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Bioaugmentation Treatment of Mature Landfill Leachate by New Isolated Ammonia Nitrogen and Humic Acid Resistant Microorganism
Bioaugmentation Treatment of Mature Landfill Leachate by New Isolated Ammonia Nitrogen and Humic Acid Resistant Microorganism
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 987-997
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 04, 2014
  • Accepted : March 25, 2014
  • Published : July 28, 2014
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About the Authors
Dahai Yu
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130012, P. R. China
Jiyu Yang
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130012, P. R. China
Fei Teng
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130012, P. R. China
Lili Feng
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130012, P. R. China
Xuexun Fang
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, 130012, P. R. China
Hejun Ren
Key Laboratory of Ground Water Resources and Environment of the Ministry of Education, College of Environment and Resources, Jilin University, Changchun, 130021, P. R. China
renhejun@126.com

Abstract
The mature landfill leachate, which is characterized by a high concentration of ammonia nitrogen (NH 3 -N) and humic acid (HA), poses a challenge to biotreatment methods, due to the constituent toxicity and low biodegradable fraction of the organics. In this study, we applied bioaugmentation technology in landfill leachate degradation by introducing a domesticated NH 3 -N and HA resistant bacteria strain, which was identified as Bacillus cereus (abbreviated as B. cereus Jlu) and Enterococcus casseliflavus (abbreviated as E. casseliflavus Jlu), respectively. The isolated strains exhibited excellent tolerant ability for NH 3 -N and HA and they could also greatly improved the COD (chemical oxygen demand), NH 3 -N and HA removal rate, and efficiency of bioaugmentation degradation of landfill leachate. Only 3 days was required for the domesticated bacteria to remove about 70.0% COD, compared with 9 days’ degradation for the undomesticated (autochthonous) bacteria to obtain a similar removal rate. An orthogonal array was then used to further improve the COD and NH 3 -N removal rate. Under the optimum condition, the COD removal rate in leachate by using E. casseliflavus Jlu and B. cereus Jlu increased to 86.0% and 90.0%, respectively after, 2 days of degradation. The simultaneous removal of NH 3 -N and HA with more than 50% and 40% removal rate in leachate by employing the sole screened strain was first observed.
Keywords
Introduction
The treatment of landfill leachate has become a major environmental concern, not only because the waste volume is growing faster than the world’s population, but also because this kind of wastewater can be a source of hazard to receiving waters and is very toxic to fish and other aquatic life [20] . Biological methods were considered as the most important methods in the landfill leachate treatment process in terms of the feasibility, economy, and effectiveness [29] . However, the implementation of traditional biological technology for landfill leachate treatment is very challenging for mature or old landfill leachate, which is characterized by high strengths of ammonia nitrogen (NH 3 -N) and humic acid (HA) [6 , 33] .
The high toxicity of NH 3 -N greatly inhibits the growth and activity of microorganisms in the leachate treatment system and then suppresses the biodegradation process [15] . Notably, a high quantity of unprocessed NH 3 -N leads to accelerate eutrophication and an increase in dissolved oxygen reduction. HA is the main fraction of the humic substances existing in fresh water sources and creating problems in treatment operations. Consequently, it is the primary target of a water treatment process, although not considered as a pollutant [32] . Not only can it adversely affect the appearance and taste of water, but it can also be halogenated to form potentially carcinogenic chlorinated organic compounds and pose a serious environmental problem [28] . It is difficult to remove HA from landfill leachate by traditional biological processes owing to it being refractory to biodegradation and oxidization. HA could also modify the bioavailability and biotoxicity of hazardous compounds, thereby impacting the biodegradation or biouptake as well [2] .
Owing to the aforementioned problems, the removal of NH 3 -N and HA from water is necessary. Currently, the main solution to solve these problems is by introducing pretreatment methods such as ammonia nitrogen stripping, coagulation–flocculation, electrocoagulation processes, oxidation, photocatalysis, and microfiltration prior to the biological step to remove nonbiodegradable and biotoxic matter [9 , 12] . All of these processes, however, consume high operational cost and cause secondary pollution problems. Hence, it is important to explore new NH 3 -N and HA treatment techniques.
