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Plant–microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil
New Biotechnology Volume 30, Number 1 November 2012
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Research Paper
Plant–microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil Veronika Kurzawova1, Petr Stursa1, Ondrej Uhlik1, Katarina Norkova1, Martin Strohalm3, Jan Lipov1, Lucie Kochankova2 and Martina Mackova1 1 Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic 2 Department of Environmental Chemistry, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
During the second half of the last century a large amount of substances toxic for higher organisms was released to the environment. Physicochemical methods of pollutant removal are difficult and prohibitively expensive. Using biological systems such as microorganisms, plants, or consortia microorganisms–plants is easier, cheaper, and more environmentally friendly. The aim of this study was to isolate, characterize and identify microorganisms from contaminated soil and to find out the effect of plants on microbial diversity in the environment. Microorganisms were isolated by two approaches with the aim to find all cultivable species and those able to utilise biphenyl as a sole source of carbon and energy. The first approach was direct extraction and the second was isolation of bacteria after enrichment cultivation with biphenyl. Isolates were biochemically characterized by NEFERMtest 24 and then the composition of ribosomal proteins in bacterial cells was determined by MALDI-TOF mass spectrometry. Ribosomal proteins can be used as phylogenetic markers and thus MALDI-TOF MS can be exploited also for taxonomic identification because the constitution of ribosomal proteins in bacterial cells is specific for each bacterial species. Identification of microorganisms using this method is performed with the help of database Bruker Daltonics MALDI BioTyper. Isolated bacteria were analyzed from the point of the bphA gene presence. Bacteria with detected bphA gene were then taxonomically identified by 16S rRNA sequence. The ability of two different plant species, tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum), to accumulate PCBs was studied as well. It was determined that various plant species differ in the PCBs accumulation from the contaminated soil. Also the content of PCBs in various plant tissues was compared. PCBs were detected in roots and aboveground biomass including leaves and berries.
Introduction Polychlorinated biphenyls (PCBs) are organic compounds that were industrially used in the last century because of their physicochemical properties, for example, thermostability, alkali resistance, acid resistance, among others [1]. PCBs were prepared by Corresponding author: Mackova, M. ([email protected]) 1871-6784/$ - see front matter ß 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nbt.2012.06.004
random chlorination of biphenyl and they differ in the position and number of chlorines on the biphenyl core. Theoretically, 209 congeners could be prepared but during manufacturing of PCB mixtures only 20–60 congeners were obtained [2]. After harmful properties of these compounds had been discovered, their use was banned. But a large amount of PCBs had already been released to the environment. PCB removal from contaminated www.elsevier.com/locate/nbt
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soil using physicochemical methods is difficult, expensive and harmful to the environment, unlike the use of biological systems (plants and microorganisms), which is easier, cheaper and environment-friendly [3]. Plants are able to absorb PCBs from contaminated soil by root system, transform them to non-phytotoxic compounds and store them in their tissues [4]. The transformation can be divided into three phases. In the first phase, enzymes-catalyzed reactions take place that lead to introducing polar groups to the molecule of xenobiotic. When the proper functional polar group is attached to the molecule, the biotransformation proceeds by reactions that belong to the second phase; these reactions are called primary conjugation. Products of these reactions are conjugates of the xenobiotic molecule with plant molecules, for example, amino acids, glutathion, glycosides [5,6]. Such a conjugation leads to a decreased phytotoxicity of the xenobiotic. These products are stored in plant tissues or are transformed by other reactions that are classified as the third phase, where conjugates interact with parts of the cell wall [7]. Plants can positively affect rhizosphere microflora by releasing various compounds to the soil, for example, amino acids, proteins, glycosides, among others. Secondary metabolites, other products of plants, are released to the rhizosphere as well and can serve as a source of carbon and energy for many bacteria. In addition, they are able to induce the expression of genes involved in bacterial PCB degradation pathway as well [8–10]. Bacteria are able to transform PCBs under aerobic or anaerobic conditions. The anaerobic process, reductive dechlorination, leads to the formation of lower chlorinated PCBs that are aerobically more easily degraded than congeners with a higher level of chlorination [11]. Aerobic bacteria that are able to degrade PCBs have been isolated and identified, for example, strains of the genera Pseudomonas, Achromobacter, Alcaligenes, Burkholderia, Ralstonia, Rhodococcus, among others. These bacteria are able to use biphenyl as a sole source of carbon and energy and PCBs are cometabolized by the enzymes of biphenyl pathway. These enzymes are encoded by genes included in biphenyl operon [12] and are marked as bph genes [13]. Polychlorinated biphenyls degradation pathway products are chlorobenzoic acids and pentadienoic acids. In this study, long time PCB-contaminated soil was vegetated with two different plant species, tobacco and nightshade, and bioaugmented with different bacterial degraders previously isolated from contaminated soils. Our aim was to find out how plants alter the spectrum of bacterial degraders to be cultured, and whether the combination of plants and bioaugmentation stimulate the PCB removal from the soil.
Materials and methods Sample preparation The soil used for the pot experiments was contaminated by PCBs (concentration of PCBs 110 mg/kg). It was procured from a dumpsite of contaminated soil in Lhenice, south Bohemia [14]. Nine different microcosms were constructed using 300 g of contaminated soil, two different plant species (tobacco and nightshade) and various bacterial strains (strains of Pseudomonas spp. and Ochrobactrum sp. strain KH-6) (Table 1). Each microcosm was prepared in three replicates. Bacteria used for bioaugmentation were previously isolated from different PCB contaminated soils – Pseudomonas sp. L15 was isolated from the nightshade rhizosphere, Pseudomonas sp. 16
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New Biotechnology Volume 30, Number 1 November 2012
TABLE 1
Prepared samples Plant species
Bacterial strains
No vegetation
No strain added
Tobacco
No strain added Pseudomonas sp. KG-3 Ochrobactrum sp. KH-6 Pseudomonas sp. JAB1
Nightshade
No strain added Pseudomonas sp. L15 Ochrobactrum sp. KH-6 Pseudomonas sp. JAB1
KG-3 and Ochrobactrum sp. KH-6 from the non-vegetated contaminated soil [14,15] and Pseudomonas sp. JAB1 was isolated from the PCB contaminated soil from the area of the ISOLIT factory in Jablonne´ v Podjesˇteˇdı´, Czech Republic [16]. The amount of added bacteria corresponded to 109 CFU. Before bioaugmentation, bacteria were cultivated in liquid mineral salt solution [17] at 288C until they reached OD650 of 1. Cells were pelleted, resuspended in physiological solution (0.85% (w/v) NaCl), and applied to soil. Microcosms were incubated under laboratory conditions (228C) for three months. Afterwards, the microcosms were destructively harvested, and the soil was used for bacterial isolation and PCB content determination.
Bacterial isolation Bacteria were isolated in two different ways. The first way was the extraction with the 1% sodium pyrophosphate (Na2H2P2O7) solution. Bacteria were extracted from 10 g of soil which was shaken with 90 ml of extraction solution at 130 rpm for 20 h. The second way was the enrichment culture with biphenyl as a sole source of carbon and energy, when a few crystals of biphenyl were added to the mineral medium. 5 g of contaminated soil was added to 50 ml of mineral medium with biphenyl. After one week, 1 ml of this suspension was used as an inoculum for a new 50 ml culture. This procedure was performed 10 times. Isolates were cultivated at 288C while shaking (130 rpm). After direct extraction or liquid enrichment cultivation with biphenyl, obtained extracts were used for the preparation of serial decimal dilutions in 0.85% NaCl. In three replicates, 100 ml of the third and fourth dilutions were spread on PCA (Plate Count Agar, Oxoid, UK) and mineral medium [18] with a few crystals of biphenyl as a sole source of carbon. Isolates were cultivated at 288C until countable colonies emerged.
