Bioremediation of the tobacco waste-contaminated soil by Pseudomonas sp. HF-1: nicotine degradation and microbial community analysis
The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented into the tobacco waste-contaminated soil to degrade nicotine and evaluate the effect of the bioremediation. Comparing with non-adding (NA) systems, the treatments with addition of strain HF-1 (TA) exhibited considerably stronger pollution disposal abilities and higher stability of pH value and moisture content, especially in groups containing a large quantity of tobacco waste. The denaturing gradient gel electrophoresis (DGGE) profiles showed that the Shannon–Wiener index decreased with increasing wastes in the NA treatments, while a gradual increase was found in the TA groups. A comparison of sequences from DGGE bands demonstrated that there were differences in the dominant microbial species between the two treatments, suggesting that strain HF-1 could persist in the soil and enhance the efficiency of tobacco waste disposal. The results of real-time fluorescence quantitative PCR (RT-qPCR) also indicated that strain HF-1 existed in the TA systems and grew with relative high quantities. In conclusion, the nicotine-degrading strain HF-1 played a leading role in the bioremediation of the tobacco waste-contaminated soil and influenced the dynamics and structure of the microbial community.
KeywordsPseudomonas sp. HF-1Tobacco waste-contaminated soilBioremediationMicrobial communityReal-time qPCR
With large quantities of tobacco products being produced and consumed, tobacco wastes which include many toxic substances, such as nicotine, aminobiphenyl, naphthylamine, and benzo(a)pyrene, are spreading to many regions (Davies et al. 2002). Among these substances, nicotine is the one which makes smoking addictive (Benowitz 1992; Heisheman et al. 1994) and it also acts as a toxic contaminant in the ecosystem. While tobacco waste is classified as “toxic and hazardous” by European Union Regulations when the nicotine content exceeds 500 mg per kg dry weight (Civilini et al. 1997), the average nicotine content reaches up to 18 g per kg dry weight. This is 35 times more than the European Union standard threshold (Novotny and Zhao 1999). Nicotine can cause cancerization, mutation, and malformation. It has a certain effect on gene expression, cell apoptosis, cell hyperplasia, hormone regulation, enzyme activity, and so on (Yildiz 2004). Besides, Nicotine can be converted into a variety of more toxic intermediate metabolites, such as N′-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, cotinine, and N-nitrosamine (Brunneman et al. 1996), which can remarkably increase cancer incidence rates (Campain 2004). Most of the tobacco wastes are stacked on the ground with inappropriate storage and processing. Owing to the water solubility of the substance, nicotine can easily permeate into the soil, thus affecting the soil’s ecological structure and polluting the groundwater. Due to its long-term existence and sustained damage in the environment, it is imperative to remove nicotine from tobacco waste-contaminated water and soil.
Among numerous technologies used for cleaning up contaminants, bioremediation by addition of exogenous bacteria is probably the most efficient way (Beškoski et al. 2011; Langer et al. 2004; Brenner et al. 2008). Early in the 1930s, several nicotine-biodegrading bacteria extracted from environmental samples were reported (Wada and Yamasaki 1953). Later, due to a growing emphasis on nicotine pollution, more and more nicotine-degrading microorganisms were isolated. These microbes can grow with nicotine as the sole source of carbon, nitrogen and energy, and can potentially be used to treat nicotine pollution. The dominant biodegradation species reported belong to the genus of Arthrobacter and Pseudomonas (Li et al. 2010), the representative strains include Arthrobacter nicotinophagum (Hylin 1958), Arthrobacter oxidans (Freudenberg et al. 1988), Arthrobacter ureafaciens, Arthrobacter nicotinovorans (Schenk et al. 1998), Pseudomonas sp. HF-1 (Ruan et al. 2005), Pseudomonas putida S16 (Wang et al. 2007), and Pseudomonas sp. HZN6 (Qiu et al. 2011).