The use of NH 3 -N and HA resistant microorganism with high biodegradable ability for mature landfill leachate to augment the traditional biology treatment might provide a solution to solve the current problems. Bioaugmentation is the introduction of indigenous, allochthonous, or genetically modified microorganisms with specific catabolic abilities into contaminated environments to enable or accelerate the degradation of targeted pollutants [11] . It has been expected as a relatively economic, environmental-friendly, straightforward, and high-efficient technology for pollutant elimination from stressed environments, which could improve the traditional biotreatment processes and reduce the energy consumption [23 , 26] . In comparison with the traditional biotreatment process, the inoculated indigenous or allochthonous microbial bacteria can enhance the biodegradation of target pollutants by strengthening or complementing the metabolic capabilities of the indigenous microbial community. The bioaugmentation technique has been successfully used in groundwater bioremediation or municipal and industrial wastewater treatment [17 , 25] . This technique has also been proved to improve degradation efficiency, enhance reactor performance, protect existing microbial communities against adverse effects, accelerate the onset of degradation, and compensate for organic or hydraulic overloading [16] . However, to date, very few examples of bioaugmentation treatment of landfill leachate with high strength of NH 3 -N and HA, by employing microorganism domesticated from indigenous microbiota in leachate, were reported.
The successful implementation of bioaugmentation depends on the efficiency of the added bacteria to degrade the pollutant under natural conditions and their ability to adapt to the indigenous bacteria [8] . These domesticated exogenous bacteria usually have the advantage to enhance the transformation or removal of specific pollutants, especially recalcitrant compounds [24] , but the competition between the exogenous and indigenous microbiotas may result in some unsuccessful bioaugmentation processes [19] . Thus, isolating, culturing, and inoculating the dominant microorganisms may enhance their survival chance relative to other microbes, and consequently could have a desirable effect on removal efficiency. Besides, it is critical to adjust conditions of the degradation to reduce the competition between the exogenous and indigenous microbiotas to make both of them remain viable and functionally active and even work in coordination.
Consequently, the aim of this study was to efficiently degrade the mature landfill leachate with high concentration of NH 3 -N and HA by using bioaugmentation. Bacteria with specific degradation capacity for NH 3 -N and HA were domesticated and isolated from autochthonous bacteria in leachate. The obtained domesticated stains were identified and their removal effectiveness of COD, NH 3 -N, and HA in the bioaugmentation treatment of landfill leachate were evaluated. An orthogonal experiment was used to further improve the bioaugmentation effectiveness of the domesticated bacteria by screening optimal landfill leachate biodegradation conditions. The growth kinetics of the biodegradation rate of COD, NH 3 -N, HA, and biomass were also determined.
Materials and Methods
- Leachate Sample and Chemicals
The leachate samples used in this study was collected from a 20-years-old municipal landfill site located near Chang Chun city in the northeast of China. The COD, BOD, NH 3 -N, and HA concentrations and pH of leachate samples were 22,000- 25,000 mg/l, 6,000 mg/l, 800 mg/l, 3,800 mg/l, and 7.5, respectively. Standard solution of NH 3 -N (1g/l) was received from the China Research Center of Certified Reference Materials. Humic acid (98%), potassium dichromate, sulfuric acid, and other reagents (A.R. grade) were obtained from Shanghai Shenggong Reagent Company (China).
- Microorganism Acclimation and Isolation
NH 3 -N and HA were used as selective pressure of specific organic pollutants for strain screening in this study. The domestication of the NH 3 -N resistant bacterial strain was first performed by adding ammonium chloride into 10 ml of landfill leachate (final NH 3 -N concentration was 1,000 mg/l). After 3 days of domestication at 30℃ and 180 rpm, the bacteria in the leachate were harvested by centrifuging at 4,000 rpm at 4℃ for 10 min. The supernatant was discarded and the pellets were resuspended in 10 ml of leachate that contained 1,200 mg/l NH 3 -N in the next round of domestication, and the mixture was incubated at the same conditions as described above. The screening process was performed for another seven rounds (final NH 3 -N concentration in domesticated system was 1,600, 2,000, 2,400, 2,800, 3,200, and 3,600 mg/l, respectively) and it was completed when the final concentration of NH 3 -N in the landfill leachate was 3,800 mg/l. The HA resistant strain was domesticated in the same way as that of the NH 3 -N resistant strain with different concentrations of HA (1,000, 2,000, 4,000, and 7,000 mg/l) during the four rounds of domestication.