Bacterial characterization Biochemical profile The biochemical characteristics of isolates obtained from contaminated soil were determined by commercially available biochemical tests NEFERMtest 24 (Lachema Group, CZ). These tests are designed for non-fermenting Gram negative bacteria. Testing was performed according to the manufacturer’s instructions.
Protein profile The protein composition of bacterial cells was determined by whole-cell MALDI-TOF mass spectrometry (Matrix Assisted Laser
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Desorption/Ionisation with the Time of Flight analyser). 1 ml of bacterial cultures grown in LB medium was centrifuged (12,000 g) for 3 min and obtained pellets were washed with 1 ml of 0.85% NaCl. This step was repeated two times. Eventually, samples were applied on MALDI target, drizzled with 1 ml of the saturated solution of a-cyano-4-hydroxycinnamic acid (Sigma– Aldrich, USA) in organic solution (50% acetonitrile, 2.5% trifluoroacetic acid), and directly screened as described earlier [19]. Flex Control software (Bruker Daltonics, Bremen, Germany) was used to record spectra in a linear positive mode at an accelerated voltage of 19 kV in the range from 2 to 20 kDa. Each final spectrum was based on collecting and summing at least 300 individual spectra (30 laser shots at 10 different spot positions). Isolates were further grouped according to closely similar patterns of proteins generated by mMass [20]. For each group, one random representative was chosen and further worked with.
Extracts were analyzed using a Hewlett-Packard 5890 gas chromatograph with an electron-capture detector and a capillary column HP-5MS (60 m, 0.25 mm inner diameter) coated with 0.25 mm immobilized phase (5% biphenyl, 95% dimethylsiloxane) with nitrogen as a carrier gas (flow rate 1 ml min1). The temperature program was as follows: 508C for 1 min, increase of temperature at a rate of 258C per min until the temperature of 1958C, increase of temperature at a rate 18C per min until the temperature of 2058C which was maintained for 5 min followed by an increase at a rate of 38C per min until the temperature of 2808C which was maintained for 5 min again. The injection volume was 1 ml. Each sample was analyzed 2 times in 3 replicates. Results were calculated relative to standards of PCB 28, 52, and 101 at a concentration 5 mg/ml [26]. Concentration of PCBs in plant tissues was analyzed as is described above for soil. Samples had been prepared by drying 6 g of plant tissues at 508C until the constant weight.
Detection of bphA gene
Results
Isolates with different protein pattern and biochemical profile were screened for the presence of bphA gene (the first gene of biphenyl operon) by polymerase chain reaction (PCR). The PCR products were generated with primers 490f: 50 -CGC GTS GMV ACC TAC AAR G-30 and 697r: 50 -GGT ACA TGT CRC TGC AGA AYT GC-30 (Burkholderia xenovorans LB400 numbering) [21] following a program of 958C for 5 min, 30 cycles of 958C for 45 s, 508C for 45 s, 728C for 60 s, and final extension at 728C for 10 min. The 25 ml reactions contained the template, 5 pmol of each primer (Generi Biotech, Czech Republic), 5 nmol of dNTPs (Finnzyme, Finland), 2.5 mg of BSA (New England BioLabs, UK), and 0.5 U of DyNAzyme II DNA polymerase with the corresponding buffer (Finnzyme, Finland). Obtained amplicons were detected by 1% agarose gel electrophoresis.
Both microorganisms and plants play an important role in the transformation of xenobiotics. Therefore, identification and characterization of bacteria degrading PCBs is a very important part of environmental microbiology. The aim of this study was (i) to compare different strategies of biphenyl-degrading bacteria isolation and (ii) to identify and characterize obtained isolates. Additionally, the effect of plants (tobacco, nightshade) and bioaugmentation by bacterial degraders on PCB removal from soil was evaluated.
Bacterial identification The identification of isolates was based on 16S rRNA gene sequence analyses. 16S rRNA gene was amplified using primers 27f: 50 -AGA GTT TGA TCM TGG CTC AG-30 and 1492r (50 -TAC GGY TAC CTT GTT ACG ACT T-30 ) [22] following a program of 958C for 5 min, 25 cycles of 958C for 45 s, 508C for 45 s, 728C for 1 min and 40 s, and final extension at 728C for 10 min. The composition of the reaction mixture was the same as for bphA amplification. Products of these reactions were used for reconditioning PCR [23] with the same primers following the same program but only 5 cycles were performed. The reconditioning PCR was prepared in three replicates for each sample and the volume of the reaction mixture was 50 ml with double amounts of chemicals. Amplicons were purified by QIAquick PCR Purification Kit (Qiagen, Germany) and were sequenced by single extension sequencing with primer 27f. The identification of isolates was performed using RDP Classifier and RDP Seqmatch [24,25].
Isolation of biphenyl-degrading bacteria The number of isolates obtained by direct extraction was 38 (Table 2). After extraction to sodium pyrophosphate, bacteria were cultivated on mineral media. Macroscopic characteristics of grown colonies were compared and for each sample colonies with different macroscopic signs were isolated. The same procedure was performed with bacteria isolated after cultivation with biphenyl, where by contrast, 18 isolates (Table 3) were obtained. These bacteria formed visible colonies on plates with mineral medium and biphenyl already after 48 h. Biochemical tests were performed for all isolates we obtained after isolation from the contaminated soil. Biochemical (metabolic) characteristics for isolates with detected bphA gene are summarized in Table 4. TABLE 2
Number of isolates obtained by direct extraction Sample
Plant
Added bacteria
Number of isolates
K
No vegetation
No strain added
5
LK
Nightshade
No strain added
2
Determination of PCB concentration in soil and plant tissue samples
LL
Nightshade
Pseudomonas sp. L15
7
LH
Nightshade
Ochrobactrum sp. KH6
4
The soils were air-dried at the laboratory temperature, sieved with 1-mm mesh, and 1 g of sieved soil was extracted with 10 ml hexane for 4 h in Soxhlet microextractor. The extract was concentrated to 1 ml by a nitrogen flow, purified on a column filled with Florisil1 Adsorbent for Chromatography, 60-100 mesh (Fluka, USA) and diluted with hexane to 10 ml.
LJ
Nightshade
Pseudomonas sp. JAB1
5
TK
Tobacco
no strain added
3
TG
Tobacco
Pseudomonas sp. KG3
4
TH
Tobacco
Ochrobactrum sp. KH6
4
TJ
Tobacco
Pseudomonas sp. JAB1
4
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New Biotechnology Volume 30, Number 1 November 2012
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New Biotechnology Volume 30, Number 1 November 2012
TABLE 3
Number of isolates obtained after cultivation with biphenyl
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Sample
Plant
Added bacteria
Number of isolates
K
No vegetation
No strain added
2
LK
Nightshade
No strain added
1
LL
Nightshade
Pseudomonas sp. L15
1
LH
Nightshade
Ochrobactrum sp. KH6
3
LJ
Nightshade
Pseudomonas sp. JAB1
2
TK
Tobacco
No strain added
2
TG
Tobacco
Pseudomonas sp. KG3
1
TH
Tobacco
Ochrobactrum sp. KH6
2
TJ
Tobacco
Pseudomonas sp. JAB1
4
obtained (Fig. 1) from the mass spectrometer were converted to gel views using mMass [20] and thereby the composition of cell proteins was compared (Fig. 2).
