.
,
Jiang
M.
,
He
J.
&
Zheng
Z.
2017
Black-odor water analysis and heavy metal distribution of Yitong River in Northeast China
.
Water Science and Technology
76
(
7-8
),
2051
2064
.
https://doi.org/10.2166/wst.2017.372
.
Le
C.
,
Zha
Y.
,
Li
Y.
,
Sun
D.
,
Lu
H.
&
Yin
B.
2010
Eutrophication of lake waters in China: cost, causes, and control
.
Environmental Management
45
(
4
),
662
668
.
https://doi.org/662-668. 10.1007/s00267-010-9440-3
.
Liang
Z. W.
,
Siegert
M.
,
Fang
W. W.
,
Sun
Y.
,
Jiang
F.
,
Lu
H.
,
Chen
G. H.
&
Wang
S. Q.
2018
Blackening and odorization of urban rivers: a bio-geochemical process
.
FEMS Microbiology Ecology
94
(
3
).
https://doi.org/10.1093/femsec/fix180
.
Liu
Y. M.
,
Chen
W.
,
Li
D. H.
,
Huang
Z. B.
,
Shen
Y. W.
&
Liu
Y. D.
2011
Cyanobacteria-/cyanotoxin-contaminations and eutrophication status before Wuxi Drinking Water Crisis in Lake Taihu, China
.
Journal of Environmental Sciences
23
(
4
),
575
581
.
https://doi.org/10.1016/s1001-0742(10)60450-0
.
Lu
X.
,
Feng
Z. Y.
,
Shang
J. G.
,
Fan
C. X.
&
Deng
J. C.
2012
Black water bloom induced by different types of organic matters and forming mechanisms of major odorous compounds
.
Huan Jing Ke Xue
33
(
9
),
3152
3159
.
https://doi.org/10.13227/j.hjkx.2012.09.047
.
Pham
T. N.
,
Nam
W. J.
,
Jeon
Y. J.
&
Yoon
H. H.
2012
Volatile fatty acids production from marine macroalgae by anaerobic fermentation
.
Bioresource Technology
124
,
500
503
.
https://doi.org/10.1016/j.biortech.2012.08.081
.
Shishir
T. A.
,
Mahbub
N.
&
Kamal
N. E.
2019
Review on bioremediation: a tool to resurrect the polluted rivers
.
Pollution
5
(
3
),
555
568
.
https://doi.org/10.22059/poll.2019.272339.558
.
Wang
G. F.
,
Li
X. N.
,
Fang
Y.
&
Huang
R.
2014
Analysis on the formation condition of the algae-induced odorous black water agglomerate
.
Saudi Journal of Biological Sciences
21
(
6
),
597
604
.
https://doi.org/10.1016/j.sjbs.2014.07.002
.
Wang
Y. L.
,
Wang
L.
,
Hua
Z. L.
&
Ding
S. M.
2016
The relationships of velocity, dissolved oxygen with Fe2+, S2− in black bloom region on Nanfei River esturay of Lake Chaohu
.
28
(
04
),
710
717
.
https://doi.org/10.18307/2016.0403
.
Wobus
A.
,
Bleul
C.
,
Maassen
S.
,
Scheerer
C.
,
Schuppler
M.
,
Jacobs
E.
&
Roske
I.
2003
Microbial diversity and functional characterization of sediments from reservoirs of different trophic state
.
FEMS Microbiology Ecology
46
(
3
),
331
347
.
https://doi.org/10.1016/S0168-6496(03)00249-6
.
Yu
H.
&
Sun
A. M.
2016
Research on carbon fiber biofilm filler in treatment of black-odorous river
.
Resources Economization & Environmental Protection
(
03
),
50
51
.
https://doi.org/10.16317/j.cnki.12-1377/x.2016.03.038
.
Yuan
Q. B.
,
Shen
Y.
,
Huang
Y. M.
&
Hu
N.
2018
A comparative study of aeration, biostimulation and bioaugmentation in contaminated urban river purification
.
Environmental Technology & Innovation
11
,
276
285
.
https://doi.org/10.1016/j.eti.2018.06.008
.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY-NC-ND 4.0), which permits copying and redistribution for non-commercial purposes with no derivatives, provided the original work is properly cited (http://creativecommons.org/licenses/by-nc-nd/4.0/).