Subsequently, the isolation of NH 3 -N and HA resistant bacterial strains was conducted by using the serial dilution method. The obtained bacterial strains were accumulated in LB medium at 37℃ for 12 h and 180 rpm. The medium was (OD was 1.0) diluted, and 20 μl of sample (diluted 10 -6 fold) was spread onto a LB (Luria-Bertani)-leachate plate (V LB :V leachate = 2:3), and the plate was incubated at 37℃ for 12 h. Pure colonies were picked, cultured, and stored for further experiments.
- Identification of the Isolated Strains
Total DNA was prepared by using a genomic DNA extraction kit (Shenggong, China) following the manufacturer’s instructions. The genes encoding 16S rDNA were amplified from the extracted genomic DNA by PCR with universal primers 27F (5’-AGAGTT TGATCCTGGCTCAG-3’) and 1492R (5’-TACCTTGTTACGACTT-3’) [10] . The target DNA fragments were purified by a TIANgen Midi Purification Kit, cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced by Shenggong Co. Ltd. (Shanghai, China). Subsequently, the fragments sequences were compared with available sequences in the GenBank database by using the BLAST program. The closet matching sequences in the GenBank database were retrieved and aligned by using Clustal W software ver. 1.7. The reconstruction of the phylogenetic tree was performed by using Mega 5.2.1 by applying the neighbor-joining method and maximum likelihood analysis to different data sets [21] .
- Bioaugmentation Degradation with Domesticated Bacterial Strains
HA and NH 3 -N resistant bacterial strains were harvested in 2 ml of LB culture medium and incubated for 24 h at 37℃ and 180 rpm, respectively. Then the bioaugmentation was performed by adding 2 ml of enrichment culture (OD 600 = 1.0) of HA or NH 3 -N resistant bacteria into 20 ml of unsterilized landfill leachate and sterilized landfill leachate (steam sterilization, autoclave for 15 min at 121℃ at 100 kPa), respectively, at 30℃ with the rotation speed of 180 rpm for 9 days. For the mixed bacteria, 1 ml of incubated HA resistant bacteria culture and 1 ml of NH 3 -N resistant bacteria culture were mixed, and then the mixed bacteria culture was added to 20 ml of unsterilized landfill leachate at 30℃ with 180 rpm for 9 days. The samples of the system were collected and analyzed at 1, 3, 5, 7, and 9 days. The degradation of landfill leachate without adding additional bacteria was used as the control. All the degradations were performed three times and the average values were presented.
- Orthogonal Design for Bioaugmentation Degradation and Data Analysis
The orthogonal array (often referred to as the Taguchi method) is a well-known technique that provides a systematic and efficient methodology for process optimization. Instead of having to test all possible combinations, this method tests pairs of combinations and uses only a fraction of all possible factor or level combinations to reduce the number of experiments, which allows the simultaneous effect of several process parameters [27] . This method has been used to achieve the best result under the given removal conditions and to improve the bioaugmentation efficiency [13] . The quantitative evaluation and statistic analysis of the effects of degradation conditions were investigated through an orthogonal experiment design. According to the preliminary experiment and references [18] , four factors were investigated to optimize the bioaugmentation, including temperature, pH, inoculums size, and the phosphorus supplement, which were found to have great effects on the degradation efficiency of landfill leachate. To simplify the experiments, the interactions between these four factors were not taken into account. The orthogonal table L 25 (5) 6 (where L is orthogonal table; 25 is the number of experimental runs; 5 is the number of levels of each factor; and 6 is the number of variables) was designed, in which two blank columns were designated for the error evaluation. The four factors were at five levels as follows: temperature of 20℃, 25℃, 30℃, 37℃, and 40℃; pH of 5, 6, 7, 8, and 9; inoculum size of 0, 0.1, 0.2, 0.3, and 0.4 ml (OD 600 = 1.0); and phosphorus supplement (KH 2 PO 4 ) of 0, 0.6, 1.2, 1.8, and 2.4 mg/ml. The orthogonal experimental table with investigation factors, the corresponding levels, and the designated boundary values are shown in Table 1 .