Biological methods using microbes for nicotine degradation have previously been used to treat environments contaminated by tobacco wastes such as burley tobacco extract leachate and tobacco solid wastes (Gravely et al. 1977). For example, nicotine was separated from the solid tobacco waste by an effective leaching method and was then decomposed by nicotine-degrading microbes (Civilini et al. 1997). A culture solution or the supernate of P. putida and Cellulomonas sp. were utilized to successfully decompose the baked leaves of burley tobacco (Newton et al. 1977; Gravely et al. 1985). Additionally, tobacco solid waste can be utilized as soil improvement or fertilizer to enhance soil fertility (Haug 1993; Kayhanian and Tchobanoglous 1993; Gies 1995). One study indicated that aerobic composting is an effective method for dealing with tobacco solid waste; this method can reduce 80 % of nicotine and 50 % of the volume and mass of total solids in the tobacco waste in 16 days (Briški et al. 2003). And 60 % of nicotine, 75.6 % of COD and 80 % of BOD can be removed by biomethanation of tobacco waste (Meher et al. 1995). Due to the complexity of the soil conditions in various areas, previous research focused on treating tobacco wastes or nicotine extracted from pollutants separately, and did not involve the bioremediation of tobacco waste-contaminated soil. In addition, the studies only paid attention to the nicotine removal but not the variations of microbial communities and the effects of bioaugmentation. Pseudomonas sp. HF-1, isolated from tobacco waste-contaminated soil in our previous work, is a highly efficient nicotine-degrading bacterium (Ruan et al. 2005) and plays a vital role in a sequencing batch reactor to treat tobacco wastewater (Wang et al. 2009b). However, whether addition of Pseudomonas sp. HF-1 is an effective way to accelerate the decomposition and utilization rate of pollutants in tobacco waste-contaminated soil is still unknown.
This study used high-performance liquid chromatography (HPLC), PCR-denaturing gradient gel electrophoresis (DGGE) and RT-qPCR to examine the nicotine degradation, microbial diversity, composition, and structure of microbial communities from treatments with addition of strain HF-1 (TA) systems and non-adding (NA) systems. The specific aims of this study were to investigate (1) the potential ability of Pseudomonas sp. HF-1 to utilize the nicotine in tobacco waste-contaminated soil; (2) the impacts of adding strain HF-1 on the microbial community structure, dynamics, and activity; and (3) the colonization of strain HF-1 during the degrading processes.
Materials and methods
Tobacco wastes and tobacco waste-contaminated soil
The tested soil, gathered from a vegetable plot, was air dried before being grinded and then passed through a 2-mm sieve. Mixing the vegetable field soil with tobacco wastes excavated from the Hangzhou LiQun cigarette factory of China, created a tobacco waste-contaminated soil which imitated the actual nicotine-contaminated condition of target remediation environment.
The experimental strain
The nicotine-degrading strain Pseudomonas sp. HF-1 (CGMCC 7.48) was previously isolated from the tobacco waste-contaminated soil by our research group (Ruan et al. 2005).
Biological activation of the tested soil
Six experimental treatments were set up in this study according to the nicotine content of 17.5 (±0.5) g per kg dry weight in the tobacco waste; 0, 25, 50, 100, 150, and 200 g tobacco waste were added to 600 g dry soil on the first day of each stage (1, 9, 17, 25, 33, 41 days; stages I–VI, respectively) imitating the continuous pollution of the environment. On the third day of stages I, III, and V (3, 19, and 35 days), the nicotine-degrading bacterium Pseudomonas sp. HF-1 was added to the tested soil at an inoculation rate of 2 % (OD550 = 1.4). Three replicates were set for each treatment. Treatments without adding strain HF-1 were carried out as controls. All of the experiments were conducted under the natural temperature, and moisture content was maintained at 20 % by adding water accordingly.