Identification of bacteria based on 16S rRNA gene
Composition of ribosomal proteins in bacterial cells The composition of ribosomal and other high-copy proteins was determined by MALDI-TOF mass spectrometry and results for each isolate were compared. Isolates were divided into groups according to ribosomal proteins of the same molecular weight. Spectra
Various isolates (different biochemical profile and different composition of ribosomal proteins in bacterial cells) were identified based on nucleotide sequence. Only 13 samples of all isolates obtained by direct extraction were positively tested for the presence of bphA gene in their genome, 9 of which were from tobacco rhizosphere and 2 from nightshade rhizosphere. There were 2 isolates in the control sample with bphA gene detected. The bphA gene was detected in 8 samples obtained after cultivation with biphenyl as well, 5 of them being from the tobacco rhizosphere, 2 from the nightshade rhizosphere and 1 from the control sample. Isolates bearing bphA gene (potential PCB degraders) were identified by 16S rRNA gene sequencing. Results are in Table 5 (isolates obtained by direct extraction) and in Table 6 (isolates obtained after repeated enrichment cultivations with biphenyl as a sole source of carbon and energy). After both isolations, Pseudomonas was the bacterial genus mostly detected.
TABLE 4
Biochemical characteristics of isolates with bphA gene detected Isolate/test
NLL1
NTK2
NTG1
TG2
TJ1
NLJ1
NTJ3
TK1
TG1
K4
TK2
TH3
NK1
NTH2
NTJ4
LL2
TG4
LL1
TJ2
OXI
+
+
+
+
+
IND
+
+
+
PHS
+
+
+
+
+
+
NO3
+
+
+
+
+
+
+
+
+
+
+
+
+
ARG
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
BGA
+
NO2
+
+
+
+
+
+
+
+
+
+
+
+
+
URE
+
+
+
+
+
+
BGL
+
+
+
+
+
+
+
+
+
ESL
+
+
+
+
+
LYS
+
+
+
+
+
+
+
NAG
+
+
+
+
+
+
+
GGT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
GLU
+
+
+
+
+
+
+
+
+
MAN
LAC
FRU
+
+
+
+
XYL
+
+
+
+
+
+
+
+
MLT
+
INO
SUC
+
+
+
+
+
CEL
+
+
GAL
+
+
+
+
+
+
TRE
SCI
+
+
+
+
+
+
+
+
+
+
+
+
+
OXI: oxidase, IND: indol production, PHS: phosphatase, NO3: nitrate reduction, ARG: argininedihydrolase, BGA: b-galactosidase, NO2: nitrite reduction, URE: urease, BGL: b-glucosidase, ESL: esculine hydrolysis, LYS: lysinedecarboxylase, NAG: N-acetyl-b-D-glucosaminidase, GGT: g-glutamyl transferase, GLU: glucosa, MAN: mannosa, LAC: lactose, FRU: fructose, XYL: xylose, MAL: maltose, INO: inositol, SUC: sucrose, CEL: cellobiose, GAL: galactose, TRE: trehalose, and SCI: Simmons’ citrate.
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FIGURE 1
Example of a spectrum from MALDI-TOF mass spectrometry of the bacterium isolated from the control sample. The top of the figure shows the spectrum converted to the gel view.
Determination of PCB concentration in plant and soil samples
Discussion
The amount of PCBs accumulated in plants was determined by gas chromatography and results are summarized in Table 7. The effect of combination of plant–microbe on biodegradation of PCBs was evaluated by determining PCB concentration in soil. Decrease of PCBs in contaminated soil is summarized in Table 8.
The removal of polychlorinated biphenyls from the environment using biological systems is an environmental friendly way for PCBcontaminated soil decontamination. In this experiment the effect of two plant species, tobacco and nightshade, on biodegradation of PCBs was studied. Plants absorb PCBs from contaminated environment and it was verified that the structure of plant roots affects the absorption of PCBs from the soil and PCB accumulation [4,27]. Both the plants used, nightshade and tobacco, belong to the family group of Solanaceae, but they differ in the amount of biomass and the ability to transport compounds absorbed from soil by roots to other tissues as stem and leaves, which is obvious from results obtained in this study (Table 7). Nightshade absorbs higher amount of PCBs, but tobacco can transfer PCBs to leaves and stem more efficiently. The accumulation of pollutants in above-ground parts of plant is important for the decontamination of polluted soils because plants containing PCBs are harvested whereas roots can be disrupted during the harvest and can remain in the soil. Plants affect bacterial degradation of PCBs in contaminated soil by excreting (among others) products of secondary metabolism [9,28], compounds from the family of flavonoids, terpenes, among others. The composition of these compounds differs in exudates of various plant species. Consequently, each plant species stimulates bacterial activity and ability to degrade PCBs with different efficiencies. Previously Mackova et al. [14] showed the results of phytoremediation studies with different plants (tobacco, nightshade, alfalfa, horseradish) in experiments carried out within several years. They summarized that tobacco and horseradish themselves mostly proved beneficial phytoremediation effect; nevertheless, this information cannot be generalized for neither all plant species nor for the same species cultivated under different conditions.
FIGURE 2
Comparison of spectra converted to gel views.
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New Biotechnology Volume 30, Number 1 November 2012
TABLE 5
Bacteria identified after direct extraction Sample
Plant
Added bacteria
16S rRNA sequence hit in BLAST NCBI
16S rRNA sequence hit in RDP
RDP score
K4
No vegetation
No strain added
Pseudomonas species
Pseudomonas amygdali
0.918
LL1
Nightshade
Pseudomonas plecoglossicida L15
Pseudomonas alcaligenes
Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri
0.81
LL2
Nightshade
Pseudomonas plecoglossicida L15
Ochrobactrum cytisi
Ochrobactrum cytisi, Ochrobactrum lupini
0.988
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TK1
Tobacco
No strain added
Pseudomonas putida
Pseudomonas monteilii
0.959
TK2
Tobacco
No strain added
Pseudomonas species
Pseudomonas jessenii
0.942
TG1
Tobacco
Pseudomonas putida KG3
Pseudomonas putida
Pseudomonas plecoglossicida
0.95
TG2
Tobacco
Pseudomonas putida KG3
Achromobacter species
Achromobacter spanius
0.956
TG4
Tobacco
Pseudomonas putida KG3
Pseudomonas stutzeri
Pseudomonas stutzeri
0.956
TH3
Tobacco
Ochrobactrum anthropi KH6
Pseudomonas species
Pseudomonas jessenii
0.959
TJ1
Tobacco
Pseudomonas species JAB1
Achromobacter species
Achromobacter spanius
0.973
TJ2
Tobacco
Pseudomonas species JAB1
Pseudomonas plecoglossicida
Pseudomonas plecoglossicida
0.832
TABLE 6
Bacteria identified after cultivation with biphenyl as a sole source of carbon Sample
Plant
Added bacteria
16S rRNA sequence hit in BLAST NCBI
16S rRNA sequence hit in RDP
RDP score
NK1
No vegetation
No strain added
Pseudomonas species
Pseudomonas alcaliphila
0.97
NLL1
Nightshade
Pseudomonas plecoglossicida L15
Pseudomonas mendocina
Pseudomonas alcaliphila
0.858
NLJ1
Nightshade
Pseudomonas species JAB1
Pseudomonas alcaliphila
Pseudomonas alcaliphila
0.977
NTK2
Tobacco
No strain added
Pseudomonas mendocina
Pseudomonas alcaliphila
0.969
NTG1
Tobacco
Pseudomonas putida KG3
Pseudomonas mendocina
Pseudomonas alcaliphila
0.965
NTH2
Tobacco
Ochrobactrum anthropi KH6
Pseudomonas species
Pseudomonas delhiensis
0.938
NTJ3
Tobacco
Pseudomonas species JAB1
Pseudomonas alcaliphila
Pseudomonas alcaliphila
0.959
NTJ4
Tobacco
Pseudomonas species JAB1
Pseudomonas species
Pseudomonas alcaliphila
0.974
TABLE 7
Amount of PCBs in nightshade (a) and tobacco(b) dry matter measured in plant tissues Plant
Added bacterial species
Plant tissues
Nightshade
No strain added
Stem + leaves Roots
Pseudomonas sp. L15
Stem + leaves Roots
Ochrobactrum sp. KH6
stem + leaves Roots
Pseudomonas sp. JAB1
Stem + leaves Roots
Tobacco
No strain added
Stem + leaves Roots
Pseudomonas sp. L15
Stem + leaves Roots
Ochrobactrum sp. KH6
Stem + leaves Roots
Pseudomonas sp. JAB1
Stem + leaves Roots
*
Amount of PCBs (mg PCB/kg dry mass)* 2.1 80.8 4.0 60.0 3.3 62.9 0.7 53.1 6.5 32.8 6.3 64.5 8.1 56.3 6.3 61.6
Small amounts of biomass at the end of experiment did not allow a sufficient number of replicate measurements of PCB concentrations. Standard analysis error of the methodology is 40%.