Design of orthogonal table L25(5)6a.
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aL = orthogonal table; 5 = factors; 6 = five levels of each; and 25 = experimental number.
The data analysis was conducted after the orthogonal array. The process/system design phase involves deciding the best values/levels for the control factors. The signal-to-noise (S/N) ratio, which results in minimization of the quality characteristic variation due to the uncontrollable parameter, is an ideal metric for that purpose. The S/N ratio also indicates the degree of predictable performance in the presence of noise factors. S/N represents the magnitude of the mean of a process compared with its variation, serves as objective functions for optimization, and helps in data analysis and prediction of optimum results. For analyzing the data of the orthogonal experiment, the S/N number used to measure the quality characteristics deviating from the desired values [5] was calculated for each experiment by using Assistant for Orthogonal Experimental Design II 3.1. The average S/N value was calculated for each factor and level. The mean S/N ratio for each variable at each level was calculated by averaging the S/N ratios for all the experiments. The S/N value ratio was classified into the smaller-the-better type, the larger-the-better type, and nominal-the-best type, based on the type of objective function [7] . The removal rate was considered as the quality characteristic with the concept of “the smaller-the-better” in this study. Regardless of the category of the performance characteristics, a smaller S/N value corresponded to a better performance. Therefore, the optimal level of the process parameters was the level that corresponds to the lowest S/N ratio. Then the range R (R = high S/N - low S/N) of the S/N for each parameter is calculated. The greater R value corresponded to a higher influence of this parameter on the degradation. The ANOVA test was used for the statistical analysis. The paired t -test was used to analyze changes in removal rate before and after degradation in the same group. A P value < 0.05 was statistically significant for ANOVA tests and t -tests.
- Experimental Validation Under the Optimum Conditions
To validate the optimization of the conditions for the culture medium, degradation of landfill leachate was carried out under the optimized conditions, to confirm the results from the analysis of the orthogonal design. To test the degradation ability of the domesticated strains and eliminate the influence of indigenous microorganism on degradation, the degradation of sterilized landfill leachate by domesticated strains was also performed under the same conditions as those for unsterilized landfill leachate. Sterilized landfill leachate without adding any microorganism was used as the control.
- Analytical Methods
The removal rates of COD, HA, and NH 3 -N were used to evaluate the effectiveness of bioaugmentation treatment of landfill leachate in this study. The COD concentration was determined by the dichromate closed refluxed and colorimetric assay method according to the Standard Methods for the Examination of Water and Wastewater [3] . Samples were diluted (if necessary) and added to the standard COD ampules. COD samples were then incubated at 150℃ for 2 h in a dry incubator (Hach Company Loveland, CO, USA). After allowing the COD tubes to cool to room temperature, COD levels were determined by measuring the absorbance of the digested assay solution at λ = 600 nm on a Shimadzu UV-2550 spectrophotometer. A 1 cm pathlength was maintained by using a standard cuvette (2.5 ml sample size). The NH 3 -N concentration was measured by the Nessler’s reagent colorimetric method according to the National Standard of the People’s Republic of China (GB 7479-87). The HA concentration was measured according to a previously used method [30] by an ultraviolet–visible spectrometer model UV–vis 2550 (Shimadzu, Japan) at 300 nm (HA exhibited the maximum UV adsorption at this wavelength). The bacterial cell biomass was detected as the optical density of samples at 600 nm. All experiments were performed in triplicate and the average values were informed.
The removal rate was calculated based on measuring the concentration of target matter before and after biotreatment by the following Eqs. (1), (2), and (3):
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where COD i , NH 3 -N i , HA i , and COD f , NH 3 -N f , and HA f were the initial and final concentrations of COD and NH 3 -N, respectively.