Test items and analytical methods
Measurements of the pH value and moisture content were performed according to the standard methods. The nicotine concentration was analyzed using HPLC (Agilent, USA) with XDB-C18 column (Wang et al. 2009b). On the basis of the GB9834-88 standard for measuring soil organic carbon (SOC), an improved protocol of sample weight and oxidant-solution preparation was implemented to determine the organic matter by potassium dichromate volumetry (Liu 2004). Both the nicotine concentration and moisture content were measured every 2 days, the pH value was measured every 3 days, and the soil organic content was measured every 4 days.
Genomic DNA extraction
At each stage (I–VI), soil samples were sieved to remove the tobacco waste. For each sample, 0.5 g of sieved soil was washed three times using sterile water and centrifuged at 12,000 rpm for 5 min. The resulting sediment was mixed with quartz sand (quartz sand/dry soil = 1:5, gram/gram) and 1 mL PBS buffer (0.1 M, pH 7.4: 19 mL 0.2 M NaH2PO4, 81 mL 0.2 M Na2HPO4), followed by oscillation for 10 min and centrifugation for 5 min. The supernatant was discarded. The genomic DNA extraction of the pellet was performed according to the soil DNA extracted kit manufacturer’s instructions (TIANDZ, China). The quality of the extracted genomic DNA was analyzed by electrophoresis on a 1.0 % (w/v) agarose gel.
The microbial community of each soil sample under different nicotine loading was examined by comparing their 16S rDNA fragments by PCR-DGGE fingerprinting. The V6–V7 regions of 16S rDNA gene fragments were amplified by PCR, using the forward primer 984F/GC (containing a GC clamp) (5′-CGCCC GGGGC GCGCC CCGGG CGGGG CGGGG GCACG GGGGG AACGC GAAGA ACCTT AC-3′) and the reverse primer 1378R (5′-CGGTG TGTAC AAGGC CCGGG AACG-3′) (Heuer et al. 1997). The expected length of the amplified fragment was about 400 bp. The amplification system was operated in 50 μL of the reaction mixture, which containing 5 μL 10 × PCR buffer with Mg2+ (TaKaRa, China), 4 μL 10 mM dNTP (TaKaRa, China), 1 μL 10 μM primer each (Sangon, China), 1 μL template DNA, 0.3 μL 5 U/μL rTaq DNA polymerase (TaKaRa, China), and 37.7 μL ddH2O. The PCR cycling conditions were as follows: denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, and extension at 72 °C for 45 s followed by a final extension of 10 min at 72 °C. 5 μL PCR products were taken to evaluate the expected size and quantified on 1.0 % (w/v) agarose gel electrophoresis.
DGGE employing a DCodeTM Universal Mutation Detection System (Bio-Rad, USA) was maintained at a constant temperature of 60 °C in 6 L of 0.5 × TAE buffer (Tris–acetate–ethylenediamine tetraacetic acid). Composite PCR products were loaded into polyacrylamide gels (6 % acrylamide–bisacrylamide: containing 38.7:1.3 of acrylamide/bisacrylamide) of 40–60 % denaturants. The DGGE gels were performed at 60 V for 30 min, then at 160 V for 6 h. The gels were visualized by silver staining (Edenborn and Sexstone 2007), documented using the GelDoc 2000 system and analyzed by Quantity One software (Bio-Rad, USA).
The DGGE gels were analyzed using Quantity One (version 4.1.1) gel analysis software (Bio-Rad, USA), and the changed bands were checked manually. The diversity of the bacterial community was reported as the Shannon–Wiener index of diversity (Ying et al. 2008). The dominant bands were excised from the DGGE gels, and incubated overnight in 50 μL TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) at 4 °C for re-amplifying with the primers 984F and 1378R in the same conditions as described above. The PCR products were purified and linked with pMD18 vector (TaKaRa, China) for TA cloning, then for sequencing (Sangon, China). All of the sequences generated from the soil samples were subjected to the NCBI nucleotide blast search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify sequences with the highest similarity. Highly similar sequences and some reference sequences of the dominant groups were imported to the data for CLUSTAL X multiple sequence alignment and the phylogenetic tree was constructed in MEGA 2.0 by applying the neighbor-joining method with 1,000 bootstrap resamplings.