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TABLE 8
Decrease of PCBs in soil vegetated by nightshade and tobacco and augmented by different bacterial strains
Nightshade
Added bacterial species
Initial amount of PCBs (mg PCBs/kg soil)
Final amount of PCBs (mg PCBs/kg soil)
Decrease of PCB (%)
No strain added
107.9 14.1
59.5 0.3
44.8
Pseudomonas sp. L15
85.5 11.2
21.7
Ochrobactrum sp. KH6
57.5 13.5
46.7
67.3 7.2
37.6
80.9 0.6
25
Pseudomonas sp. L15
51.1 7.5
52.8
Ochrobactrum sp. KH6
81.2 6.3
24.7
Pseudomonas sp. JAB1
73.4 0.1
32
Pseudomonas sp. JAB1 Tobacco
No strain added
107.9 14.1
Interactions of plant–microorganisms are not the only one factor affecting PCB degradation. Mutual relations between microorganisms play an important role in the nature. Bacteria can positively or negatively stimulate each other. Therefore, we studied how the addition of various bacterial species (bioaugmentation) to vegetated contaminated soil can affect PCB degradation. Bacteria previously isolated from PCB contaminated soils with verified ability to degrade PCBs were used for the bioaugmentation. Table 8 suggests that the combination of tobacco and Pseudomonas sp. KG3 leads to the best biodegradation results under given conditions. Decrease of PCBs in contaminated soil with added bacterial strain Pseudomonas sp. KG3 was over 50% whereas in control sample the decrease was only 25% within 3 months. In the sample vegetated by tobacco with Pseudomonas sp. JAB1 the decrease was 32% and with Ochrobactrum sp. KH6, the decrease of PCBs was comparable to the control sample. Different results, however, were achieved after vegetation with nightshade, where the highest decrease of PCBs concentration in soil was obtained after bioaugmentation with Ochrobactrum sp. KH6 and with Pseudomonas sp. JAB1. Vegetation by different plant species is known to influence rhizosphere microbial diversity [17,29–31]. However, we are showing here that diversity of cultured bacteria is also dependent on the way they were isolated. Two ways of isolation were performed in this study, the first was direct extraction and the second one was after long-term cultivation with biphenyl as a sole source of carbon and energy. Both ways of isolation were aimed to obtain only bacteria capable of using biphenyl as a sole source of carbon and energy and potentially degrading PCBs. The first difference in
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Plant
these two ways of isolation is the number of morphologically different isolates; 38 isolates were obtained after direct extraction and 18 after long-term cultivation with biphenyl (Tables 2 and 3). After direct extraction three different bacterial genera were identified, but after long-term cultivation with biphenyl as a sole carbon source only bacterial genus Pseudomonas was detected. Bacteria from the genus Pseudomonas form natural soil microflora and belong to the PCBs degraders [32–36] which is also the reason why this bacterial genus was used for bioaugmentation. Pseudomonads were also the most frequently identified isolates obtained from the contaminated soil. Interestingly, when the contaminated soil from the same site was vegetated by horseradish (Armoracia rusticana), among 54 isolates only one was identified as Pseudomonas spp. [18]. This is the other evidence that different bacterial genera are favoured in rhizospheres of different plants. Isolation and identification of cultivable microorganisms from contaminated soil is the important part of rhizoremediation research. However, to find appropriate conditions for these degraders and make degradation of PCBs more efficient is important as well. In summary, the results of this study show how different strategies of bacterial isolation influence the diversity of culturable bacteria and demonstrate how combination of plants and bacteria can increase PCB removal from contaminated soil.
Acknowledgements This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project ME 10041), and Czech Science Foundation (project 525/09/1058).
References 1 Mackova´, M. et al. (2010) Bacterial degradation of polychlorinated biphenyls, in geomicrobiology: molecular and environmental perspective. In Geomicrobiology: Molecular and Environmental Perspective (Loy, A., Mandl, M., Barton, L.L., eds), pp. 347–366, Springer 2 Abraham, W-R. et al. (2002) Polychlorinated biphenyl-degrading microbial communities in soils and sediments. Curr. Opin. Microbiol. 5, 246–253 3 Demnerova´, K. et al. (2005) Two approaches to biological decontamination of groundwater and soil polluted by aromatics – characterization of microbial populations. Int. Microbiol. 8, 205–211 4 Van Aken, B. et al. (2010) Phytoremediation of polychlorinated biphenyls: new trends and promises. Environ. Sci. Technol. 44, 2767–2776 5 Rezek, J. et al. (2008) Hydroxy-PCBs, methoxy-PCBs and hydroxy-methoxy-PCBs: metabolites of polychlorinated biphenyls formed in vitro by tobacco cells. Environ. Sci. Technol. 42, 5746–5751
6 Rezek, J. et al. (2007) Plant metabolites of polychlorinated biphenyls in hairy root culture of black nightshade Solanum nigrum SNC-9O. Chemosphere 69, 1221–1227 7 Sandermann, H. (1994) Higher-plant metabolism of xenobiotics – the green liver concept. Pharmacogenetics 4, 225–241 8 Leigh, M.B. et al. (2006) Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site. Appl. Environ. Microbiol. 72, 2331–2342 9 Leigh, M.B. et al. (2002) Root turnover: an important source of microbial substrates in rhizosphere remediation of recalcitrant contaminants. Environ. Sci. Technol. 36, 1579–1583 10 Gilbert, E.S. and Crowley, D.E. (1997) Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. strain B1B. Appl. Environ. Microbiol. 63, 1933–1938
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11 Furukawa, K. (2006) Oxygenases and dehalogenases: molecular approaches to efficient degradation of chlorinated environmental pollutants. Biosci. Biotech. Biochem. 70, 2335–2348 12 Abramowicz, D.A. (1990) Aerobic and anaerobic biodegradation of PCBs – a review. Crit. Rev. Biotechnol. 10, 241–249 13 Furukawa, K. and Fujihara, H. (2008) Microbial degradation of polychlorinated biphenyls: biochemical and molecular features. J. Biosci. Bioeng. 105, 433–449 14 Mackova´, M. et al. (2009) Phyto/rhizoremediation studies using long-term PCBcontaminated soil. Environ. Sci. Pollut. Res. 16, 817–829 15 Beranova, K. et al. (2007) Rhizoremediation for decontamination of long-term PCB contaminated soil with focus on microbial diversity. J. Biotechnol. 131 S243-S243 16 Ryslava, E. et al. (2003) Study of PCB degradation in real contaminated soil. Fresenius Environ. Bull. 12, 296–301 17 Uhlı´k, O. et al. (2009) Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing. Appl. Environ. Microbiol. 75, 6471–6477 18 Uhlı´k, O. et al. (2011) Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil. Appl. Environ. Microbiol. 77, 6858–6866 19 Koubek, J. et al. (2012) Whole-cell MALDI-TOF: rapid screening method in environmental microbiology. Int. Biodeterior. Biodegrad. 69, 82–86 20 Strohalm, M. et al. (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal. Chem. 82, 4648–4651 21 Rysˇlava´, E. et al. (2004) Detection of Polychlorinated Biphenyl-degrading Bacteria in Soil (Verstraete, W., ed.), pp. 839–842, Taylor & Francis Group plc 22 Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M., eds), pp. 115–175, John Wiley and Sons 23 Thompson, J.R. et al. (2002) Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res. 30, 2083–2088