Results and Discussion
- Isolation and Identification of NH3-N and HA Resistant Bacterial Strain
The colonies of NH 3 -N resistant strains were gram-positive, yellow, circular shaped, with semitransparent, slabby, wet, and smooth surfaces on LB plates. The colonies of HA resistant strains were gram-positive, yellow, coccoid, small, and somewhat transparent cells on LB plates. Ten individual isolates were collected for sequencing on NH 3 -N resistant bacteria plate and their 16S rDNA sequences were found to be identical. Ten individual isolates were also collected for sequencing on HA resistant bacteria plate, and their 16S rDNA sequences were identical. Thus one NH 3 -N (abbreviated as B. cereus Jlu) and one HA (abbreviated as E. casseliflavus Jlu) resistant bacterial strain were domesticated and isolated from landfill leachate, respectively. To identify the strains, the near-complete 16S rDNA sequences of strain B. cereus Jlu (1,427 bp, Accession No. KF857221) and strain E. casseliflavus Jlu (1,413 bp, Accession No. KF857222) were determined, and the similarity search performed against the GenBank sequence database indicated that B. cereus Jlu was 99% identical to the Bacillus cereus group and E. casseliflavus Jlu was 97% identical to Enterococcus casseliflavus species. The neighbor-joining phylogenetic trees ( Fig. 1 ) of the two 16S rDNA sequences were conducted and the results indicated that NH 3 -N and HA resistant strains were most closely related to Bacillus cereus and Enterococcus casseliflavus , respectively.
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Neighbor-joining phylogenetic tree of the 16S rDNA gene sequences of strain (A) Jlu BC and (B) Jlu EC (shown in bold face) and most closely related species. The GenBank accession numbers for the corresponding sequences are given in parenthesis after the strain name.
NH 3 -N and HA tolerance tests for the selected strains were performed in order to investigate the endurance ability of B. cereus Jlu to NH 3 -N and E. casseliflavus Jlu to HA. The test was performed by incubating strain pellets (prepared by centrifuging 0.5 ml OD 600 = 1.0 strain broth at 4,000 rpm for 10 min) in 3 ml of M9 minimal medium (Sigma China) containing different concentrations of NH 3 -N (from 1 to 100 g/l) and HA (1 to 15 g/l) at 37℃, 180 rpm for 2da ys. Control experiments without NH 3 -N and HA stresses in M9 minimal medium were also performed. The results of the tolerance test showed that B. cereus Jlu could survive even when the NH 3 -N concentration was as high as 50 g/l. As far as E. casseliflavus Jlu was concerned, could survive in 10 g/l HA. This result confirmed that our screened strains possessed high endurance ability for the specific environmental pressure. It had been reported that Bacillus cereus predominated in wastewater treatment systems to remove heavy metal, nitrogen, and phosphorus as well as organic matter in some countries [14 , 31] . Enterococcus casseliflavus had also been reported to reduce BOD and COD in tannery effluents [22] . To our knowledge, however, none of the members of Bacillus cereus and Enterococcus casseliflavus have been reported to possess simultaneous COD, NH 3 -N, and HA removal abilities and such high NH 3 -N and HA tolerance abilities. Therefore, it was necessary to investigate the augmented performances of B. cereus Jlu and E. casseliflavus Jlu to treat high NH 3 -Nand HA-containing landfill leachate.
- Bioaugmentation Treatment of the Landfill Leachate by Domesticated Bacterial Strain
The COD, NH3-N, and HA removal efficiencies in unsterilized landfill leachate. The success of bioaugmentation depends, to a large degree, on the ability of the introduced microbes to survive and display their activities in the mixed culture [1] . B. cereus Jlu and E. casseliflavus Jlu were used to evaluate the effectiveness of bioaugmentation treatment of landfill leachate, respectively. In addition, the removal efficiency of landfill leachate by using the mixture of B. cereus Jlu and E. casseliflavus Jlu was studied to evaluate if there was any synergy or inhibition effects of these two domesticated bacteria on the pollution removal in the bioaugmentation treatment process.