The primary nucleotide sequences for the bands have been deposited in the GenBank database under accession no. JX433024–JX433063.
Fluorescence qPCR analysis
A Mastercycler®ep realplex (Enppendorf, Germany) was used to quantify total bacteria and the strain HF-1 in the tobacco waste-contaminated soil with SYBR green detection. The 16S rRNA gene, amplified by the total bacterial primers, 338f (5′-CCTACGGGAGGCAGCAG-3′) and 518r (5′-ATTACCGCGGCTGCTGG-3′) (Edenborn and Sexstone 2007), was used as a housekeeping gene or control gene targeting an approximate 180-bp region. The hsp gene was amplified by a pair of specific oligonucleotide primers, hspF (5′-GTCGC TCTGT TTCTC CTCCA-3′) and hspR (5′-GCTCT CTACC ACACC GCTTT-3′), and the size of the amplified segment was about 173 bp. The specificity of the designed primer pair was confirmed by electrophoresis of the PCR products using the genomic DNA of the soil samples as a template. Real-time PCR was performed in a 10-μL reaction mixture system containing the following components: 5 μL 2× conc (LightCycler® 480 SYBR Green I Master, Roche), 0.5 μL each primer (10 μM), 0.5 μL genomic DNA template, and 3.5 μL PCR-grade H2O (LightCycler® 480 SYBR Green I Master, Roche). Amplification conditions were as follows: 95 °C for 5 min; 45 cycles at 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 30 s; Melting Curve: 95 °C for 5 s, 65 °C for 1 min and 97 °C for continuous.
The data was presented in the form of average and mean standard error (SE). Statistical analyses were performed in SPSS (version 11.5) using ANOVA and the Pearson correlation coefficient. Differences were considered significant when p < 0.05.
T test of the geochemical parameters between the treatments with addition of strain HF-1 (TA) and its non-added (NA) groups
25 g TA vs. NA
50 g TA vs. NA
100 g TA vs. NA
150 g TA vs. NA
200 g TA vs. NA
Characterization of the tobacco waste-contaminated soil during bioremediation
According to the experimental design of moisture evaporation for keeping soil moist and powerful humidity maintaining function of the tobacco wastes, the moisture content of all groups fluctuated between 15 and 20 % throughout the processes (shown in ESM Figs. S4 and S5), except for the control without addition.
Above all, an increase in content of tobacco wastes ranging from 100 to 200 mg/L resulted in a remarkable decrease in nicotine removal rates in the treatments, and bioaugmentation of strain HF-1 enhanced both nicotine degradation and pollution disposal rates, as indicated by SOC decline.
DGGE profiles analysis
The Shannon–Wiener biodiversity indices of microbial community in different treatments
25 g tobacco waste
100 g tobacco waste
200 g tobacco waste
3.06 ± 0.03
3.12 ± 0.02
3.48 ± 0.04
3. 47 ± 0.02
2.74 ± 0.02
2.88 ± 0.02
3.24 ± 0.05
3.07 ± 0.03
3.47 ± 0.03
3. 21 ± 0.01
3.15 ± 0.03
2.54 ± 0.02
3.56 ± 0.02
3.04 ± 0.04
3.75 ± 0.01
3.10 ± 0.01
2.94 ± 0.02
2.21 ± 0.02
Identities of bands obtained from the V6-V7 regions of the 16S rRNA gene using DGGE analysis in the TA and NA systems
No. of band
Pseudomonas sp. HF-1
Terribacillus sp. VB25
Pseudomonas sp. WO1_S1
Roseomonas sp. PN1
Pseudomonas sp. PNS-15
Pseudomonas sp. HF-1
Uncultured Pantoea sp.