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24 Cole, J.R. et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37, D141–D145 25 Wang, Q. et al. (2007) Naı¨ve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 26 Burkhard, J. et al. (1997) Analytical procedure for the estimation of polychlorinated biphenyl transformation by plant tissue cultures. Anal. Commun. 34, 287–290 27 Macek, T. et al. (2000) Exploitation of plants for the removal of organics in environmental remediation. Biotechnol. Adv. 18, 23–34 28 Singer, A.C. et al. (2003) Secondary plant metabolites in phytoremediation and biotransformation. Trends Biotechnol. 21, 123–130 29 Ionescu, M. et al. (2009) Isolation and characterization of different plant associated bacteria and their potential to degrade polychlorinated biphenyls. Int. Biodeterior. Biodegrad. 63, 667–672 30 Slater, H. et al. (2011) Assessing the potential for rhizoremediation of PCB contaminated soils in northern regions using native tree species. Chemosphere 84, 199–206 31 Mackova´, M. et al. (2006) Phytoremediation and Rhizoremediation. Theoretical Background. Springer 32 Furukawa, K. et al. (1989) Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon. J. Bacteriol. 171, 5467–5472 33 Nova´kova´, H. et al. (2002) PCB metabolism by Pseudomonas sp. P2. Int. Biodeterior. Biodegrad. 50, 47–54 34 Rysˇlava´, E. et al. (2003) Study of PCB degradation in real contaminated soil. Fresenius Environ. Bull. 12, 296–301 35 Singer, A.C. et al. (2002) Differential enantioselective transformation of atropisomeric polychlorinated biphenyls by multiple bacterial strains with different inducing compounds. Appl. Environ. Microbiol. 68, 5756–5759 36 Ve´zina, J. et al. (2008) Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit. J. Mol. Microbiol. Biotechnol. 15, 139–151
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Plant–microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil Veronika Kurzawova1, Petr Stursa1, Ondrej Uhlik1, Katarina Norkova1, Martin Strohalm3, Jan Lipov1, Lucie Kochankova2 and Martina Mackova1 1 Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic 2 Department of Environmental Chemistry, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
During the second half of the last century a large amount of substances toxic for higher organisms was released to the environment. Physicochemical methods of pollutant removal are difficult and prohibitively expensive. Using biological systems such as microorganisms, plants, or consortia microorganisms–plants is easier, cheaper, and more environmentally friendly. The aim of this study was to isolate, characterize and identify microorganisms from contaminated soil and to find out the effect of plants on microbial diversity in the environment. Microorganisms were isolated by two approaches with the aim to find all cultivable species and those able to utilise biphenyl as a sole source of carbon and energy. The first approach was direct extraction and the second was isolation of bacteria after enrichment cultivation with biphenyl. Isolates were biochemically characterized by NEFERMtest 24 and then the composition of ribosomal proteins in bacterial cells was determined by MALDI-TOF mass spectrometry. Ribosomal proteins can be used as phylogenetic markers and thus MALDI-TOF MS can be exploited also for taxonomic identification because the constitution of ribosomal proteins in bacterial cells is specific for each bacterial species. Identification of microorganisms using this method is performed with the help of database Bruker Daltonics MALDI BioTyper. Isolated bacteria were analyzed from the point of the bphA gene presence. Bacteria with detected bphA gene were then taxonomically identified by 16S rRNA sequence. The ability of two different plant species, tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum), to accumulate PCBs was studied as well. It was determined that various plant species differ in the PCBs accumulation from the contaminated soil. Also the content of PCBs in various plant tissues was compared. PCBs were detected in roots and aboveground biomass including leaves and berries.
Introduction Polychlorinated biphenyls (PCBs) are organic compounds that were industrially used in the last century because of their physicochemical properties, for example, thermostability, alkali resistance, acid resistance, among others [1]. PCBs were prepared by Corresponding author: Mackova, M. ([email protected]) 1871-6784/$ - see front matter ß 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nbt.2012.06.004
random chlorination of biphenyl and they differ in the position and number of chlorines on the biphenyl core. Theoretically, 209 congeners could be prepared but during manufacturing of PCB mixtures only 20–60 congeners were obtained [2]. After harmful properties of these compounds had been discovered, their use was banned. But a large amount of PCBs had already been released to the environment. PCB removal from contaminated www.elsevier.com/locate/nbt
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soil using physicochemical methods is difficult, expensive and harmful to the environment, unlike the use of biological systems (plants and microorganisms), which is easier, cheaper and environment-friendly [3]. Plants are able to absorb PCBs from contaminated soil by root system, transform them to non-phytotoxic compounds and store them in their tissues [4]. The transformation can be divided into three phases. In the first phase, enzymes-catalyzed reactions take place that lead to introducing polar groups to the molecule of xenobiotic. When the proper functional polar group is attached to the molecule, the biotransformation proceeds by reactions that belong to the second phase; these reactions are called primary conjugation. Products of these reactions are conjugates of the xenobiotic molecule with plant molecules, for example, amino acids, glutathion, glycosides [5,6]. Such a conjugation leads to a decreased phytotoxicity of the xenobiotic. These products are stored in plant tissues or are transformed by other reactions that are classified as the third phase, where conjugates interact with parts of the cell wall [7]. Plants can positively affect rhizosphere microflora by releasing various compounds to the soil, for example, amino acids, proteins, glycosides, among others. Secondary metabolites, other products of plants, are released to the rhizosphere as well and can serve as a source of carbon and energy for many bacteria. In addition, they are able to induce the expression of genes involved in bacterial PCB degradation pathway as well [8–10]. Bacteria are able to transform PCBs under aerobic or anaerobic conditions. The anaerobic process, reductive dechlorination, leads to the formation of lower chlorinated PCBs that are aerobically more easily degraded than congeners with a higher level of chlorination [11]. Aerobic bacteria that are able to degrade PCBs have been isolated and identified, for example, strains of the genera Pseudomonas, Achromobacter, Alcaligenes, Burkholderia, Ralstonia, Rhodococcus, among others. These bacteria are able to use biphenyl as a sole source of carbon and energy and PCBs are cometabolized by the enzymes of biphenyl pathway. These enzymes are encoded by genes included in biphenyl operon [12] and are marked as bph genes [13]. Polychlorinated biphenyls degradation pathway products are chlorobenzoic acids and pentadienoic acids. In this study, long time PCB-contaminated soil was vegetated with two different plant species, tobacco and nightshade, and bioaugmented with different bacterial degraders previously isolated from contaminated soils. Our aim was to find out how plants alter the spectrum of bacterial degraders to be cultured, and whether the combination of plants and bioaugmentation stimulate the PCB removal from the soil.