The COD removal rate is shown in Fig. 2 A. The results showed that even a small amount of the indigenous bacteria possessed a relatively high COD removal ability, which confirmed that the microorganism source for domestication was in good condition. Bioaugmentation treatments for unsterilized landfill leachate proved to be effective, and no inhibition of indigenous bacteria by the domesticated strains was observed. As shown in Fig. 2 A, the highest COD removal rates (72.3% for E. casseliflavus Jlu, 76.2% for B. cereus Jlu) were obtained in 3 days and then decreased in the following days for the domesticated bacteria. As far as the degradation by using autochthonous bacteria was concerned, the COD removal rate kept increasing for 9 days, and the maximum COD removal rate was 72.0%. Although indigenous bacteria also exhibited good ability to remove COD from the landfill leachate, a higher COD removal rate was observed by using the B. cereus Jlu, E. casseliflavus Jlu, and the mixed domesticated bacteria in less degradation time, respectively. The result also showed that the maximum COD removal rate increased slightly (79.8%) by using the mixed bacteria compared with those of E. casseliflavus Jlu (72.3%) and B. cereus Jlu (76.2%), which suggested no competition or inhibition between the two domesticated bacteria. The reason might be that a mixture of microorganisms had a synergy effect on increasing the biomass activity, growth efficiency, and enzyme production. In addition, mixed cultures served to overcome feedback regulation and catabolic repression, as the products of one microorganism acted as a substrate for the other [4] . No big difference of COD removal rate was observed between the control experiment (no additional microorganism added in the degradation system) and by adding additional autochthonous bacteria in the biodegradation of unsterilized landfill leachate, which suggested that the inoculum size of autochthonous bacteria did not have a great effect on the COD removal rate.
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COD (A), NH3-N (B), and HA (C) removal rates of unsterilized landfill leachate by using different bacteria at different hydraulic retention times. Columns with a different color stand for (white) Control, (black) Autochthonous bacteria, (dark gray) E. casseliflavus Jlu, (light gray) B. cereus Jlu, and (spotted) Mixed bacteria.
The variation trends of NH 3 -N and HA removal rate by using different bacteria were similar with that of COD removal rate, as shown in Figs. 2 B and 2 C. The highest NH 3 -N and HA removal rate by using B. cereus Jlu was 50.1% and 20.3%, respectively. The highest NH 3 -N and HA removal rate by using E. casseliflavus Jlu was 51.2% and 21.3%, respectively. The mixture of B. cereus Jlu and E. casseliflavus Jlu slightly decreased the maximum NH 3 -N removal rate to 47.8%, and increased the maximum HA removal rate to 22.8%. The maximum NH 3 -N removal rate by using B. cereus Jlu and E. casseliflavus Jlu was higher than that of the indigenous bacteria (42.7%), as shown in Fig. 2 B. However, B. cereus Jlu and E. casseliflavus Jlu did not increase the maximum HA removal rate compared with the indigenous bacteria, as shown in Fig. 2 C. The above results indicated that both B. cereus Jlu and E. casseliflavus Jlu possessed COD, NH 3 -N, and HA removal abilities for landfill leachate, and their pollutions removal abilities were in the order of COD, NH 3 -N, and HA.
The COD, NH3-N, and HA removal efficiencies in sterilized landfill leachate. To eliminate the effect of indigenous bacteria on the domesticated strains in bioaugmentation treatment of landfill leachate, the degradation of sterilized landfill leachate was investigated. The COD, NH 3 -N, and HA removal efficiencies of autochthonous bacteria enriched from landfill leachate were also studied to compare with those of the domesticated bacteria. Sterilized landfill leachate without addition of the isolated bacteria was used as the control.
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COD (A), NH3-N (B), and HA (C) removal rates of sterilized landfill leachate by using different bacteria at different hydraulic retention times. Columns with different fills stand for (white) Control, (black) Autochthonous bacteria, (dark gray) E. casseliflavus Jlu, and (light gray) B. cereus Jlu.