Bacteria have evolved a diversity of potential to utilize numerous organic compounds of both natural and xenobiotic origin (Margesin and Schinner 2001; Fetzner 1998). Several studies have been published on the removal of nicotine by microbes, but most of them ignore the response of bacterial communities and the corresponding behavior of microflora to bioremediation of polluted soils. Therefore, this study aimed to characterize the microbial communities during the nicotine degradation with both indigenous and inoculated microorganisms.
Based on the nicotine degradation curve of different samples in this study, it can be found that strain HF-1 had great biodegradation ability towards the nicotine in the tobacco waste-contaminated soils, and the performance of Pseudomonas sp. HF-1 showed to be better with higher nicotine concentration. An increase in content of tobacco wastes ranging from 25 to 150 mg/L resulted in a remarkable decrease in nicotine removal rates in the treatments. However, a relatively high amount of nicotine was accumulated both in the treatments and controls when the amount of tobacco waste added was higher than 150 g. The probable reason is that some indigenous microbes in soil could utilize the nicotine as a carbon and nitrogen source with low concentration, but a high concentration of tobacco waste can inhibit their growth and activity.
The maximum polluted concentration of this experiment was set as 334 g tobacco wastes per kg soil, corresponding to 6 g nicotine per kg dry soil, but strain HF-1 was still able to survive in a high concentration of nicotine and even showed high metabolic rates. Therefore, the strain has the potential to maintain stable metabolic ability and adapt to the sudden increase of nicotine content caused by external influences in the actual polluted environment. In practical conditions, the nicotine concentration in the soil increased continuously since tobacco wastes of the cigarette factory are piled on the ground during the manufacturing processes. The aspect of the experiment which involved repeated addition of the pollutant closely reflects the realistic application and has significant meaning for the bioremediation of tobacco waste-contaminated soil.
The variations of pH value in the systems were probably mainly due to the acidic materials in the tobacco waste, such as some alcohols, benzene, and unsaturated fatty acids (Wang et al. 2012). The pH value decreased as the acidic materials dissolved out of the tobacco waste, and increased due to the formation of basic compounds (i.e. amines) which result from the degradation of organic compounds. Another hypothesis is that the toxicity of tobacco wastes to other genera in the contaminated soil system was reduced as a result of nicotine degradation by strain HF-1, causing the system to be more favorable for the removal of other pollutants and the acidic metabolic intermediates (Wang et al. 2009b). This suggests that the metabolic potential of the microbial communities were probably improved owing to the inoculation of strain HF-1.
The addition of tobacco wastes brought abundant organic carbons to the soil (De Neve and Hofman 2000; Madejón et al. 2001). During bioremediation, the changes of the SOC could indirectly reflect the metabolic potential of the microbial communities. Bioaugmentation of strain HF-1 enhanced both nicotine degradation and pollution disposal rates, as indicated by SOC decline. Although strain HF-1 and indigenous soil microbes demonstrated to have certain metabolic effects on the tobacco waste, some substances and toxicants of the wastes could not be totally degraded in a short period. To simulate the actual preservation method of tobacco wastes, the wastes were added to the systems regularly. The differences between the treatments with fewer amounts of wastes and their controls were not significant, and the possible reason may be that the concentration was just under the tolerance which can successfully induce the microbe’s metabolic activity. Furthermore, the tobacco wastes composed of tobacco stems and tobacco leaves, which are rich in cellulose and lignin, and are good for maintaining the moisture of the soils (Li et al 2010) (see ESM Figs. S4 and S5). Also, it could be that the relatively low level of contaminants did not reduce the metabolism of indigenous microbes, thus increasing competition for the HF-1.