Materials and methods Sample preparation The soil used for the pot experiments was contaminated by PCBs (concentration of PCBs 110 mg/kg). It was procured from a dumpsite of contaminated soil in Lhenice, south Bohemia [14]. Nine different microcosms were constructed using 300 g of contaminated soil, two different plant species (tobacco and nightshade) and various bacterial strains (strains of Pseudomonas spp. and Ochrobactrum sp. strain KH-6) (Table 1). Each microcosm was prepared in three replicates. Bacteria used for bioaugmentation were previously isolated from different PCB contaminated soils – Pseudomonas sp. L15 was isolated from the nightshade rhizosphere, Pseudomonas sp. 16
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TABLE 1
Prepared samples Plant species
Bacterial strains
No vegetation
No strain added
Tobacco
No strain added Pseudomonas sp. KG-3 Ochrobactrum sp. KH-6 Pseudomonas sp. JAB1
Nightshade
No strain added Pseudomonas sp. L15 Ochrobactrum sp. KH-6 Pseudomonas sp. JAB1
KG-3 and Ochrobactrum sp. KH-6 from the non-vegetated contaminated soil [14,15] and Pseudomonas sp. JAB1 was isolated from the PCB contaminated soil from the area of the ISOLIT factory in Jablonne´ v Podjesˇteˇdı´, Czech Republic [16]. The amount of added bacteria corresponded to 109 CFU. Before bioaugmentation, bacteria were cultivated in liquid mineral salt solution [17] at 288C until they reached OD650 of 1. Cells were pelleted, resuspended in physiological solution (0.85% (w/v) NaCl), and applied to soil. Microcosms were incubated under laboratory conditions (228C) for three months. Afterwards, the microcosms were destructively harvested, and the soil was used for bacterial isolation and PCB content determination.
Bacterial isolation Bacteria were isolated in two different ways. The first way was the extraction with the 1% sodium pyrophosphate (Na2H2P2O7) solution. Bacteria were extracted from 10 g of soil which was shaken with 90 ml of extraction solution at 130 rpm for 20 h. The second way was the enrichment culture with biphenyl as a sole source of carbon and energy, when a few crystals of biphenyl were added to the mineral medium. 5 g of contaminated soil was added to 50 ml of mineral medium with biphenyl. After one week, 1 ml of this suspension was used as an inoculum for a new 50 ml culture. This procedure was performed 10 times. Isolates were cultivated at 288C while shaking (130 rpm). After direct extraction or liquid enrichment cultivation with biphenyl, obtained extracts were used for the preparation of serial decimal dilutions in 0.85% NaCl. In three replicates, 100 ml of the third and fourth dilutions were spread on PCA (Plate Count Agar, Oxoid, UK) and mineral medium [18] with a few crystals of biphenyl as a sole source of carbon. Isolates were cultivated at 288C until countable colonies emerged.
Bacterial characterization Biochemical profile The biochemical characteristics of isolates obtained from contaminated soil were determined by commercially available biochemical tests NEFERMtest 24 (Lachema Group, CZ). These tests are designed for non-fermenting Gram negative bacteria. Testing was performed according to the manufacturer’s instructions.
Protein profile The protein composition of bacterial cells was determined by whole-cell MALDI-TOF mass spectrometry (Matrix Assisted Laser
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Desorption/Ionisation with the Time of Flight analyser). 1 ml of bacterial cultures grown in LB medium was centrifuged (12,000 g) for 3 min and obtained pellets were washed with 1 ml of 0.85% NaCl. This step was repeated two times. Eventually, samples were applied on MALDI target, drizzled with 1 ml of the saturated solution of a-cyano-4-hydroxycinnamic acid (Sigma– Aldrich, USA) in organic solution (50% acetonitrile, 2.5% trifluoroacetic acid), and directly screened as described earlier [19]. Flex Control software (Bruker Daltonics, Bremen, Germany) was used to record spectra in a linear positive mode at an accelerated voltage of 19 kV in the range from 2 to 20 kDa. Each final spectrum was based on collecting and summing at least 300 individual spectra (30 laser shots at 10 different spot positions). Isolates were further grouped according to closely similar patterns of proteins generated by mMass [20]. For each group, one random representative was chosen and further worked with.
Extracts were analyzed using a Hewlett-Packard 5890 gas chromatograph with an electron-capture detector and a capillary column HP-5MS (60 m, 0.25 mm inner diameter) coated with 0.25 mm immobilized phase (5% biphenyl, 95% dimethylsiloxane) with nitrogen as a carrier gas (flow rate 1 ml min1). The temperature program was as follows: 508C for 1 min, increase of temperature at a rate of 258C per min until the temperature of 1958C, increase of temperature at a rate 18C per min until the temperature of 2058C which was maintained for 5 min followed by an increase at a rate of 38C per min until the temperature of 2808C which was maintained for 5 min again. The injection volume was 1 ml. Each sample was analyzed 2 times in 3 replicates. Results were calculated relative to standards of PCB 28, 52, and 101 at a concentration 5 mg/ml [26]. Concentration of PCBs in plant tissues was analyzed as is described above for soil. Samples had been prepared by drying 6 g of plant tissues at 508C until the constant weight.
Detection of bphA gene
Results
Isolates with different protein pattern and biochemical profile were screened for the presence of bphA gene (the first gene of biphenyl operon) by polymerase chain reaction (PCR). The PCR products were generated with primers 490f: 50 -CGC GTS GMV ACC TAC AAR G-30 and 697r: 50 -GGT ACA TGT CRC TGC AGA AYT GC-30 (Burkholderia xenovorans LB400 numbering) [21] following a program of 958C for 5 min, 30 cycles of 958C for 45 s, 508C for 45 s, 728C for 60 s, and final extension at 728C for 10 min. The 25 ml reactions contained the template, 5 pmol of each primer (Generi Biotech, Czech Republic), 5 nmol of dNTPs (Finnzyme, Finland), 2.5 mg of BSA (New England BioLabs, UK), and 0.5 U of DyNAzyme II DNA polymerase with the corresponding buffer (Finnzyme, Finland). Obtained amplicons were detected by 1% agarose gel electrophoresis.
Both microorganisms and plants play an important role in the transformation of xenobiotics. Therefore, identification and characterization of bacteria degrading PCBs is a very important part of environmental microbiology. The aim of this study was (i) to compare different strategies of biphenyl-degrading bacteria isolation and (ii) to identify and characterize obtained isolates. Additionally, the effect of plants (tobacco, nightshade) and bioaugmentation by bacterial degraders on PCB removal from soil was evaluated.
Bacterial identification The identification of isolates was based on 16S rRNA gene sequence analyses. 16S rRNA gene was amplified using primers 27f: 50 -AGA GTT TGA TCM TGG CTC AG-30 and 1492r (50 -TAC GGY TAC CTT GTT ACG ACT T-30 ) [22] following a program of 958C for 5 min, 25 cycles of 958C for 45 s, 508C for 45 s, 728C for 1 min and 40 s, and final extension at 728C for 10 min. The composition of the reaction mixture was the same as for bphA amplification. Products of these reactions were used for reconditioning PCR [23] with the same primers following the same program but only 5 cycles were performed. The reconditioning PCR was prepared in three replicates for each sample and the volume of the reaction mixture was 50 ml with double amounts of chemicals. Amplicons were purified by QIAquick PCR Purification Kit (Qiagen, Germany) and were sequenced by single extension sequencing with primer 27f. The identification of isolates was performed using RDP Classifier and RDP Seqmatch [24,25].