As shown in Fig. 3 A, the COD removal rate kept increasing with the increase of hydraulic retention time for the control experiment. The biodegradation proved to be effective, since the COD removal rates by using all of the autochthonous bacteria, B. cereus Jlu, and E. casseliflavus Jlu were much higher than that of the control experiment during the whole treatment process. Similar to the control experiment results, the COD removal rate increased gradually with the increase of hydraulic retention time by adding autochthonous bacteria. As far as the B. cereus Jlu and E. casseliflavus Jlu were concerned, the COD removal rate for the sterilized landfill leachate gradually increased to 81.0% for B. cereus Jlu and 74.1% for E. casseliflavus Jlu after 9 days (with the extension of the degradation time, the COD removal rate could not be further increased for all the tested samples, data not shown). As shown in Fig. 4 , the biomass increased with the hydraulic retention time, and the growth kinetics of biomass suggested that the COD removal rate varied according to the biomass growth. The higher maximum COD removal rate by using E. casseliflavus Jlu (74.1 ± 1.0%) and B. cereus Jlu (81.0 ± 2.5%) compared with that of control (34.6 ± 1.3%), as shown in Fig. 3 A, indicated the success of bioaugmentation for treatment of the sterilized landfill leachate. This was verified by repeated-measures analysis of variance (ANOVA), which revealed a statistical significant effect on COD removal for E. casseliflavus Jlu (paired t -tests: t (3) = 23.6, p < 0.001) and B. cereus Jlu (paired t -tests: t (3) = 10.7, p < 0.01) from control. There was no big differences in the COD removal between autochthonous bacteria (73.2 ± 1.6%) and domesticated strains (paired t -tests: t (3) = 0.8, p > 0.5 for E. casseliflavus Jlu, and t (3) = 3.4, 0.05 < p < 0.1 for B. cereus Jlu).
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Growth kinetics of microorganism biomass in (A) unsterilized and (B) sterilized landfill leachate for (×) B. cereus Jlu; (■) autochthonous bacteria; (▲) E. casseliflavus Jlu and (◆) control (no strain added) during the biodegradation.
The variation trends of COD removal rate in biodegradation of unsterilized and sterilized landfill leachates were similar by using autochthonous bacteria and E. casseliflavus Jlu, which suggested that sterilization had little effect on the degradation efficiency of these bacteria. The maximum COD removal rates by using autochthonous bacteria, E. casseliflavus Jlu, and B. cereus Jlu were 72.8%, 72.3%, and 76.2% for unsterilized landfill leachate, respectively, and 73.2%, 74.1%, and 81.0% for the sterilized landfill leachate, respectively. The variation trends of NH 3 -N and HA removal rate by using different bacteria were similar with that of COD removal rate, as shown in Fig. 3 B and 3 C, where 46.8% NH 3 -N and 41.0% HA removal rates were obtained by using E. casseliflavus Jlu, and 50.8% NH 3 -N and 42.3% HA removal rates by using B. cereus Jlu. The maximum NH 3 -N and HA removal rate by using either B. cereus Jlu or E. casseliflavus Jlu was much improved compared with that of the indigenous bacteria, which was 31.7% for NH 3 -N (as shown in Fig. 3 B) and 34.5% for HA (as shown in Fig. 3 C). The above results confirmed that both B. cereus Jlu and E. casseliflavus Jlu possessed COD, NH 3 -N, and HA removal abilities for landfill leachate. It was expected that NH 3 -N resistant strain B. cereus Jlu would show higher NH 3 -N removal ability, and HA resistant strain E. casseliflavus Jlu would show higher HA removal ability. However, the removal rates of NH 3 -N and HA by using B. cereus Jlu and E. casseliflavus Jlu were similar. The results also indicated that the presence of high strength of NH 3 -N had no influence on the degradation of HA and vice versa . The great increase of HA removal rate after sterilization was likely due to the fact that biorefractory humic acid was decomposed into more smaller substances as a result of sterilization, and thus improved the HA removal ability by using B. cereus Jlu and E. casseliflavus Jlu.