Community profile data obtained by PCR-DGGE are useful for assessing the effects of different environmental factors on the change of the microbial community composition (Yang and Crowley 2000). If the band exists or does not exist in different samples, it indicates the existing situation of this species in the microbial community. Therefore, the existence of the bands could be taken as an important factor to investigate the relationships between microbial community compositions and physicochemical parameters in our research. DGGE results indicated that the microbial communities of the samples changed with the tobacco wastes and strain supplementation, especially for those with high concentration of added tobacco waste. This may be due to the stable augmentation of Pseudomonas sp. HF-1 in the treated groups and the negative effect of tobacco wastes on the microbial communities. The rapid nicotine degradation by strain HF-1 may provide a relatively suitable environment for other strains, which contributes to the increase of biodiversity. Otherwise, strain HF-1 appeared to be a nutritional generalist that can use multi-organic substrates, and thus affected the microbial community structure and the dynamics of the environmental samples (Couling et al. 2010; Wang et al. 2009b). It formed an inherent harmonious relationship among specific species (Peng et al. 2010). In addition, Actinobacteria and Firmicutes consistently accounted for a large proportion and existed stably in different treatment systems. This indicates that they also played important roles in bioremediation of contaminated soil and demonstrate high adaptability in relatively larger concentrations of tobacco waste.
The fluorescence qPCR was used to detect strain HF-1 augmentation and colonization in the process of bioremediation of tobacco waste-polluted soil and whether HF-1 existed in the control treatment during all periods (Wang et al. 2009b). The hsp gene is located on the nicotine-degrading gene cluster of the degradative plasmid pMH1. It plays a large part in nicotine degradation and can be used as a marker for strain HF-1; meanwhile, no similar gene sequence was found in NCBI (Wang et al. 2009a). Although RT-qPCR is sensitive enough to detect single copy target cell in some cases (Almeida et al. 2010; Garneau et al. 2011), the method may be biased by the DNA extraction and specificity primers. For example, DNA isolated from soil contains inhibitory substances that may interfere with PCR amplification (König et al. 2010; Riley et al. 2010). In this study, template DNA with high purity and high specificity primers were chosen to eliminate PCR inhibition and decrease background (Du et al. 2006).
After addition of the tobacco wastes, the organics-degrading bacteria grew rapidly. In this case, the tobacco wastes functioned as carbon/nitrogen and energy sources for the degradation bacteria while acting as toxicants for the indigenous when the amount of tobacco wastes was high. The TAs supplemented with nicotine-degrading bacteria could reduce the nicotine concentration and provide a better living environment for the indigenous microorganisms. On the contrary, the NA groups maintained at a higher nicotine level and a lower bacterial quantity. The presence of larger number of appropriate microorganisms is a key to successful bioremediation (Devinny and Chang 2000). The kinetics of gene expression indicated that strain HF-1 possessing hsp exhibited a rapid response to the presence of nicotine. In the high tobacco waste groups, the total bacteria remained at a low level while strain HF-1, with strong tolerance to nicotine, could still survive. In the following period, most of the microbes adapted to the adversity and utilized the soils organic matters, especially in polluted soils with low concentrations of tobacco waste. Therefore, the abundance of strain HF-1 in the high concentration groups was relatively less than the lower ones. A successful colonization of a specific strain during bioaugmentation was due to that the specific functional gene of the inoculated strain could always be detected in the system (Cunliffe et al. 2006; Saito and Minamisawa 2006). Both DGGE and RT-qPCR analysis strongly suggested that strain HF-1 could successfully colonize in the tobacco waste-contaminated soil for a long time and showed vital functions in nicotine degradation.
In conclusion, our results indicate that the highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 had great nicotine biodegradation ability in soil and is an environmentally friendly alternative to the tobacco waste-contaminated soils. The disposal rates of the pollutants were enhanced and pH was moderately adjusted in the TA treatments by successful colonization of strain HF-1 through bioaugmentation. Moreover, it also suggested that strain HF-1 had a positive impact on the biodiversity and the structure of the microbial communities by minimizing nicotine toxicity.
This work was financially supported by the National Natural Science Foundation of China (no. 31170115, 31100032) and the Major Science and Technology Program for Water Pollution Control and Treatment (no. 2012ZX07101-012). We are sincerely grateful to Mr. Philip Alexzander Lorhrmann for greatly improving the use of English.