Isolation of biphenyl-degrading bacteria The number of isolates obtained by direct extraction was 38 (Table 2). After extraction to sodium pyrophosphate, bacteria were cultivated on mineral media. Macroscopic characteristics of grown colonies were compared and for each sample colonies with different macroscopic signs were isolated. The same procedure was performed with bacteria isolated after cultivation with biphenyl, where by contrast, 18 isolates (Table 3) were obtained. These bacteria formed visible colonies on plates with mineral medium and biphenyl already after 48 h. Biochemical tests were performed for all isolates we obtained after isolation from the contaminated soil. Biochemical (metabolic) characteristics for isolates with detected bphA gene are summarized in Table 4. TABLE 2
Number of isolates obtained by direct extraction Sample
Plant
Added bacteria
Number of isolates
K
No vegetation
No strain added
5
LK
Nightshade
No strain added
2
Determination of PCB concentration in soil and plant tissue samples
LL
Nightshade
Pseudomonas sp. L15
7
LH
Nightshade
Ochrobactrum sp. KH6
4
The soils were air-dried at the laboratory temperature, sieved with 1-mm mesh, and 1 g of sieved soil was extracted with 10 ml hexane for 4 h in Soxhlet microextractor. The extract was concentrated to 1 ml by a nitrogen flow, purified on a column filled with Florisil1 Adsorbent for Chromatography, 60-100 mesh (Fluka, USA) and diluted with hexane to 10 ml.
LJ
Nightshade
Pseudomonas sp. JAB1
5
TK
Tobacco
no strain added
3
TG
Tobacco
Pseudomonas sp. KG3
4
TH
Tobacco
Ochrobactrum sp. KH6
4
TJ
Tobacco
Pseudomonas sp. JAB1
4
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New Biotechnology Volume 30, Number 1 November 2012
TABLE 3
Number of isolates obtained after cultivation with biphenyl
Research Paper
Sample
Plant
Added bacteria
Number of isolates
K
No vegetation
No strain added
2
LK
Nightshade
No strain added
1
LL
Nightshade
Pseudomonas sp. L15
1
LH
Nightshade
Ochrobactrum sp. KH6
3
LJ
Nightshade
Pseudomonas sp. JAB1
2
TK
Tobacco
No strain added
2
TG
Tobacco
Pseudomonas sp. KG3
1
TH
Tobacco
Ochrobactrum sp. KH6
2
TJ
Tobacco
Pseudomonas sp. JAB1
4
obtained (Fig. 1) from the mass spectrometer were converted to gel views using mMass [20] and thereby the composition of cell proteins was compared (Fig. 2).
Identification of bacteria based on 16S rRNA gene
Composition of ribosomal proteins in bacterial cells The composition of ribosomal and other high-copy proteins was determined by MALDI-TOF mass spectrometry and results for each isolate were compared. Isolates were divided into groups according to ribosomal proteins of the same molecular weight. Spectra
Various isolates (different biochemical profile and different composition of ribosomal proteins in bacterial cells) were identified based on nucleotide sequence. Only 13 samples of all isolates obtained by direct extraction were positively tested for the presence of bphA gene in their genome, 9 of which were from tobacco rhizosphere and 2 from nightshade rhizosphere. There were 2 isolates in the control sample with bphA gene detected. The bphA gene was detected in 8 samples obtained after cultivation with biphenyl as well, 5 of them being from the tobacco rhizosphere, 2 from the nightshade rhizosphere and 1 from the control sample. Isolates bearing bphA gene (potential PCB degraders) were identified by 16S rRNA gene sequencing. Results are in Table 5 (isolates obtained by direct extraction) and in Table 6 (isolates obtained after repeated enrichment cultivations with biphenyl as a sole source of carbon and energy). After both isolations, Pseudomonas was the bacterial genus mostly detected.
TABLE 4
Biochemical characteristics of isolates with bphA gene detected Isolate/test
NLL1
NTK2
NTG1
TG2
TJ1
NLJ1
NTJ3
TK1
TG1
K4
TK2
TH3
NK1
NTH2
NTJ4
LL2
TG4
LL1
TJ2
OXI
+
+
+
+
+
IND
+
+
+
PHS
+
+
+
+
+
+
NO3
+
+
+
+
+
+
+
+
+
+
+
+
+
ARG
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
BGA
+
NO2
+
+
+
+
+
+
+
+
+
+
+
+
+
URE
+
+
+
+
+
+
BGL
+
+
+
+
+
+
+
+
+
ESL
+
+
+
+
+
LYS
+
+
+
+
+
+
+
NAG
+
+
+
+
+
+
+
GGT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
GLU
+
+
+
+
+
+
+
+
+
MAN
LAC
FRU
+
+
+
+
XYL
+
+
+
+
+
+
+
+
MLT
+
INO
SUC
+
+
+
+
+
CEL
+
+
GAL
+
+
+
+
+
+
TRE
SCI
+
+
+
+
+
+
+
+
+
+
+
+
+
OXI: oxidase, IND: indol production, PHS: phosphatase, NO3: nitrate reduction, ARG: argininedihydrolase, BGA: b-galactosidase, NO2: nitrite reduction, URE: urease, BGL: b-glucosidase, ESL: esculine hydrolysis, LYS: lysinedecarboxylase, NAG: N-acetyl-b-D-glucosaminidase, GGT: g-glutamyl transferase, GLU: glucosa, MAN: mannosa, LAC: lactose, FRU: fructose, XYL: xylose, MAL: maltose, INO: inositol, SUC: sucrose, CEL: cellobiose, GAL: galactose, TRE: trehalose, and SCI: Simmons’ citrate.
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FIGURE 1
Example of a spectrum from MALDI-TOF mass spectrometry of the bacterium isolated from the control sample. The top of the figure shows the spectrum converted to the gel view.
Determination of PCB concentration in plant and soil samples
Discussion
The amount of PCBs accumulated in plants was determined by gas chromatography and results are summarized in Table 7. The effect of combination of plant–microbe on biodegradation of PCBs was evaluated by determining PCB concentration in soil. Decrease of PCBs in contaminated soil is summarized in Table 8.
The removal of polychlorinated biphenyls from the environment using biological systems is an environmental friendly way for PCBcontaminated soil decontamination. In this experiment the effect of two plant species, tobacco and nightshade, on biodegradation of PCBs was studied. Plants absorb PCBs from contaminated environment and it was verified that the structure of plant roots affects the absorption of PCBs from the soil and PCB accumulation [4,27]. Both the plants used, nightshade and tobacco, belong to the family group of Solanaceae, but they differ in the amount of biomass and the ability to transport compounds absorbed from soil by roots to other tissues as stem and leaves, which is obvious from results obtained in this study (Table 7). Nightshade absorbs higher amount of PCBs, but tobacco can transfer PCBs to leaves and stem more efficiently. The accumulation of pollutants in above-ground parts of plant is important for the decontamination of polluted soils because plants containing PCBs are harvested whereas roots can be disrupted during the harvest and can remain in the soil. Plants affect bacterial degradation of PCBs in contaminated soil by excreting (among others) products of secondary metabolism [9,28], compounds from the family of flavonoids, terpenes, among others. The composition of these compounds differs in exudates of various plant species. Consequently, each plant species stimulates bacterial activity and ability to degrade PCBs with different efficiencies. Previously Mackova et al. [14] showed the results of phytoremediation studies with different plants (tobacco, nightshade, alfalfa, horseradish) in experiments carried out within several years. They summarized that tobacco and horseradish themselves mostly proved beneficial phytoremediation effect; nevertheless, this information cannot be generalized for neither all plant species nor for the same species cultivated under different conditions.