- Bioaugmentation Optimization Based on Orthogonal Experimental Design
Considering the effectiveness, energy consumption, and operability, bioaugmentation treatments of unsterilized landfill leachate were performed by using E. casseliflavus Jlu and B. cereus Jlu. Four factors, including temperature (A), pH (B), inoculum size (C), and phosphorus supplement (D), were investigated at five levels to optimize the bioaugmentation treatment of landfill leachate by using E. casseliflavus Jlu and B. cereus Jlu based on orthogonal experimental design. The COD removal rate was used to evaluate the effectiveness of bioaugmentation treatment of landfill leachate in this part. Orthogonal optimization results for landfill leachate COD removal by using B. cereus Jlu and E. casseliflavus Jlu are shown in Table 2 . The minimum level of average S/N for B. cereus Jlu was A3, B3, C1, and D2, respectively, which suggested that the optimal test conditions were 30℃, pH 7, inoculum size = 0 ml, and phosphorus supplement = 0.6 mg. According to the value of R, the influences of factors A, B, C, and D on COD removal decreased in the order B > A> D > C. The pH was shown to yield the largest effect on COD removal efficiency, with the highest removal of COD being at neutral pH. The COD removal rate changed with fluctuations of temperature, and 30℃ was found to be the optimum temperature for degradation. The influence of inoculum size was the least important factor and there were no big differences of COD removal rate by variation of phosphorus supplement. This result was further supported by statistic analysis. The F ratio obtained by using B. cereus Jlu decreased in the order B (4.72) > A (1.54) > D (0.54) > C (0.09), which corresponded well with the R result. pH was found to be a significant factor for the degradation because its F ratio (4.72) was higher than the critical F value (3.84) when the P value was lower than 0.05. The minimum level of S/N average for E. casseliflavus Jlu was A5, B4, C2, and D1, respectively, which suggested that the optimal conditions were 40℃, pH 8, inoculum size = 0.1 ml, and phosphorus supplement = 0 mg/ml. In terms of R values, the influences of factor A, B, C, and D on COD removal decreased in the order B > A > C > D. pH was the dominant factor over the others, followed by temperature, inoculum size, and phosphorus supplement. The statistical analysis results also confirmed the above result. The F ratio obtained by using E. casseliflavus Jlu decreased in the order B (8.64) > A (3.02) > C (1.02) > D (0.21), which corresponded well with the R result. pH was found to be a highly significant factor for the degradation because its F ratio (8.64) was higher than the critical F value (7.01) when the P value was lower than 0.01.
The results and statistical analysis of the orthogonal experiment by usingB. cereusJlu andE. casseliflavusJlu for landfill leachate degradation.
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aF ratio; bF0.05(4,8): Critical F value; **highly significant; *significant; NA: not significant.
- Experimental Validation of the Mathematic Model
The optimal degradation conditions for B. cereus Jlu and E. casseliflavus Jlu were at pH 7 and 8, temperature 40℃ and 30℃, with 0 and 0.1 ml inoculum size, and 0.6 and 0 mg/ml phosphorus supplement, respectively. The bioaugmentation treatments of landfill leachate by using the domesticated bacteria were performed under the optimum condition, respectively. The results showed that the bioaugmentation efficiency was further improved by the orthogonal experimental design. The maximum COD removal rates, of leachate by using B. cereus Jlu and E. casseliflavus Jlu was 90.0% and 86.0%, respectively, after 2 days of degradation at 180 rpm. No big differences of removal rates of NH 3 -N and HA were observed under these conditions (data not shown).
Our results showed that the bioaugmented system was a powerful tool for enhancing the biodegradability of mature landfill leachate, and the specific domesticated bacteria have the potential to improve the efficiency of the conventional biological treatment facilities currently. The maximum COD, NH 3 -N, and HA removal efficiencies were much higher by using domesticated bacteria compared with those of autochthonous bacteria. Orthogonal experimental design proved to be a good method for degradation condition optimization. The obtained B. cereus Jlu and E. casseliflavus Jlu have the potential to render mature landfill leachate more biodegradable, which is of much value to treatment of wastewater with a high strength of NH 3 -N and HA. The application of these bacteria could lower the cost for the pretreatment and the membrane treatment after the biotreatment process. Large-scale treatment and component analysis during the bioaugmentation are under way.
Acknowledgements
The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (No. 31100574 and No. 41101226) and Fund from Science and Technology Department of Jilin Province (No. 20110407).
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