FIGURE 2
Comparison of spectra converted to gel views.
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TABLE 5
Bacteria identified after direct extraction Sample
Plant
Added bacteria
16S rRNA sequence hit in BLAST NCBI
16S rRNA sequence hit in RDP
RDP score
K4
No vegetation
No strain added
Pseudomonas species
Pseudomonas amygdali
0.918
LL1
Nightshade
Pseudomonas plecoglossicida L15
Pseudomonas alcaligenes
Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri
0.81
LL2
Nightshade
Pseudomonas plecoglossicida L15
Ochrobactrum cytisi
Ochrobactrum cytisi, Ochrobactrum lupini
0.988
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TK1
Tobacco
No strain added
Pseudomonas putida
Pseudomonas monteilii
0.959
TK2
Tobacco
No strain added
Pseudomonas species
Pseudomonas jessenii
0.942
TG1
Tobacco
Pseudomonas putida KG3
Pseudomonas putida
Pseudomonas plecoglossicida
0.95
TG2
Tobacco
Pseudomonas putida KG3
Achromobacter species
Achromobacter spanius
0.956
TG4
Tobacco
Pseudomonas putida KG3
Pseudomonas stutzeri
Pseudomonas stutzeri
0.956
TH3
Tobacco
Ochrobactrum anthropi KH6
Pseudomonas species
Pseudomonas jessenii
0.959
TJ1
Tobacco
Pseudomonas species JAB1
Achromobacter species
Achromobacter spanius
0.973
TJ2
Tobacco
Pseudomonas species JAB1
Pseudomonas plecoglossicida
Pseudomonas plecoglossicida
0.832
TABLE 6
Bacteria identified after cultivation with biphenyl as a sole source of carbon Sample
Plant
Added bacteria
16S rRNA sequence hit in BLAST NCBI
16S rRNA sequence hit in RDP
RDP score
NK1
No vegetation
No strain added
Pseudomonas species
Pseudomonas alcaliphila
0.97
NLL1
Nightshade
Pseudomonas plecoglossicida L15
Pseudomonas mendocina
Pseudomonas alcaliphila
0.858
NLJ1
Nightshade
Pseudomonas species JAB1
Pseudomonas alcaliphila
Pseudomonas alcaliphila
0.977
NTK2
Tobacco
No strain added
Pseudomonas mendocina
Pseudomonas alcaliphila
0.969
NTG1
Tobacco
Pseudomonas putida KG3
Pseudomonas mendocina
Pseudomonas alcaliphila
0.965
NTH2
Tobacco
Ochrobactrum anthropi KH6
Pseudomonas species
Pseudomonas delhiensis
0.938
NTJ3
Tobacco
Pseudomonas species JAB1
Pseudomonas alcaliphila
Pseudomonas alcaliphila
0.959
NTJ4
Tobacco
Pseudomonas species JAB1
Pseudomonas species
Pseudomonas alcaliphila
0.974
TABLE 7
Amount of PCBs in nightshade (a) and tobacco(b) dry matter measured in plant tissues Plant
Added bacterial species
Plant tissues
Nightshade
No strain added
Stem + leaves Roots
Pseudomonas sp. L15
Stem + leaves Roots
Ochrobactrum sp. KH6
stem + leaves Roots
Pseudomonas sp. JAB1
Stem + leaves Roots
Tobacco
No strain added
Stem + leaves Roots
Pseudomonas sp. L15
Stem + leaves Roots
Ochrobactrum sp. KH6
Stem + leaves Roots
Pseudomonas sp. JAB1
Stem + leaves Roots
*
Amount of PCBs (mg PCB/kg dry mass)* 2.1 80.8 4.0 60.0 3.3 62.9 0.7 53.1 6.5 32.8 6.3 64.5 8.1 56.3 6.3 61.6
Small amounts of biomass at the end of experiment did not allow a sufficient number of replicate measurements of PCB concentrations. Standard analysis error of the methodology is 40%.
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TABLE 8
Decrease of PCBs in soil vegetated by nightshade and tobacco and augmented by different bacterial strains
Nightshade
Added bacterial species
Initial amount of PCBs (mg PCBs/kg soil)
Final amount of PCBs (mg PCBs/kg soil)
Decrease of PCB (%)
No strain added
107.9 14.1
59.5 0.3
44.8
Pseudomonas sp. L15
85.5 11.2
21.7
Ochrobactrum sp. KH6
57.5 13.5
46.7
67.3 7.2
37.6
80.9 0.6
25
Pseudomonas sp. L15
51.1 7.5
52.8
Ochrobactrum sp. KH6
81.2 6.3
24.7
Pseudomonas sp. JAB1
73.4 0.1
32
Pseudomonas sp. JAB1 Tobacco
No strain added
107.9 14.1
Interactions of plant–microorganisms are not the only one factor affecting PCB degradation. Mutual relations between microorganisms play an important role in the nature. Bacteria can positively or negatively stimulate each other. Therefore, we studied how the addition of various bacterial species (bioaugmentation) to vegetated contaminated soil can affect PCB degradation. Bacteria previously isolated from PCB contaminated soils with verified ability to degrade PCBs were used for the bioaugmentation. Table 8 suggests that the combination of tobacco and Pseudomonas sp. KG3 leads to the best biodegradation results under given conditions. Decrease of PCBs in contaminated soil with added bacterial strain Pseudomonas sp. KG3 was over 50% whereas in control sample the decrease was only 25% within 3 months. In the sample vegetated by tobacco with Pseudomonas sp. JAB1 the decrease was 32% and with Ochrobactrum sp. KH6, the decrease of PCBs was comparable to the control sample. Different results, however, were achieved after vegetation with nightshade, where the highest decrease of PCBs concentration in soil was obtained after bioaugmentation with Ochrobactrum sp. KH6 and with Pseudomonas sp. JAB1. Vegetation by different plant species is known to influence rhizosphere microbial diversity [17,29–31]. However, we are showing here that diversity of cultured bacteria is also dependent on the way they were isolated. Two ways of isolation were performed in this study, the first was direct extraction and the second one was after long-term cultivation with biphenyl as a sole source of carbon and energy. Both ways of isolation were aimed to obtain only bacteria capable of using biphenyl as a sole source of carbon and energy and potentially degrading PCBs. The first difference in
Research Paper
Plant
these two ways of isolation is the number of morphologically different isolates; 38 isolates were obtained after direct extraction and 18 after long-term cultivation with biphenyl (Tables 2 and 3). After direct extraction three different bacterial genera were identified, but after long-term cultivation with biphenyl as a sole carbon source only bacterial genus Pseudomonas was detected. Bacteria from the genus Pseudomonas form natural soil microflora and belong to the PCBs degraders [32–36] which is also the reason why this bacterial genus was used for bioaugmentation. Pseudomonads were also the most frequently identified isolates obtained from the contaminated soil. Interestingly, when the contaminated soil from the same site was vegetated by horseradish (Armoracia rusticana), among 54 isolates only one was identified as Pseudomonas spp. [18]. This is the other evidence that different bacterial genera are favoured in rhizospheres of different plants. Isolation and identification of cultivable microorganisms from contaminated soil is the important part of rhizoremediation research. However, to find appropriate conditions for these degraders and make degradation of PCBs more efficient is important as well. In summary, the results of this study show how different strategies of bacterial isolation influence the diversity of culturable bacteria and demonstrate how combination of plants and bacteria can increase PCB removal from contaminated soil.
Acknowledgements This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project ME 10041), and Czech Science Foundation (project 525/09/1058).
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